Nucleases with improved salt tolerance and / or temperature performance

Abstract

The present invention relates to nucleases with improved properties such as enzymatic activity at high, low, and / or high salt temperatures. The present invention also relates to methods for hydrolyzing polynucleotide substrates using nucleases at high, low, or high salt temperatures. The present invention further relates to the use of nucleases for hydrolyzing polynucleotides in the manufacture of biopharmaceuticals, pharmaceutical compositions, vaccines, or viral vectors. Furthermore, the present invention relates to kits comprising nucleases.

Description

[Technical Field]

[0001] The present invention relates to nucleases with improved properties, such as enzymatic activity, at high temperatures, low temperatures, and / or high salt concentrations. The present invention also relates to methods for hydrolyzing polynucleotide substrates using nucleases at high temperatures, low temperatures, or high salt concentrations. The present invention further relates to the use of nucleases to hydrolyze polynucleotides in the production of biopharmaceuticals, pharmaceutical compositions, vaccines, or viral vectors. Furthermore, the present invention relates to kits comprising nucleases. [Background technology]

[0002] Nucleases are hydrolytic enzymes that cleave nucleic acids (DNA or RNA) and are widely and economically important. Exonucleases are distinct from endonucleases. Exonucleases cleave mononucleotides from the 5' or 3' end of nucleic acids, whereas endonucleases occur within nucleic acids, producing oligonucleotides, respectively. Furthermore, specific nucleases (e.g., restriction enzymes) are distinct from nonspecific nucleases (e.g., RNase A). Specific nucleases specifically recognize and cleave nucleic acids at defined sequences or structures, whereas nonspecific nucleases cleave nucleic acids randomly. Restriction enzymes have become essential tools in molecular biology, specifically dividing various DNA molecules into fragments that can then be selectively religated with other fragments using ligases to form new DNA molecular constructs. Nonspecific nucleases are primarily used for nucleic acid degradation in various processes. If a nuclease cleaves only DNA it is called a "DNase" and if it cleaves only RNA it is called an "RNase."

[0003] Removal of DNA and RNA from samples by nonspecific hydrolysis under the catalysis of nonspecific nucleases, particularly endonucleases, is a critical processing step in the production of several economically valuable biotechnology products, such as antibodies or enzymes, polysaccharides, lipids, small molecules such as antibiotics, metabolic end products, intermediates, or chemicals. The need to remove nucleic acids is particularly important when the production of the biotechnology product occurs intracellularly in production cells and nucleic acids are released during the production process, or when some of the production cells are lysed during production. As a result, significant amounts of nucleic acids are released into the preparation (fermentation broth), potentially contaminating the desired biotechnology product or making further purification more difficult. For example, a similar problem occurs in the production of proteins (biotechnology products) using cell-free in vitro translation. Nucleic acids increase the viscosity of the preparation to the extent that subsequent steps for separation or purification of the biotechnology product, such as filtration or chromatography, become impossible or more difficult. The use of endonucleases can degrade nucleic acids into small fragments, thereby reducing the viscosity of the preparation and allowing the resulting cleaved nucleic acids and other undesired components of the preparation to be separated from the biotechnology product using simple methods such as ultrafiltration.

[0004] The endonuclease from the species Serratia marcescens (SEQ ID NO: 1), also referred to as "Serratia marcescens nuclease" or "Serratia marcescens endonuclease," is classified as EC 3.1.30.2 according to the International Union of Biochemistry and Molecular Biology enzyme class numbering. This endonuclease catalyzes the nonspecific hydrolysis of phosphodiester bonds in both double-stranded and single-stranded DNA and RNA to generate smaller 5'-phosphorylated oligonucleotides. Serratia marcescens endonuclease is commercially available under the trade names Benzonase® or DENARASE®. This endonuclease has been "engineered" for ease of manufacture, but its amino acid sequence remains identical to wild-type Serratia marcescens endonuclease. This endonuclease has an optimum temperature of 37°C, an optimum pH of 8.0-9.0, and an optimum concentration of 1-2 mM Mg. 2+ It exhibits high activity and stability with ionic requirements and optimal performance at monovalent cation concentrations (e.g., Na+, K+) of 0-20 mM. Inherently, this endonuclease performs poorly or even non-functionally at reaction conditions outside these optimal ranges.

[0005] Therefore, the industrial use of enzymes is limited under reaction conditions outside the optimal range but still relevant to industrial manufacturing processes, particularly at high, low, or high salt concentrations. For example, enzymatic cleavage of nucleic acids in certain industrial manufacturing processes for biotechnology products requires the hydrolysis activity of nucleases under reaction conditions outside the optimal range of wild-type enzymes. Such conditions, such as the presence of salts such as NaCl or KCl at salt concentrations as low as 0.05 M or as high as 1.00 M, or at temperatures as low as 4 ° C or as high as 60 ° C, can be relatively harsh.

[0006] Because conventional nucleases known from the prior art do not meet these reaction requirements associated with various industrial applications, there is a need for nucleases that offer advantages over known nucleases, in particular nucleases that have improved resistance to high temperatures, low temperatures, or high salt concentrations. Summary of the Invention

[0007] [Objective of the Invention] It is an object of the present invention to provide an improved nuclease, preferably an endonuclease, more preferably a non-specific endonuclease, that has advantages over prior art nucleases, particularly compared to Serratia marcescens nuclease (SEQ ID NO: 1). The improved nuclease should exhibit sufficient enzymatic performance under conditions of high temperature, low temperature, or high salt concentration. The nuclease should be suitable for use in industrial hydrolysis of polynucleotides (RNA and / or DNA) into mononucleotides (e.g., by exonuclease activity) or oligonucleotides (e.g., by non-specific endonuclease activity).

[0008] This object is achieved by the subject matter of the patent claims.

[0009] [Summary of the Invention] In summary, the present invention relates to improved modified nucleases, in particular endonuclease variants, characterized by a broader range of industrial applicability. In particular, the present invention relates to variants of the endonuclease of the organism Serratia marcescens (SEQ ID NO: 1), which have amino acid substitutions at sequence positions within defined subsequences of the amino acid sequence of the wild-type enzyme.

[0010] Surprisingly, it has been found that improved modified nucleases, in particular endonuclease variants, can be provided which are characterized by high activity at salt concentrations in the range of 0.05M to 1.00M.

[0011] It has further surprisingly been found that improved modified nucleases, in particular endonuclease variants, can be provided which are characterized by high activity at temperatures in the range of 40° C. to 60° C. Even more surprisingly, it has been found that improved modified nucleases, in particular endonuclease variants, can be provided which are characterized by high activity at temperatures in the range of 4° C. to 25° C. [Brief explanation of the drawings]

[0012] [Figure 1] 1 shows a three-dimensional representation of the final model calculated by the method set forth in Example 8. [Figure 2] 1 shows a three-dimensional representation of the final model calculated by the method set forth in Example 8. [Figure 3] 1 shows a three-dimensional representation of the final model calculated by the method set forth in Example 8. [Figure 4] 1 shows a three-dimensional representation of the final model calculated by the method set forth in Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0013] A first aspect of the present invention relates to a nuclease comprising an amino acid sequence having at least 65% identity to SEQ ID NO: 1, wherein the amino acid sequence has a substitution at least one amino acid position selected from the group consisting of P51, A52, S53, G54, K55, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, K84, D86, A94, S116, D117, N119, Q120, A124, D128, Q129, K132, D135, E151, K162, D191, P195, K196, G197, D199, R204, I218, A228, and E239 compared to SEQ ID NO: 1.

[0014] A particularly preferred embodiment of the invention comprises an amino acid sequence having at least 75% identity to SEQ ID NO: 1, (i) a substitution at position P51, where P is replaced by an amino acid selected from the group consisting of L, G, A, V, and I; (ii) a substitution at position T77, where T is replaced by an amino acid selected from the group consisting of R, H, and K; (iii) (a) A124, A52, G54, P73, A74, G78, A81, and P195; or (b) T56, S53, N58, N80, N119, and Q120 wherein in each case of (a) or (b), an amino acid of SEQ ID NO: 1 is replaced by an amino acid selected from the group consisting of R, H, and K; and The present invention relates to a nuclease, preferably an endonuclease, more preferably a non-specific endonuclease, comprising:

[0015] Another particularly preferred embodiment of the present invention relates to a nuclease, preferably an endonuclease, more preferably a non-specific endonuclease, comprising an amino acid sequence having at least 65% identity with SEQ ID NO: 1, wherein the amino acid sequence is substituted at least at one amino acid position selected from the group consisting of E239, T56, K84, D191, T77, and N58 compared to SEQ ID NO: 1, in each case the native amino acid at the selected position is selected from the group consisting of A, G, V, L, I, and P; preferably selected from the group consisting of A, G, V, L, and P; more preferably selected from the group consisting of A, G, L, and P; even more preferably selected from the group consisting of A, G, and P; even more preferably selected from the group consisting of A and G; most preferably replaced by A.

[0016] Yet another particularly preferred embodiment of the present invention relates to a nuclease, preferably an endonuclease, having at least 70% identity to SEQ ID NO: 1, wherein the amino acid sequence has been substituted in at least two amino acid positions selected from the group consisting of P51, T56, K84, and N58 compared to SEQ ID NO: 1, in each case the native amino acid at the selected position is substituted by an amino acid selected from the group consisting of L, P, A, G, V, and I; preferably selected from the group consisting of L, P, I, G, and V; more preferably selected from the group consisting of L, P, I, and G; even more preferably selected from the group consisting of L, P, and G; even more preferably selected from the group consisting of L and P.

[0017] [Definition] For purposes of this specification, a nuclease (also known as a nucleodepolymerase or polynucleotidase) is a hydrolytic enzyme capable of cleaving the phosphodiester bond between nucleotides of nucleic acids (DNA or RNA). Exonucleases digest nucleic acids from the 5' or 3' end to form mononucleotides, while endonucleases act on the middle region of nucleic acids to form oligonucleotides. Furthermore, specific nucleases are distinguished from nonspecific nucleases, in that specific nucleases recognize and cleave nucleic acids with a defined sequence or structure, while nonspecific nucleases randomly cleave nucleic acids. Certain nucleases can cleave only DNA, others can cleave only RNA, and others can cleave both DNA and RNA. The nuclease according to the present invention is preferably an endonuclease, more preferably a nonspecific endonuclease. In a preferred embodiment, the nuclease is a deoxyribonuclease, preferably a deoxyriboendonuclease. In another preferred embodiment, the nuclease is a ribonuclease, preferably a riboendonuclease.

[0018] Within the meaning of the present invention, whenever an embodiment of any aspect of the invention refers to a nuclease, it is intended that any such embodiment also refers to an endonuclease and / or a polypeptide that provides the same embodiment. In this respect, the present invention also relates to an endonuclease and / or polypeptide comprising an amino acid sequence having at least 65% identity to SEQ ID NO: 1, wherein the amino acid sequence has been substituted in at least one amino acid position selected from the group consisting of P51, A52, S53, G54, K55, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, K84, D86, A94, S116, D117, N119, Q120, A124, D128, Q129, K132, D135, E151, K162, D191, P195, K196, G197, D199, R204, I218, A228, and E239 compared to SEQ ID NO: 1. Thus, all embodiments of any aspect of the invention that refer to a nuclease shall also refer correspondingly to an endonuclease and / or polypeptide.

[0019] The nuclease according to the present invention comprises an amino acid sequence having a defined identity with the amino acid sequence of SEQ ID NO: 1. This means that any nuclease according to the present invention may comprise the amino acid sequence as a subsequence of the entire amino acid sequence, or may consist essentially of the amino acid sequence. When a nuclease according to the present invention comprises the amino acid sequence as a subsequence of the entire amino acid sequence, the entire amino acid sequence may be extended, i.e., may contain additional amino acid residues at the N-terminus and / or C-terminus of the subsequence. Such extension may be advantageous, for example, when the nuclease is immobilized on a solid support for purification.

[0020] For purposes herein, percent identity is calculated as follows: sequence identity [%] = number of matches / L × 100, where L is the number of aligned positions, i.e., identities and non-identities (including gaps, if any). Identity is preferably calculated using BLASTP with the following algorithm parameters: Matrix: BLOSUM62; Gap Costs: Existence: 11; Extension: 1; Expect threshold: 0.05; and Word size: 6 (see, e.g., Altschul SF et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402; or Altschul SF (2005) "Protein database searches using compositionally adjusted substitution matrices," FEBS J. 272:5101-5109). BlastP is accessible online at the NCBI homepage (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). Other program settings can be adjusted as desired, for example, using the following settings: -Field "Enter Query Sequence":Query subrange:None; -Field "Choose Search Set":Database:non-redundant protein sequences(nr);Optional parameters:none; -Field "Program Selection":Algorithm:blastp(protein-protein BLAST) -Algorithm parameters:Field "General parameters":Max target sequences:5000;Short queries:Automatically adjust parameters for short input sequences;Expect threshold:0.05;Word size:6;Max matches in a query range:0; -Algorithm parameters:Field "Scoring parameters":Matrix:BLOSUM62;Gap Costs:Existence:11 Extension:1;Compositional adjustments:Conditional compositional score matrix adjustment; -Algorithm parameters:Field "Filters and Masking":Filter: None;Mask: None.

[0021] In addition to the default parameters for calculating percent identity, when a reference sequence, referred to as a "query sequence," is aligned with another sequence, referred to as a "subject sequence," particularly when the query sequence is any sequence provided by the present invention, preferably SEQ ID NO: 1, and the subject sequence is any other sequence, such as a sequence disclosed in a database, the subject sequence must be aligned over a sequence range covering at least 70% of the entire length of the query sequence (at least 70% "sequence coverage"), and the alignment of both sequences must cover at least 70% of the query sequence. For purposes of this specification, sequence coverage is calculated as the number of aligned positions, i.e., the number of identical and non-identical positions (including gaps, if any), divided by the entire length of the query sequence and multiplied by 100. Alignments with low sequence coverage of the query sequence by the subject sequence are excluded from the determination of sequence identity for purposes of this application. However, the subject sequence may be longer than the length of the alignment.

[0022] During expression of a nuclease, whether naturally or recombinantly expressed in a particular host organism, the nuclease gene may be linked to a so-called signal peptide ("SP") gene sequence. The SP gene sequence is expressed along with the nuclease to form a single precursor protein containing the SP and nuclease protein portions. The SP element of the SP-nuclease fusion protein typically serves as a secretion signal and is cleaved from the nuclease during the secretion process. For purposes of this specification, the gene or peptide sequence of the nuclease's signal peptide is ignored in calculating the identity of the nuclease according to the present invention. Tools for identifying SP gene or protein sequences are known to those skilled in the art and are described, for example, in section 2 of https: / / www.uniprot.org / help / signal, where the prediction tools Phobius, Predotar, SignalP, and TargetP are given as examples.

[0023] For the purposes of this specification, a particular target amino acid sequence being substituted at "a particular amino acid position number compared to SEQ ID NO: 1" means that such target amino acid sequence aligned with "amino acid position number of SEQ ID NO: 1" in an alignment made in accordance with the sequence identity requirements is substituted at such target amino acid position number of the target amino acid sequence, regardless of whether the amino acid position number of SEQ ID NO: 1 and the target amino acid position number are identical or different.

[0024] For the purposes of this specification, a particular target amino acid sequence being substituted "at a particular amino acid position number compared to SEQ ID NO: 1" further means that at any particular amino acid position number in SEQ ID NO: 1, the amino acid of SEQ ID NO: 1 at such position is substituted by any different amino acid selected from the group of naturally occurring proteinogenic amino acids, i.e., A, R, N, D, C, Q, E, G, H, I, L, K, M, F, T, W, Y, V, P or S.

[0025] For purposes herein, substituted at least at an amino acid position in a nuclease means that at least one amino acid position is substituted with either (i) a positively charged amino acid selected from R, K, and H, or (ii) a negatively charged amino acid selected from E and D, or (iii) an aliphatic non-polar amino acid selected from G, A, V, L, I, and P, or (iv) an aromatic amino acid selected from W, F, and Y, or (v) an aliphatic polar uncharged amino acid selected from N, Q, S, T, C, and M.

[0026] For purposes herein, the amino acid position of the nuclease that is substituted may be at least (i) a positively charged amino acid that is substituted with another positively charged amino acid, or (ii) a negatively charged amino acid that is substituted with another negatively charged amino acid, or (iii) an aliphatic nonpolar amino acid that is substituted with another aliphatic nonpolar amino acid, or (iv) an aromatic amino acid that is substituted with another aromatic amino acid, or (v) an aliphatic polar uncharged amino acid that is substituted with another aliphatic polar uncharged amino acid.

[0027] For purposes herein, the amino acid positions of the nuclease that are substituted are at least (i) a positively charged amino acid that is substituted with an aliphatic nonpolar amino acid, or an aromatic amino acid, or an aliphatic polar uncharged, or negatively charged amino acid; or (ii) a negatively charged amino acid that is substituted with an aliphatic nonpolar amino acid, or an aromatic amino acid, or an aliphatic polar uncharged, or positively charged amino acid; or (iii) an aliphatic nonpolar amino acid that is substituted with an aromatic amino acid, or an aliphatic polar uncharged, or positively charged, or negatively charged amino acid; or (iv) an aromatic amino acid that is substituted with an aliphatic nonpolar amino acid, or an aliphatic polar uncharged, or positively charged, or negatively charged amino acid; or (v) an aliphatic polar uncharged amino acid that is substituted with an aliphatic nonpolar amino acid, or an aromatic amino acid, or a positively charged, or negatively charged amino acid.

[0028] For purposes of this specification, the charge state of any amino acid substituted at any position compared to SEQ ID NO: 1, in particular the charge of the positively charged amino acids R, K, and H and the charge of the negatively charged amino acids E and D, is determined at a pH of 7.

[0029] For purposes of this specification, the specific substitution positions disclosed herein are described in accordance with common practice in the field of amino acid substitution, and the format [unsubstituted amino acid - sequence position] is chosen, where the sequence position is located immediately adjacent to the amino acid letter (single-letter code) of the amino acid not yet mutated to the left, and no specific amino acid is chosen as a substitution for such amino acid position. For purposes of clarity only, the substitution "P51" indicates the substitution of the amino acid proline (P) at sequence position 51 with any substitution.

[0030] For purposes of this specification, the specific substitutions disclosed herein are described in accordance with common practice in the field of amino acid substitutions, and are presented in the format [unsubstituted amino acid-sequence position-substituted amino acid], with the sequence position immediately adjacent to the amino acid letter (single-letter code) on the left or right side. For purposes of clarity only, the substitution "P51R" indicates the substitution of the amino acid proline (P) at sequence position 51 with the amino acid arginine (R), i.e., P is replaced by R.

[0031] <Structural characteristics of the nuclease according to the present invention> In a preferred embodiment of the nuclease according to the invention, the nuclease is present in a liquid formulation.

[0032] In a preferred embodiment of the nuclease according to the present invention, the nuclease is present in a liquid formulation comprising (i) a buffer, (ii) glycerol, sucrose, or other stabilizer, and / or (iii) a benzoate, or other preservative.

[0033] In a preferred embodiment of the nuclease according to the invention, the nuclease is solid or is present in a solid formulation, preferably a powder.

[0034] In a preferred embodiment of the nuclease according to the invention, the nuclease is present as or contained in a powder obtained by freeze-drying, spray-drying or other techniques for drying known in the state of the art.

[0035] In a preferred embodiment of the nuclease according to the invention, the nuclease is contained in a crude cell lysate, or a crude cell supernatant, or a partially purified enzyme derived from any of the foregoing, or a purified enzyme derived from any of the foregoing.

[0036] In a preferred embodiment of the nuclease according to the invention, the nuclease is present immobilized on a support.

[0037] In preferred embodiments of nucleases according to the invention, the amino acid sequence of the nuclease has at least 66%, preferably at least 69%, more preferably at least 72%, even more preferably at least 75%, even more preferably at least 78%, even more preferably at least 80%, even more preferably at least 81%, most preferably at least 84%, and especially at least 87% identity to SEQ ID NO: 1. In preferred embodiments of nucleases according to the invention, the amino acid sequence has at least 88%, preferably at least 89%, more preferably at least 90%, even more preferably at least 91%, even more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and especially at least 95% identity to SEQ ID NO: 1.

[0038] In a preferred embodiment of the nuclease according to the invention, the identity of the amino acid sequence of the nuclease with the sequence of SEQ ID NO: 1 is at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%, or at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or is at least 85%; preferably at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%; more preferably at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%; even more preferably at least 96%, or at least 97%, or at least 98%, or at least 99%; even more preferably at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4, or at least 99.5%; even more preferably at least 99.6%.

[0039] In preferred embodiments of the nuclease according to the invention, the identity of the amino acid sequence of the nuclease with the sequence of SEQ ID NO: 1 is at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%; preferably at least 96%, or at least 97%, or at least 98%, or at least 99%; more preferably at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4, or at least 99.5%; even more preferably at least 99.6%.

[0040] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence is selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 , 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 6, 187, 188, 189, 190, 191, 192, 193, 194, or 195, preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, most preferably at least 97%, most preferably at least 98%, and especially at least 99%, or 100% identical to

[0041] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence is selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, or 147, preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, most preferably at least 97%, most preferably at least 98%, and especially at least 99%, or 100% identical to

[0042] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has been substituted at position P51 compared to SEQ ID NO: 1, the substitutions being P51A, P51R, P51N, P51D, P51C, P51Q, P51E, P51G, P51H, P51I, P51L, P51K, P51M, P51F, P51T, P51W, P51Y, P51V and P51S, preferably are selected from the group consisting of P51D, P51E, P51R, P51K, P51H, P51G, P51A, P51V, P51L, and P51I, more preferably P51L, P51R, P51G, P51D, P51A, P51V, P51I, even more preferably P51D, P51R, P51G, and P51L, and most preferably P51R, P51G, and P51L.

[0043] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at position A52 compared to SEQ ID NO: 1, wherein the substitution is selected from the group consisting of A52R, A52N, A52D, A52C, A52Q, A52E, A52G, A52H, A52I, A52L, A52K, A52M, A52F, A52P, A52T, A52W, A52Y, A52V and A52S, preferably A52R, A52K, A52H, more preferably A52R.

[0044] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has been substituted at position S53 compared to SEQ ID NO: 1, the substitutions being S53A, S53R, S53N, S53D, S53C, S53Q, S53E, S53G, S53H, S53I, S53L, S53K, S53M, S53F, S53P, S53T, S53W, S53Y and S53V, preferably S S53R, S53K, S53H, S53G, S53A, S53V, S53L, S53I, S53P, S53W, S53F, S53Y, and S53D, more preferably S53R, S53K, S53H, S53G, S53A, S53V, S53L, S53I, and S53P, even more preferably S53K, and S53A, and most preferably S53K.

[0045] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at position G54 compared to SEQ ID NO: 1, wherein the substitution is selected from the group consisting of G54A, G54R, G54N, G54D, G54C, G54Q, G54E, G54H, G54I, G54L, G54K, G54M, G54F, G54P, G54T, G54W, G54Y, G54V and G54S, preferably G54D, G54E, G54R, G54K, G54H, G54A, G54V, G54L, G54I and G54P, preferably G54K, G54H and G54R, more preferably G54R.

[0046] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has a substitution at position K55 compared to SEQ ID NO: 1, wherein the substitution is selected from the group consisting of K55A, K55R, K55N, K55D, K55C, K55Q, K55E, K55G, K55H, K55I, K55L, K55M, K55F, K55P, K55T, K55W, K55Y, K55V and K55S, preferably K55G, K55A, K55V, K55L, K55I, K55P, K55R, K55H, K55N, K55Q, K55S, K55T, K55C and K55M, more preferably K55H and K55R, most preferably K55R.

[0047] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has been substituted at position T56 compared to SEQ ID NO: 1, the substitutions being T56A, T56R, T56N, T56D, T56C, T56Q, T56E, T56G, T56H, T56I, T56L, T56K, T56M, T56F, T56P, T56W, T56Y, T56V, and T 56S, preferably T56P, T56D, T56R, T56K, T56S, T56G, T56A, T56V, T56I, T56F, T56L, and T56M, even more preferably T56P, T56D, T56R, T56K, T56S, and T56G, most preferably T56R, T56K, T56S, and T56G.

[0048] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has a substitution at position N58 compared to SEQ ID NO: 1, the substitution being N58A, N58R, N58D, N58C, N58Q, N58E, N58G, N58H, N58I, N58L, N58K, N58M, N58F, N58P, N58T, N58W, N58Y, N58V and N58S, preferably N58R, N58R N58K, N58H, N58G, N58A, N58V, N58L, N58I, N58P, N58W, N58F, N58Y, and N58D, more preferably N58R, N58K, N58H, N58G, N58A, N58V, N58L, N58I, and N58P, more preferably N58K, N58R, and N58V, and most preferably N58K and N58R.

[0049] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at position A72 compared to SEQ ID NO: 1, wherein the substitution is selected from the group consisting of A72R, A72N, A72D, A72C, A72Q, A72E, A72G, A72H, A72I, A72L, A72K, A72M, A72F, A72P, A72T, A72W, A72Y, A72V and A72S, preferably A72D, A72E, A72R, A72K, A72H, A72G, A72V, A72L, A72I and A72P, more preferably A72K, A72H and A72R, most preferably A72K.

[0050] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has a substitution at position P73 compared to SEQ ID NO: 1, wherein the substitution is selected from the group consisting of P73A, P73R, P73N, P73D, P73C, P73Q, P73E, P73G, P73H, P73I, P73L, P73K, P73M, P73F, P73T, P73W, P73Y, P73V and P73S, preferably P73D, P73E, P73R, P73K, P73H, P73G, P73V, P73L, P73I and P73A, more preferably P73K, P73H and P73R, most preferably P73K and P73R.

[0051] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at position A74 compared to SEQ ID NO: 1, wherein the substitution is selected from the group consisting of A74R, A74N, A74D, A74C, A74Q, A74E, A74G, A74H, A74I, A74L, A74K, A74M, A74F, A74P, A74T, A74W, A74Y, A74V and A74S, preferably A74D, A74E, A74R, A74K, A74H, A74G, A74V, A74L, A74I and A74P, more preferably A74K, A74H and A74R, most preferably A74K and A74R.

[0052] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at position T77 compared to SEQ ID NO: 1, wherein the substitution is selected from the group consisting of T77A, T77R, T77N, T77D, T77C, T77Q, T77E, T77G, T77H, T77I, T77L, T77K, T77M, T77F, T77P, T77W, T77Y, T77V and T77S, preferably T77R, T77K, T77H, T77G, T77A, T77V, T77L, T77I, T77P, T77W, T77F, T77Y and T77D, more preferably T77K, T77R, T77H, T77F and T77L, most preferably T77K, T77R, T77F and T77L.

[0053] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at position G78 compared to SEQ ID NO: 1, wherein the substitution is selected from the group consisting of G78A, G78R, G78N, G78D, G78C, G78Q, G78E, G78H, G78I, G78L, G78K, G78M, G78F, G78P, G78T, G78W, G78Y, G78V and G78S, preferably G78R, G78K and G78H, more preferably G78R.

[0054] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at position N80 compared to SEQ ID NO: 1, wherein the substitution is selected from the group consisting of N80A, N80R, N80D, N80C, N80Q, N80E, N80G, N80H, N80I, N80L, N80K, N80M, N80F, N80P, N80T, N80W, N80Y, N80V and N80S, preferably N80R, N80K, N80H, N80G, N80A, N80V, N80L, N80I, N80P, N80W, N80F, N80Y and N80D, preferably N80K, N80R and N80H, more preferably N80H and N80R, most preferably N80H.

[0055] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has a substitution at position A81 compared to SEQ ID NO: 1, wherein the substitution is selected from the group consisting of A81R, A81N, A81D, A81C, A81Q, A81E, A81G, A81H, A81I, A81L, A81K, A81M, A81F, A81P, A81T, A81W, A81Y, A81V and A81S, preferably A81K, A81H and A81R, more preferably A81K.

[0056] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at position A82 compared to SEQ ID NO: 1, wherein the substitution is selected from the group consisting of A82R, A82N, A82D, A82C, A82Q, A82E, A82G, A82H, A82I, A82L, A82K, A82M, A82F, A82P, A82T, A82W, A82Y, A82V and A82S, preferably A82K, A82H and A82R, more preferably A82R.

[0057] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has a substitution at position K84 compared to SEQ ID NO: 1, wherein the substitution is selected from the group consisting of K84A, K84R, K84N, K84D, K84C, K84Q, K84E, K84G, K84H, K84I, K84L, K84M, K84F, K84P, K84T, K84W, K84Y, K84V and K84S, preferably K84G, K84A, K84V, K84L, K84I, K84P, K84R, K84H, K84N, K84Q, K84S, K84T, K84C and K84M, preferably K84G, K84A, K84V, K84L, K84I and K84P, more preferably K84G.

[0058] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has been substituted at position D86 compared to SEQ ID NO: 1, the substitution being selected from the group consisting of D86A, D86R, D86N, D86C, D86Q, D86E, D86G, D86H, D86I, D86L, D86K, D86M, D86F, D86P, D86T, D86W, D86Y, D86V and D86S; D86G, D86A, D86V, D86L, D86I, D86P, D86C, D86M, D86S, D86T and D86Q, preferably D86C, D86M, D86S, D86T and D86Q, more preferably D86S.

[0059] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at position A94 compared to SEQ ID NO: 1, wherein the substitution is selected from the group consisting of A94R, A94N, A94D, A94C, A94Q, A94E, A94G, A94H, A94I, A94L, A94K, A94M, A94F, A94P, A94T, A94W, A94Y, A94V, and A94S; preferably A94G, A94V, A94L, A94I, and A94P; more preferably A94G.

[0060] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has a substitution at position S116 compared to SEQ ID NO: 1, the substitution being S116A, S116R, S116N, S116D, S116C, S116Q, S116E, S116G, S116H, S116I, S116L, S116K, S116M, S116F, S116P, S116T , S116W, S116Y, and S116V, preferably S116R, S116K, S116H, S116G, S116A, S116V, S116L, S116I, S116P, S116W, S116F, S116Y, and S116D, preferably S116K, S116H, and S116R, more preferably S116K.

[0061] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has been substituted at position D117 compared to SEQ ID NO: 1, the substitutions being D117G, D117A, D117V, D117L, D117I, D117P, D117W, D117F, D117Y, D117C, D117M, D117S, D117Q, D117T, D117N, D117E, D117H, D117R and D117K, preferably D117G, D117A, D117V, D117L, D117I, D117P, D117W, D117F, D117Y, D117C, D117M, D117S, D117Q, D117T, D117N, D117H, D117R and D117K, more preferably D117K, D117A, D117S, D117N, D117R, D117H, and D117Y, even more preferably D117A, D117N, D117R, D117K, and D117S, even more preferably D117A, D117N, D117K, and D117S, most preferably D117A, D117N, and D117S.

[0062] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at position N119 compared to SEQ ID NO: 1, wherein the substitution is selected from the group consisting of N119A, N119R, N119D, N119C, N119Q, N119E, N119G, N119H, N119I, N119L, N119K, N119M, N119F, N119P, N119T, N119W, N119Y, N119V and N119S, preferably N119R, N119K and N119H, more preferably N119R.

[0063] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has a substitution at position Q120 compared to SEQ ID NO: 1, the substitution being Q120A, Q120R, Q120N, Q120D, Q120C, Q120E, Q120G, Q120H, Q120I, Q120L, Q120K, Q120M, Q120F, Q120P, Q120T, Q120W, Q120Y, Q120V, and Q120S, preferably Q120R, Q120K, Q120H, Q120G, Q120A, Q120V, Q120L, Q120I, Q120P, Q120W, Q120F, Q120Y, and Q120D, more preferably Q120K, Q120H, and Q120R, most preferably Q120R.

[0064] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at position A124 compared to SEQ ID NO: 1, wherein the substitution is selected from the group consisting of A124R, A124N, A124D, A124C, A124Q, A124E, A124G, A124H, A124I, A124L, A124K, A124M, A124F, A124P, A124T, A124W, A124Y, A124V and A124S, preferably A124D, A124E, A124R, A124K, A124H, A124G, A124V, A124L, A124I and A124P, preferably A124K, A124H and A124R, more preferably A124K and A124R.

[0065] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has been substituted at position D128 compared to SEQ ID NO: 1, the substitutions being D128A, D128R, D128N, D128C, D128Q, D128E, D128G, D128H, D128I, D128L, D128K, D128M, D128F, D128P, D128T, D128W, D128Y, D128V and D128S, preferably Preferably, it is selected from the group consisting of D128G, D128A, D128V, D128L, D128I, D128P, D128W, D128F, D128Y, D128C, D128M, D128N, D128Q, D128S, D128T, D128H, D128R and D128K, most preferably D128G, D128Y, D128F, D128M, D128N, D128S, D128R and D128K.

[0066] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has a substitution at position Q129 compared to SEQ ID NO: 1, the substitution being Q129A, Q129R, Q129N, Q129D, Q129C, Q129E, Q129G, Q129H, Q129I, Q129L, Q129K, Q129M, Q129F, Q129P, Q129T, Q129W, Q129Y, Q129 V, and Q129S, preferably Q129R, Q129K, Q129H, Q129G, Q129A, Q129V, Q129L, Q129I, Q129P, Q129W, Q129F, Q129Y, and Q129D, preferably Q129K, Q129H, and Q129R, more preferably Q129K, Q129R, even more preferably Q129R.

[0067] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at position K132 compared to SEQ ID NO: 1, wherein the substitution is selected from the group consisting of K132A, K132R, K132N, K132D, K132C, K132Q, K132E, K132G, K132H, K132I, K132L, K132M, K132F, K132P, K132T, K132W, K132Y, K132V and K132S, preferably K132R and K132H, most preferably K132R.

[0068] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has been substituted at position D135 compared to SEQ ID NO: 1, the substitutions being D135A, D135R, D135N, D135C, D135Q, D135E, D135G, D135H, D135I, D135L, D135K, D135M, D135F, D135P, D135T, D135W, D135Y, D135V and D135S, preferably D135G, D135A, D135V, D135L, D135I, D135P, D135W , D135F, D135Y, D135C, D135M, D135N, D135Q, D135S, D135T, D135R, D135H, and D135K, more preferably D135G, D135A, D135V, D135L, D135I, D135P, D135C, D135M, D135N, D135Q, D135S, D135T, D135R, D135H, and D135K, most preferably D135A, D135M, D135G, D135N, D135R, and D135K.

[0069] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has been substituted at position E151 compared to SEQ ID NO: 1, the substitutions being E151A, E151R, E151N, E151D, E151C, E151Q, E151G, E151H, E151I, E151L, E151K, E151M, E151F, E151P, E151T, E151W, E151Y, E151V, and E151R. E151S, E151G, E151A, E151V, E151L, E151I, E151P, E151W, E151F, E151Y, E151C, E151M, E151N, E151Q, E151S, E151T, E151H and E151K, preferably E151A, E151G, E151V, E151L, E151I and E151P, more preferably E151L.

[0070] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has a substitution at position K162 compared to SEQ ID NO: 1, wherein the substitution is selected from the group consisting of K162A, K162R, K162N, K162D, K162C, K162Q, K162E, K162G, K162H, K162I, K162L, K162M, K162F, K162P, K162T, K162W, K162Y, K162V and K162S, preferably K162N, K162Q, K162S, K162T, K162C and K162M, most preferably K162N.

[0071] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has been substituted at position D191 compared to SEQ ID NO: 1, the substitutions being D191G, D191A, D191V, D191L, D191I, D191P, D191W, D191F, D191Y, D191C, D191M, D191S, D191Q, D191T, D191N, D191E, D191F, ...F, D191F, D191G, D191A, D191G, D191A, D191G, D191H, D191H, D191H, D191H, D191H, D191H, D191H, D191H, D191H, D191H, D191H, D191H, D191H, D191H, D191H, D191H, D191H, D191H, D191H, D191R and D191K, preferably D191G, D191A, D191V, D191L, D191I, D191P, D191C, D191M, D191S, D191Q and D191T, more preferably D191G, D191A, D191V, D191L, D191I and D191P, most preferably D191P.

[0072] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has a substitution at position P195 compared to SEQ ID NO: 1, wherein the substitution is selected from the group consisting of P195A, P195R, P195N, P195D, P195C, P195Q, P195E, P195G, P195H, P195I, P195L, P195K, P195M, P195F, P195T, P195W, P195Y, P195V and P195S, preferably P195K, P195H and P195R, most preferably P195R.

[0073] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has been substituted at position K196 compared to SEQ ID NO: 1, the substitution being K196A, K196R, K196N, K196D, K196C, K196Q, K196E, K196G, K196H, K196I, K196L, K196M, K196F, K196P, K196T, K196W, K196Y, K196V, and K196S, preferably K196G, K196A, K196V, K196L, K196I, K196P, K196R, K196H, K196N, K196Q, K196S, K196T, K196C, and K196M, more preferably K196R, and K196H, most preferably K196R.

[0074] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at position G197 compared to SEQ ID NO: 1, the substitution being selected from the group consisting of G197A, G197R, G197N, G197D, G197C, G197Q, G197E, G197H, G197I, G197L, G197K, G197M, G197F, G197P, G197T, G197W, G197Y, G197V and G197S, preferably G197D, G197E, G197R, G197K, G197H, G197A, G197V, G197L, G197I and G197P, preferably G197K, G197H and G197R, more preferably G197K.

[0075] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has been substituted at position D199 compared to SEQ ID NO: 1, the substitutions being D199G, D199A, D199V, D199L, D199I, D199P, D199W, D199F, D199Y, D199C, D199M, D199S, D199Q, D199T, D199N, D199E, D199H, D199R and D199K, preferably D199G. , D199A, D199V, D199L, D199I, D199P, D199W, D199F, D199Y, D199C, D199M, D199N, D199S, D199Q, D199T, and D199K, preferably D199N, D199S, D199Q, D199T, D199C, D199M, D199H, and D199K, more preferably D199N, D199S, and D199K.

[0076] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has a substitution at position R204 compared to SEQ ID NO: 1, the substitution being R204A, R204N, R204D, R204C, R204Q, R204E, R204G, R204H, R204I, R204L, R204K, R204M, R204F, R204P, R204T, R204W, R204Y, R204V , and R204S, preferably R204G, R204A, R204V, R204L, R204I, R204P, R204K, R204H, R204N, R204Q, R204S, R204T, R204C, and R204M, more preferably R204Q, R204N, R204S, R204T, R204C and R204M, most preferably R204Q.

[0077] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has a substitution at position I218 compared to SEQ ID NO: 1, wherein the substitution is selected from the group consisting of I218R, I218N, I218D, I218C, I218Q, I218E, I218G, I218H, I218A, I218L, I218K, I218M, I218F, I218P, I218T, I218W, I218Y, I218V and I218S, preferably I218F, I218W and I218Y, more preferably I218F.

[0078] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at position A228 compared to SEQ ID NO: 1, wherein the substitution is selected from the group consisting of A228R, A228N, A228D, A228C, A228Q, A228E, A228G, A228H, A228I, A228L, A228K, A228M, A228F, A228P, A228T, A228W, A228Y, A228V and A228S, preferably A228K, A228H and A228R, more preferably A228R.

[0079] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has been substituted at position E239 compared to SEQ ID NO: 1, the substitutions being E239A, E239R, E239N, E239D, E239C, E239Q, E239G, E239H, E239I, E239L, E239K, E239M, E239F, E239P, E239T, E239W, E239Y, E239V, and E239 S, preferably E239G, E239A, E239V, E239L, E239I, E239P, E239W, E239F, E239Y, E239C, E239M, E239N, E239Q, E239S, E239T, E239H and E239K, preferably E239A, E239G, E239V, E239L, E239I, and E239P, more preferably E239A.

[0080] In a preferred embodiment of the nuclease according to the invention, A substitution at position P51, P73, or P195 means that P is replaced by an amino acid selected from the group consisting of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, T, W, Y, V, or S; and / or A substitution at position S53, or S116 means that S is replaced by an amino acid selected from the group consisting of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, T, W, Y, or V; and / or A substitution at position K55, K84, K132, K162, or K196 means that K is replaced by an amino acid selected from the group consisting of A, R, N, D, C, Q, E, G, H, I, L, M, F, P, T, W, Y, V, or S; and / or A substitution at position T56, or T77 means that T is replaced by an amino acid selected from the group consisting of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, W, Y, V, or S; and / or A substitution at position N58, N80, or N119 means that N is replaced by an amino acid selected from the group consisting of A, R, D, C, Q, E, G, H, I, L, K, M, F, P, T, W, Y, V, or S; and / or A substitution at position A52, A72, A74, A81, A82, A94, A124, or A228 means that A is replaced by an amino acid selected from the group consisting of R, N, D, C, Q, E, G, H, I, L, K, M, F, P, T, W, Y, V, or S; and / or A substitution at position G54, G78, or G197 means that G is replaced by an amino acid selected from the group consisting of A, R, N, D, C, Q, E, H, I, L, K, M, F, P, T, W, Y, V, or S; and / or A substitution at position D86, D117, D128, D135, D191 or D199 means that D is replaced by an amino acid selected from the group consisting of A, R, N, C, Q, E, G, H, I, L, K, M, F, P, T, W, Y, V or S; and / or A substitution at position Q120, or Q129 means that Q is substituted by an amino acid selected from the group consisting of A, R, N, D, C, E, G, H, I, L, K, M, F, P, T, W, Y, V, or S; and / or A substitution at position R204 means that R is substituted by an amino acid selected from the group consisting of A, N, D, C, Q, E, G, H, I, L, K, M, F, P, T, W, Y, V, or S; and / or A substitution at position 1218 means that I is replaced by an amino acid selected from the group consisting of A, R, N, D, C, Q, E, G, H, L, K, M, F, P, T, W, Y, V, or S. A substitution at position E151 or E239 means that E is replaced by an amino acid selected from the group consisting of A, R, N, D, C, Q, G, H, I, L, K, M, F, P, T, W, Y, V, or S.

[0081] In a preferred embodiment of the nuclease according to the invention, at least one amino acid position is substituted by: a positively charged amino acid selected from R, K, and H; or a negatively charged amino acid selected from E and D; an aliphatic nonpolar amino acid selected from G, A, V, L, I, and P; or an aromatic amino acid selected from W, F, and Y; or an aliphatic polar uncharged amino acid selected from N, Q, S, T, C, and M;

[0082] In a preferred embodiment of the nuclease according to the invention, at least one amino acid position is is a positively charged amino acid and is substituted by another positively charged amino acid; or is a negatively charged amino acid and is substituted by another negatively charged amino acid; or an aliphatic nonpolar amino acid that has been substituted by another aliphatic nonpolar amino acid; or is an aromatic amino acid and is substituted by another aromatic amino acid; or It is an aliphatic polar uncharged amino acid that is substituted by another aliphatic polar uncharged amino acid.

[0083] In a preferred embodiment of the nuclease according to the invention, at least one amino acid position is a positively charged amino acid that is substituted by an aliphatic nonpolar amino acid, or an aromatic amino acid, or an aliphatic polar uncharged or negatively charged amino acid; or a negatively charged amino acid that is substituted by an aliphatic nonpolar amino acid, or an aromatic amino acid, or an aliphatic polar uncharged or positively charged amino acid; or an aliphatic nonpolar amino acid substituted with an aromatic amino acid, or an aliphatic polar uncharged, positively charged, or negatively charged amino acid; or an aromatic amino acid substituted with an aliphatic nonpolar amino acid, or an aliphatic polar uncharged, positively charged, or negatively charged amino acid; or It is an aliphatic polar uncharged amino acid, substituted by an aliphatic nonpolar amino acid, or an aromatic amino acid, or a positively charged amino acid, or a negatively charged amino acid.

[0084] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence is substituted at amino acid position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38 compared to SEQ ID NO: 1.

[0085] In a preferred embodiment of the nuclease according to the present invention, the amino acid sequence is substituted at amino acid positions 1 to 86 compared to SEQ ID NO: 1; preferably at amino acid positions 1 to 84; more preferably at amino acid positions 1 to 50; even more preferably at amino acid positions 1 to 39; even more preferably at amino acid positions 1 to 27; even more preferably at amino acid positions 1 to 11; and even more preferably at amino acid positions 1 to 6.

[0086] A preferred embodiment of the present invention relates to a nuclease, preferably an endonuclease, more preferably a non-specific endonuclease, even more preferably a nuclease according to any of the above inventions, comprising an amino acid sequence having at least 65% identity to SEQ ID NO: 1, wherein the amino acid sequence has a substitution in at least one amino acid position selected from the group consisting of P51, S53, K55, T56, N58, A72, P73, A74, T77, N80, K84, D86, S116, D117, A124, D128, D135, D191, K196, G197, D199, R204, and E239 compared to SEQ ID NO: 1; Substitutions at positions P51, A72, P73, A74, A124, or G197 are a positively charged amino acid, or a negatively charged amino acid, or means that it is substituted by a different aliphatic nonpolar amino acid; Substitutions at positions S53, T56, N58, T77, N80, or S116 are those where S, T, or N is a positively charged amino acid, or an aliphatic nonpolar amino acid, or aromatic amino acids, or aliphatic polar uncharged amino acids, or means that it is substituted by a negatively charged amino acid; Substitutions at positions K55, K84, R204, or K196 are an aliphatic nonpolar amino acid, or a different positively charged amino acid, or means that it is substituted by an aliphatic polar uncharged amino acid; The substitution at position D86 or D191 is an aliphatic nonpolar amino acid, or means that it is substituted by a polar uncharged amino acid; Substitutions at positions D128, D135, D117, E239 or D199 are an aliphatic nonpolar amino acid, or aromatic amino acids, or aliphatic polar uncharged amino acids, or It means that it is substituted with a positively charged amino acid.

[0087] Another preferred embodiment of the present invention relates to a nuclease, preferably an endonuclease, more preferably a non-specific endonuclease, and even more preferably a nuclease according to the invention as described above, comprising an amino acid sequence having at least 65% identity to SEQ ID NO: 1, wherein the amino acid sequence has been substituted in at least one amino acid position selected from the group consisting of P51, S53, K55, T56, N58, A72, P73, A74, T77, N80, K84, D86, S116, D117, A124, D128, D135, D191, K196, G197, D199, R204, and E239 compared to SEQ ID NO: 1; Substitutions at positions P51, A72, P73, A74, A124, or G197 are a positively charged amino acid selected from R, K, and H, or a negatively charged amino acid selected from D and E, or is substituted by a different aliphatic nonpolar amino acid selected from G, A, V, L, I, and P; Substitutions at positions S53, T56, N58, T77, N80, or S116 are those where S, T, or N is a positively charged amino acid selected from R, K, and H, or an aliphatic nonpolar amino acid selected from G, A, V, L, I, and P; or an aromatic amino acid selected from W, F, and Y; or an aliphatic polar uncharged amino acid selected from N, Q, S, T, C, and M, preferably S and M; or means replaced by D; Substitutions at positions K55, K84, R204, or K196 are an aliphatic nonpolar amino acid selected from G, A, V, L, I, and P; or a different positively charged amino acid selected from R, K, and H, or means substituted by an aliphatic polar uncharged amino acid selected from N, Q, S, T, C, and M; The substitution at position D86 or D191 is an aliphatic nonpolar amino acid selected from G, A, V, L, I, and P; or means substituted by a polar uncharged amino acid selected from N, Q, S, T, C, and M, preferably Q, S, T, C, and M; Substitutions at positions D128, D135, D117, E239 or D199 are an aliphatic nonpolar amino acid selected from G, A, V, L, I, and P; or an aromatic amino acid selected from W, F, and Y; or an aliphatic polar uncharged amino acid selected from N, Q, S, T, C, and M; or It means that it is substituted with a positively charged amino acid selected from R, K, and H, preferably K and H.

[0088] Another preferred embodiment of the present invention relates to a nuclease, preferably an endonuclease, more preferably a non-specific endonuclease, and even more preferably a nuclease according to the present invention, comprising an amino acid sequence having at least 67% identity with SEQ ID NO: 1, wherein the amino acid sequence has been substituted in at least one amino acid position selected from the group consisting of P51, T56, N58, P73, T77, A124, D128, D135, G197, R204, and E239 compared to SEQ ID NO: 1.

[0089] Another preferred embodiment of the present invention relates to a nuclease, preferably an endonuclease, more preferably a non-specific endonuclease, even more preferably a nuclease according to the invention as described above, comprising an amino acid sequence having at least 89% identity to SEQ ID NO: 1, the amino acid sequence is substituted at at least one amino acid position selected from the group consisting of P51, S53, K55, T56, N58, A72, P73, A74, T77, N80, K84, D86, S116, D117, A124, D128, D135, D191, K196, G197, D199, R204, and E239 compared to SEQ ID NO: 1; Substitutions at positions P51, A72, P73, A74, A124, or G197 are a positively charged amino acid, or a negatively charged amino acid, or means that it is substituted by a different aliphatic nonpolar amino acid; Substitutions at positions S53, T56, N58, T77, N80, or S116 are those where S, T, or N is a positively charged amino acid, or an aliphatic nonpolar amino acid, or aromatic amino acids, or aliphatic polar uncharged amino acids, or means that it is substituted by a negatively charged amino acid; Substitutions at positions K55, K84, R204, or K196 are an aliphatic nonpolar amino acid, or a different positively charged amino acid, or means that it is substituted by an aliphatic polar uncharged amino acid; The substitution at position D86 or D191 is an aliphatic nonpolar amino acid, or means that it is substituted by a polar uncharged amino acid; Substitutions at positions D128, D135, D117, E239, or D199 are an aliphatic nonpolar amino acid, or aromatic amino acids, or aliphatic polar uncharged amino acids, or It means that it is substituted with a positively charged amino acid.

[0090] Another preferred embodiment of the present invention relates to a nuclease, preferably an endonuclease, more preferably a non-specific endonuclease, even more preferably a nuclease according to the invention as described above, comprising an amino acid sequence having at least 89% identity to SEQ ID NO: 1, the amino acid sequence is substituted at at least one amino acid position selected from the group consisting of P51, S53, K55, T56, N58, A72, P73, A74, T77, N80, K84, D86, S116, D117, A124, D128, D135, D191, K196, G197, D199, R204, and E239 compared to SEQ ID NO: 1; Substitutions at positions P51, A72, P73, A74, A124, or G197 are a positively charged amino acid selected from R, K, and H, or a negatively charged amino acid selected from D and E, or is substituted by a different aliphatic nonpolar amino acid selected from G, A, V, L, I, and P; Substitutions at positions S53, T56, N58, T77, N80, or S116 are those where S, T, or N is a positively charged amino acid selected from R, K, and H, or an aliphatic nonpolar amino acid selected from G, A, V, L, I, and P; or an aromatic amino acid selected from W, F, and Y; or an aliphatic polar uncharged amino acid selected from N, Q, S, T, C, and M, preferably S and M, more preferably M; or means replaced by D; Substitutions at positions K55, K84, R204, or K196 are an aliphatic nonpolar amino acid selected from G, A, V, L, I, and P; or a different positively charged amino acid selected from R, K, and H, or means substituted by an aliphatic polar uncharged amino acid selected from N, Q, S, T, C, and M; The substitution at position D86 or D191 is an aliphatic nonpolar amino acid selected from G, A, V, L, I, and P; or means substituted by a polar uncharged amino acid selected from N, Q, S, T, C, and M, preferably Q, S, T, C, and M; Substitutions at positions D128, D135, D117, E239, or D199 are an aliphatic nonpolar amino acid selected from G, A, V, L, I, and P; or an aromatic amino acid selected from W, F, and Y; or aliphatic polar uncharged amino acids selected from N, Q, S, T, C, and M, preferably Q, S, T, C, and M; or It means that it is substituted with a positively charged amino acid selected from R, K, and H, preferably K and H.

[0091] Another preferred embodiment of the present invention relates to a nuclease, preferably an endonuclease, more preferably a non-specific endonuclease, even more preferably a nuclease according to the invention as described above, comprising an amino acid sequence having at least 67%, preferably at least 89% identity with SEQ ID NO: 1, the amino acid sequence is substituted at at least one amino acid position selected from the group consisting of P51, S53, K55, T56, N58, A72, P73, A74, T77, N80, K84, D86, S116, D117, A124, D128, D135, D191, G197, D199, R204, and E239 compared to SEQ ID NO: 1; A substitution at position P51, A72, P73, A74, A124, or G197 means that P, A, or G is replaced by a positively charged amino acid selected from R, K, and H; P51 is substituted by an aliphatic nonpolar amino acid selected from G, A, V, L, and I; P51 is substituted by a negatively charged amino acid selected from D and E; A substitution at position S53, T56, N58, T77, N80, or S116 means that S, T, or N is replaced by a positively charged amino acid selected from R, K, and H; A substitution at position S53, T56, N58, or T77 means that S, T, or N is replaced by an aliphatic nonpolar amino acid selected from G, A, V, L, I, and P; T77 is substituted by an aromatic amino acid selected from W, F, and Y; T56 is replaced by D; K55 is replaced by R and H; K84 is replaced by G, A, V, L, I, and P; R204 is replaced by N, Q, S, T, C, and M; A substitution at position D117, D128, D135, D191, or E239 means that D or E is replaced by an aliphatic nonpolar amino acid selected from G, A, V, L, I, and P; A substitution at position D86 means that D is replaced by a polar uncharged amino acid selected from Q, S, T, C, and M; Substitutions at positions D117, D128, D135, or D199 are an aliphatic polar uncharged amino acid selected from N, Q, S, T, C, and M; or K, and H; D128 is substituted by an aromatic amino acid selected from W, F, and Y.

[0092] Another preferred embodiment of the present invention relates to a nuclease, preferably an endonuclease, more preferably a non-specific endonuclease, even more preferably a nuclease according to the invention as described above, comprising an amino acid sequence having at least 67%, preferably at least 89% identity with SEQ ID NO: 1, the amino acid sequence is substituted at at least one amino acid position selected from the group consisting of P51, S53, K55, T56, N58, A72, P73, A74, T77, N80, K84, D86, S116, D117, A124, D128, D135, D191, G197, D199, R204, and E239 compared to SEQ ID NO: 1; A substitution at position S53, D117, or E239 means that S, D, or E is replaced by A; A substitution at position P51, or T56 means that P or T is replaced by D; A substitution at position T77, or D128 means that T or D is replaced by F; A substitution at position P51, K84, D128, or D135 means that P, K, or D is replaced by G; N80 is replaced by H; A substitution at position S53, T56, N58, A72, P73, A74, T77, S116, D117, A124, D128, D135, G197, or D199 means that A, D, G, N, P, S, or T is replaced by K; A substitution at position P51, or T77 means that P or T is replaced by L; D128 is replaced by M; A substitution at position D117, D128, D135, or D199 means that D is replaced by N; A substitution at position T56, or D191 means that D or T is replaced by P; R204 is replaced by Q; A substitution at position P51, K55, T56, N58, P73, A74, T77, or A124 means that A, K, N, P, or T is replaced by R; A substitution at position D86, D117, D128, or D199 means that D is replaced by S; N58 is replaced by V; D128 is replaced by Y.

[0093] In a preferred embodiment, the nuclease according to the invention comprises an amino acid sequence having at least 67%, preferably at least 89%, identity to SEQ ID NO: 1, The amino acid sequences are as follows compared to SEQ ID NO: 1: P51L, P51G, P51R, P51D, S53A, S53K, K55R, T56D, T56P, T56R, T56K, T56S, T56M, D56G, D56A, D56V, D56I, D56L, D56F, N58V, N58R, N58K, A72K, P73R, P73K, A74R, A74K, T77L, T77F, T77R, T77K, N80H, K84G, D86S, S116K, D117H, D117I, D117 R, D117K, D117A, D117N, D117S, A124R, A124K, D128K, D128N, D128M, D128S, D128Y, D128G, D128F, D135A, D135M, D135K, D135N, D135G, D191P, G197K, D199K, D199N, D199S, R204Q, and E239A; preferably P51L, P51G, P51R, P51D, S53A, S53K, K55R, T56D, T56P, T56R, T5 6K, N58V, N58R, N58K, A72K, P73R, P73K, A74R, A74K, T77L, T77F, T77R, T77K, N80H, K84G, D86S, S116K, D117K, D117A, D117N, D117 S, A124R, A124K, D128K, D128N, D128M, D128S, D128Y, D128G, D128F, D135K, D135N, D135G, D191P, G197K, D199K, D199N, D199S, R2 04Q, and E239A at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 amino acid positions selected from the group consisting of E239A, E239B, E239C, E239D, E239E, E239F, E239G, E239H, E239I ...

[0094] In another preferred embodiment, the nuclease according to the invention comprises an amino acid sequence having at least 67%, preferably at least 80%, more preferably at least 89%, and most preferably at least 95% identity to SEQ ID NO: 1, The amino acid sequences are P51L, P51R, P51G, P51D, P51A, P51V, P51I, A52R, S53K, S53A, G54R, K55R, T56P, T56D, T56R, T56K, T56S, T56G, T56A, T56V, T56I, T56F, T56L, T56M, N58V, N58R, N58K, A72K, P73K, P73R, A74R, A74K, T77L, T77K, T77F, T77R, T77H, G ... 8R, N80H, N80R, A81K, A82R, K84G, D86S, A94G, S116K, D117K, D117A, D117S, D117N, D117R, D117H, D117Y, N119R, Q120R, A124K , A124R, A124H, D128M, D128Y, D128K, D128S, D128N, D128F, D128G, D128R, Q129R, K132R, D135N, D135K, D135G, D135R, D135A, D at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, or at least 38 amino acid positions selected from the group consisting of 135M, E151L, K162N, D191P, P195R, K196R, G197K, D199N, D199K, D199S, R204Q, I218F, A228R, and E239A.

[0095] In another preferred embodiment, the nuclease according to the invention comprises an amino acid sequence having at least 67%, preferably at least 80%, more preferably at least 89%, and most preferably at least 95% identity to SEQ ID NO: 1, The amino acid sequences are as follows compared to SEQ ID NO: 1: P51L, P51R, P51G, P51D, P51A, P51V, P51I, S53K, S53A, K55R, T56P, T56D, T56R, T56K, T56S, T56G, T56A, T56V, T56I, T56F, T56L, T56M, N58V, N58R, N58K, A72K, P73K, P73R, A74K, T77L, T77K, T77F, T77R, T77 H, N80H, A81K, A82R, K84G, D86S, A94G, S116K, D117K, D117A, D117S, D117N, D117R, D117H, D117Y, Q120R, A124K, A 124R, A124H, D128M, D128Y, D128K, D128S, D128N, D128F, D128G, D128R, Q129R, K132R, D135N, D135K, D135G, D135R , D135A, D135M, E151L, K162N, D191P, K196R, G197K, D199N, D199K, D199S, R204Q, I218F, A228R, and E239A.

[0096] Another preferred embodiment of the present invention relates to a nuclease, preferably an endonuclease, more preferably a non-specific endonuclease, even more preferably a nuclease according to the invention as described above, comprising an amino acid sequence having at least 67%, preferably at least 89% identity with SEQ ID NO: 1, the amino acid sequence is substituted at at least one amino acid position selected from the group consisting of P51, S53, K55, T56, N58, A72, P73, A74, T77, N80, K84, D86, S116, D117, A124, D128, D135, D191, G197, D199, R204, and E239 compared to SEQ ID NO: 1; an aliphatic nonpolar amino acid selected from G, A, V, L, I, and P replaces P51, S53, T56, N58, T77, K84, D117, D128, D135, D191, or E239; a positively charged amino acid selected from R, K, and H replaces P51, S53, K55, T56, N58, A72, P73, A74, T77, N80, S116, A124, or G197; a positively charged amino acid selected from R, K and H, preferably K and H, replaces D117, D128, D135, or D199; an aromatic amino acid selected from W, F, and Y replaces T77, or D128; D replaces P51, or T56; an aliphatic polar uncharged amino acid selected from N, Q, S, T, C, and M replaces D117, D128, D135, D199, or R204; An aliphatic polar uncharged amino acid selected from Q, S, T, C, and M replaces D86.

[0097] In a preferred embodiment, the nuclease according to the invention comprises an amino acid sequence having at least 67%, preferably at least 89%, identity to SEQ ID NO: 1, the amino acid sequence is substituted at at least one amino acid position selected from the group consisting of P51, S53, K55, T56, N58, A72, P73, A74, T77, N80, K84, D86, S116, D117, A124, D128, D135, D191, G197, D199, R204, and E239 compared to SEQ ID NO: 1; Amino acid A replaces S53, D117, or E239; Amino acid D replaces P51, or T56; Amino acid F replaces T77 or D128; the amino acid G replaces P51, K84, D128, or D135; Amino acid H replaces N80; amino acid K replaces S53, T56, N58, A72, P73, A74, T77, S116, D117, A124, D128, D135, G197, or D199; amino acid L replaces P51, or T77; Amino acid M replaces D128; Amino acid N replaces D117, D128, D135, or D199; Amino acid P replaces T56, or D191; Amino acid Q replaces R204; amino acid R replaces P51, K55, T56, N58, P73, A74, T77, or A124; Amino acid S replaces D86, D117, D128, or D199; Amino acid V replaces N58; Amino acid Y replaces D128.

[0098] Another preferred embodiment of the present invention relates to a nuclease, preferably an endonuclease, more preferably a non-specific endonuclease, comprising an amino acid sequence having at least 67%, preferably at least 89%, identity to SEQ ID NO: 1, the amino acid sequence has substitutions at least at the first and second amino acid positions compared to SEQ ID NO: 1; the first amino acid position is selected from the group consisting of P51, T56, T77, and A124; The second amino acid position is selected from the group consisting of P51, S53, K55, T56, N58, A72, P73, A74, T77, N80, D86, D117, Q120, A124, D128, Q129, D135, D191, K196, G197, D199, R204, and E239.

[0099] Another preferred embodiment of the present invention relates to a nuclease, preferably an endonuclease, more preferably a non-specific endonuclease, comprising an amino acid sequence having at least 89% identity to SEQ ID NO: 1, the amino acid sequence has substitutions at least at the first and second amino acid positions compared to SEQ ID NO: 1; the first amino acid position is selected from the group consisting of P51, T56, T77, and A124; the second amino acid position is selected from the group consisting of P51, S53, K55, T56, N58, A72, P73, A74, T77, N80, D86, D117, A124, D128, D135, D191, K196, G197, D199, R204, and E239; If the amino acid sequence is substituted at amino acid position P51, the substitution is P51R, P51K, P51H, P51D, P51E, P51G, P51A, P51V, P51L, P51I, preferably P51L, P51G, P51R, P51D, When the amino acid sequence is substituted at amino acid position T56, the substitutions are T56R, T56K, T56H, T56G, T56A, T56V, T56L, T56I, T56P, T56S, T56M, and T56D, preferably T56K, T56R, T56P, and T56D, more preferably T56K, T56R, and T56D; when the amino acid sequence is substituted at amino acid position T77, the substitutions are T77R, T77K, T77H, T77G, T77A, T77V, T77L, T77I, T77P, T77W, T77F, T77Y, and T77D, preferably T77K, T77R, T77F, and T77L; When the amino acid sequence is substituted at amino acid position A124, the substitutions are A124D, A124E, A124R, A124K, A124H, A124G, A124V, A124L, A124I, and A124P, preferably A124K, A124H and A124R, more preferably A124K and A124R.

[0100] In a preferred embodiment of the nuclease according to the invention, the first amino acid position and the second amino acid position are both selected from the group consisting of P51, T56, T77, and A124.

[0101] In a preferred embodiment of the nuclease according to the invention, Substitutions at positions P51, A72, P73, A74, A124, or G197 are a positively charged amino acid selected from R, K, and H, or different aliphatic nonpolar amino acids selected from G, A, V, L, I, and P; or means replaced by D; Substitutions at positions S53, T56, N58, T77, N80, Q120, or Q129 are those where S, T, Q, or N is a positively charged amino acid selected from R, K, and H, or an aliphatic nonpolar amino acid selected from G, A, V, L, I, and P; or an aromatic amino acid selected from W, F, and Y; or an aliphatic polar uncharged amino acid selected from N, Q, S, T, C, and M, preferably S and M, more preferably M; or means replaced by D; Substitutions at positions K55, R204, or K196 are an aliphatic nonpolar amino acid selected from G, A, V, L, I, and P; or a different positively charged amino acid selected from R, K, and H, or means substituted by an aliphatic polar uncharged amino acid selected from N, Q, S, T, C, and M; The substitution at position D86 or D191 is an aliphatic nonpolar amino acid selected from G, A, V, L, I, and P; or means substituted by a polar uncharged amino acid selected from N, Q, S, T, C, and M, preferably Q, S, T, C, and M; Substitutions at positions D128, D135, D117, E239, or D199 are an aliphatic nonpolar amino acid selected from G, A, V, L, I, and P; or an aromatic amino acid selected from W, F, and Y; or aliphatic polar uncharged amino acids selected from N, Q, S, T, C, and M, preferably Q, S, T, C, and M; or It means that it is substituted with a positively charged amino acid selected from R, K, and H, preferably K and H.

[0102] Another preferred embodiment of the present invention relates to the following. [1]: A nuclease, preferably an endonuclease, comprising an amino acid sequence having at least 67%, preferably at least 89%, identity to SEQ ID NO: 1 (the amino acid sequence is substituted at least at a first amino acid position and a second amino acid position compared to SEQ ID NO: 1, wherein the first amino acid position is selected from the group consisting of P51, T56, T77, and A124, and the second amino acid position is selected from the group consisting of P51, S53, K55, T56, N58, A72, P73, A74, T77, N80, D86, D117, Q120, A124, D128, Q129, D135, D191, K196, G197, D199, R204, and E239). [2]: The amino acid sequence has at least two or more amino acids selected from the group consisting of P51, S53, K55, T56, N58, A72, P73, A74, T77, N80, D86, D117, Q120, A124, D128, Q129, K132, D135, D191, K196, G197, D199, R204, and E239, preferably P51, T56, N58, T77, Q120, A124, D128, Q129, D135, K196, and G197, compared with SEQ ID NO: 1. The nuclease according to [1], wherein at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 amino acid positions are substituted. [3]: The amino acid sequence is P51D, P51R, P51G, P51L, S53K, S53A, K55H, K55R, T56S, T56M, T56R, T56K, T56G, T56A, T56V, T56L, T56I, T56F, T56P, T56D, N58K, N58R, N58V, A72K, A72H, A72R, P73K, P73R, A74K, A74H, A74R, T77K, T77R, T77F, T77L, N80K, N80R, N80H, D86S, D117A, D117B, D117C, D117D, D117E, D117F, D117F, D117H ... 7N, D117Y, D117K, D117H, D117R, D117S, Q120R, A124K, A124R, D128G, D128Y, D128F, D128M, D128N, D128S, D128R, D128K, Q129R, K132R, D135A, D135M, D135G, D135N, D135R, D135K, D191P, K196R, G197K, D199N, D199S, D199K, R204Q, and E239A, preferably P51R, P51G, P51L, T56S, T a minority selected from the group consisting of N56M, T56R, T56K, T56G, T56A, T56V, T56L, T56I, T56F, T56P, T56D, N58K, N58R, N58V, T77K, T77R, T77F, T77L, Q120R, A124K, A124R, D128G, D128Y, D128F, D128M, D128N, D128S, D128R, D128K, Q129R, D135A, D135M, D135G, D135N, D135R, D135K, K196R, and G197K The nuclease according to [1] or [2], wherein the nuclease is substituted at at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 amino acid positions. [4]: The amino acid sequences are SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 1 3, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 6, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, or 102, having at least 90%, preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, most preferably at least 97%, most preferably at least 98%, and particularly at least 99%, or 100% identity. [5]: (A) improved thermostability compared to the wild-type nuclease of SEQ ID NO: 1; and / or (B) improved relative salt activity compared to the relative salt activity of the wild-type nuclease of SEQ ID NO: 1; and / or (C) improved S of the wild-type nuclease of SEQ ID NO: 1. 高 Improved activity compared to S 高 activity; and / or (D) an improved relative low-temperature activity compared to the relative low-temperature activity of the wild-type nuclease of SEQ ID NO: 1; and / or (E) a T 低 Improved T activity compared to 低 The nuclease according to any one of [1] to [4], characterized by any one of the following activities: [6]: has improved thermal stability compared to SEQ ID NO: 1, and the thermal stability is at a temperature T 安定性 The nuclease according to any one of [1] to [5], characterized by a higher residual activity after 15 minutes of incubation at 0.5°C. [7]: A nuclease according to any one of [1] to [6], which is capable of hydrolyzing the phosphodiester of a polynucleotide substrate into a cleaved polynucleotide or oligonucleotide. [8]: The nuclease according to any one of [1] to [7], wherein the polynucleotide substrate is selected from the group consisting of single-stranded RNA, single-stranded DNA, double-stranded DNA, double-stranded RNA, and hybrid-stranded DNA / RNA, preferably double-stranded DNA. [9]: A method for hydrolyzing a polynucleotide substrate, preferably a polynucleotide substrate contained in a sample, comprising the steps of: (a) preparing a nuclease according to any one of [1] to [8]; (b) preparing a composition containing the polynucleotide substrate; and (c) contacting the polynucleotide substrate in the composition prepared in step (b) with the nuclease prepared in step (a) to allow the nuclease to hydrolyze the polynucleotide substrate, thereby obtaining a composition containing cleaved polynucleotides.

[10] : The method of [9], wherein step (c) is carried out at a temperature of (i) between 4°C and 90°C; preferably between 10°C and 80°C; more preferably between 20°C and 65°C; even more preferably between 25°C and 65°C; even more preferably between 30°C and 50°C; even more preferably between 34°C and 42°C; most preferably 37°C; or (ii) between 20°C and 90°C; preferably between 23°C and 80°C; more preferably between 25°C and 65°C; even more preferably between 30°C and 65°C; even more preferably between 37°C and 65°C; even more preferably between 50°C and 65°C; most preferably 51°C; or (iii) between 4°C and 65°C; preferably between 4°C and 38°C; more preferably between 4°C and 25°C; even more preferably between 4°C and 20°C; even more preferably between 4°C and 15°C; even more preferably between 4°C and 10°C; most preferably between 4°C and 6°C, and most preferably at 5°C.

[11] : The method according to [9] or

[10] , wherein step (c) is carried out in the presence of a salt, the salt being NaCl or KCl, preferably NaCl.

[12] : The method of [9] or

[11] , wherein step (c) is carried out at a salt concentration of 0.00 M to 1.00 M, or 0.01 M to 1.00 M, preferably 0.15 M to 0.85 M, more preferably 0.20 M to 0.70 M, even more preferably 0.25 M to 0.60 M, even more preferably 0.30 M to 0.60 M, even more preferably 0.35 M to 0.60 M, most preferably 0.40 M to 0.60 M, and especially 0.50 M.

[13] : The method according to [9] or

[12] , wherein step (c) is carried out in the absence of a salt.

[14] : (d) the additional step of inactivating the nuclease after step (c), thereby obtaining a composition comprising the cleaved polynucleotide and the inactivated nuclease.

[15] : The method of [9] or

[14] , comprising the additional step of (e) separating the nuclease from the composition comprising the cleaved polynucleotide obtained in step (c) and, optionally, the inactivated nuclease obtained in step (d).

[0103] Another preferred embodiment of the invention comprises an amino acid sequence having at least 75% identity to SEQ ID NO: 1, (i) a substitution at position P51, where P is replaced by an amino acid selected from the group consisting of L, G, A, V, and I; (ii) a substitution at position T77, where T is replaced by an amino acid selected from the group consisting of R, H, and K; (iii) (a) A124, A52, G54, P73, A74, G78, A81, and P195; or (b) T56, S53, N58, N80, N119, and Q120 (in each case of (a) or (b), an amino acid of SEQ ID NO: 1 is replaced with an amino acid selected from the group consisting of R, H, and K); and The present invention relates to a nuclease, preferably an endonuclease, more preferably a non-specific endonuclease, comprising:

[0104] In a preferred embodiment of the nuclease according to the invention, the nuclease, preferably the endonuclease, (i) a substitution at position P51, where P is replaced by an amino acid selected from the group consisting of L, G, A, V, and I, preferably L; (ii) a substitution at position T77, where T is replaced by an amino acid selected from the group consisting of R, H, and K, preferably R; (iii) a substitution at position A124, where A is replaced by an amino acid selected from the group consisting of R, H, and K, preferably K.

[0105] In a preferred embodiment of the nuclease according to the invention, the nuclease, preferably the endonuclease, (i) a substitution at position P51, where P is replaced by an amino acid selected from the group consisting of L, G, A, V, and I, preferably L; (ii) a substitution at position T77, where T is replaced by an amino acid selected from the group consisting of R, H, and K, preferably R; (iii) a substitution at position A81, where A is replaced by an amino acid selected from the group consisting of R, H, and K, preferably K.

[0106] In a preferred embodiment of the nuclease according to the present invention, the nuclease, preferably the endonuclease, comprises at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49 ... At least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, or at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, or at least 38 positions.

[0107] Preferably, the nuclease, preferably the endonuclease according to the present invention comprises at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 8 At least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, or at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, or at least 38 positions are substituted.

[0108] In a preferred embodiment of the nuclease according to the present invention, the nuclease, preferably the endonuclease, is selected from the group consisting of A52, S53, G54, K55, T56, N58, A72, P73, A74, G78, N80, A81, A82, K84, D86, A94, S116, D117, N119, Q120, A124, D128, Q129, K132, D135, E151, K162, D191, P195, K196, G197, D199, R204, I218, A228, and E239, preferably S53, K55, T56, N58, A72, P73, A74, G78, N80, A81, A82, K84, D86, A94, S116, D117, N119, Q120, A124, D128, Q129, K132, D135, E151, K162, D191, P195, K196, G197, D199, R204, I218, A228, and E239, compared to SEQ ID NO: 1. 73, A74, G78, N80, A81, A82, K84, D86, A94, S116, D117, Q120, A124, D128, Q129, K132, D135, E151, K162, D191, K196, G197, D199, R204, I218, A228, and E239.

[0109] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence of the nuclease, preferably the endonuclease, is (iii) a third substitution at a position selected from the group consisting of: (a) A124, A52, G54, P73, A74, G78, A81, and P195; or (b) T56, S53, N58, N80, N119, and Q120 (in each case of (a) or (b), an amino acid of SEQ ID NO: 1 is replaced with an amino acid selected from the group consisting of R, H, and K); and (iv) a fourth substitution at another position selected from the group consisting of: (a) A124, A52, G54, P73, A74, G78, A81, and P195; or (b) T56, S53, N58, N80, N119, and Q120 (in each case of (a) or (b), an amino acid of SEQ ID NO: 1 is replaced with an amino acid selected from the group consisting of G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S, and T).

[0110] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence of the nuclease, preferably the endonuclease, is (iv) comprising a fourth substitution, wherein the fourth substitution is selected from the group consisting of: A124R, A124N, A124D, A124C, A124Q, A124E, A124G, A124H, A124I, A124L, A124K, A124M, A124F, A124P, A124T, A124W, A124Y, A124V, and A124S, preferably A124D, A124E, A124R, A124K, A124H, A124G, A124V, A124L, A124I, and A124P, preferably A124K, A124H and A124R, more preferably A124K, and A124R; A52R, A52N, A52D, A52C, A52Q, A52E, A52G, A52H, A52I, A52L, A52K, A52M, A52F, A52P, A52T, A52W, A52Y, A52V, and A52S, preferably A52R, A52K, A52H, more preferably A52R; G54A, G54R, G54N, G54D, G54C, G54Q, G54E, G54H, G54I, G54L, G54K, G54M, G54F, G54P, G54T, G54W, G54Y, G54V, and G54S, preferably G54D, G54E, G54R, G54K, G54H, G54A, G54V, G54L, G54I, and G54P, preferably G54K, G54H, and G54R, more preferably G54R; P73A, P73R, P73N, P73D, P73C, P73Q, P73E, P73G, P73H, P73I, P73L, P73K, P73M, P73F, P73T, P73W, P73Y, P73V, and P73S, preferably P73D, P73E, P73R, P73K, P73H, P73G, P73V, P73L, P73I, and P73A, more preferably P73K, P73H and P73R, most preferably P73K, and P73R; A74R, A74N, A74D, A74C, A74Q, A74E, A74G, A74H, A74I, A74L, A74K, A74M, A74F, A74P, A74T, A74W, A74Y, A74V, and A74S, preferably A74D, A74E, A74R, A74K, A74H, A74G, A74V, A74L, A74I, and A74P, more preferably A74K, A74H, and A74R, most preferably A74K, and A74R; G78A, G78R, G78N, G78D, G78C, G78Q, G78E, G78H, G78I, G78L, G78K, G78M, G78F, G78P, G78T, G78W, G78Y, G78V, and G78S, preferably G78R, G78K, and G78H, more preferably G78R; A81R, A81N, A81D, A81C, A81Q, A81E, A81G, A81H, A81I, A81L, A81K, A81M, A81F, A81P, A81T, A81W, A81Y, A81V, and A81S, preferably A81K, A81H, and A81R, more preferably A81K; P195A, P195R, P195N, P195D, P195C, P195Q, P195E, P195G, P195H, P195I, P195L, P195K, P195M, P195F, P195T, P195W, P195Y, P195V, and P195S, preferably P195K, P195H and P195R, most preferably P195R; T56A, T56R, T56N, T56D, T56C, T56Q, T56E, T56G, T56H, T56I, T56L, T56K, T56M, T56F, T56P, T56W, T56Y, T56V, and T56S, preferably T56P, T56D, T56R, T56K, T56S, T56G, T56A, T56V, T56I, T56F, T56L, and T56M, even more preferably T56P, T56D, T56R, T56K, T56S, and T56G, most preferably T56R, T56K, T56S, and T56G; S53A, S53R, S53N, S53D, S53C, S53Q, S53E, S53G, S53H, S53I, S53L, S53K, S53M, S53F, S53P, S53T, S53W, S53Y, and S53V, preferably S53R, S53K, S53H, S53G, S53A, S53V, S53L, S53I, S53 P, S53W, S53F, S53Y, and S53D, more preferably S53R, S53K, S53H, S53G, S53A, S53V, S53L, S53I, and S53P, even more preferably S53R, S53K, S53H, and S53A, even more preferably S53K, and S53A, most preferably S53K; N58A, N58R, N58D, N58C, N58Q, N58E, N58G, N58H, N58I, N58L, N58K, N58M, N58F, N58P, N58T, N58W, N58Y, N58V, and N58S, preferably N58R, N58K, N58H, N58G, N58A, N58V, N58L, N58I, N58P, N58W, N58F, N58Y, and N58D, more preferably N58R, N58K, N58H, N58G, N58A, N58V, N58L, N58I, and N58P, more preferably N58K, N58R, N58H, and N58V, most preferably N58K, and N58R; N80A, N80R, N80D, N80C, N80Q, N80E, N80G, N80H, N80I, N80L, N80K, N80M, N80F, N80P, N80T, N80W, N80Y, N80V, and N80S, preferably N80R, N80K, N80H, N80G, N80A, N80V, N80L, N80I, N80P, N80W, N80F, N80Y, and N80D, preferably N80K, N80R, and N80H, more preferably N80H and N80R, most preferably N80H; N119A, N119R, N119D, N119C, N119Q, N119E, N119G, N119H, N119I, N119L, N119K, N119M, N119F, N119P, N119T, N119W, N119Y, N119V, and N119S, preferably N119R, N119K, and N119H, more preferably N119R; and Q120A, Q120R, Q120N, Q120D, Q120C, Q120E, Q120G, Q120H, Q120I, Q120L, Q120K, Q120M, Q120F, Q120P, Q120T, Q120W, Q120Y, Q120V, and Q120S, preferably Q120R, Q120K, Q120H, Q120G, Q120A, Q120V, Q120L, Q120I, Q120P, Q120W, Q120F, Q120Y, and Q120D, more preferably Q120K, Q120H, and Q120R, and most preferably Q120R.

[0111] In a preferred embodiment of the nuclease according to the invention, the nuclease, preferably the endonuclease, has the following amino acid sequence(s) relative to SEQ ID NO: 1: P51L, P51G, P51A, P51V, P51I, A52R, A52K, A52H, S53R, S53K, S53H, G54R, G54K, G54H, K55R, T56R, T56K, T56H, N58R, N58K, N58H, A72K, P73K, P73R, P73H, A74R, A74K, A74H, T77K, T77R, T77H, G78R, G79R, G80R, G81R, G82R, G83R, G84R, G85R, G86R, G87R, G88R, G89 ...9R, G80R, G81R, G81R, G82R, G83R, G84R, G85R, G85R, G86R, G87R, G88R, G89R, G89R, G89R, G80 8K, G78H, N80R, N80K, N80H, A81R, A81K, A81H, A82R, K84G, D86S, A94G, S116K, D117K, D117A, D117S, D117N, D117R, D117H, D117Y, N119 R, N119K, N119H, Q120R, Q120K, Q120H, A124K, A124R, A124H, D128M, D128Y, D128K, D128S, D128N, D128F, D128G, D128R, Q129R, K132R, At least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 selected from the group consisting of D135N, D135K, D135G, D135R, D135A, D135M, E151L, K162N, D191P, P195R, P195K, P195H, K196R, G197K, D199N, D199K, D199S, R204Q, I218F, A228R, and E239A At least 4, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, or at least 38 positions.

[0112] Preferably, the nuclease, preferably the endonuclease, is selected from the group consisting of P51L, P51G, P51A, P51V, P51I, A52R, S53K, G54R, K55R, T56R, T56K, N58R, N58K, A72K, P73K, P73R, A74R, A74K, T77K, T77R, T77H, G78R, N80R, A81K, A82R, K84G, D86S, A94G, S116K, D117K, D117A, D117S, D117N, D117R, D117H, D117Y, N119R, Q120R, A124K, A124R, A124H, D128M, D128Y, D128K, D12 8S, D128N, D128F, D128G, D128R, Q129R, K132R, D135N, D135K, D135G, D135R, D135A, D135M, E151L, K162N, D191P, P at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, or at least 38 positions selected from the group consisting of 195R, K196R, G197K, D199N, D199K, D199S, R204Q, I218F, A228R, and E239A.

[0113] Preferably, the nuclease, preferably the endonuclease, is selected from the group consisting of SEQ ID NOs: 2, 3, 4, 6, 7, 8, 11, 12, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 0, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 107, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156 , 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, and 195, preferably 55, 57, 58, 59, 61, 64, 66, 69, 86, 128, 131, 132, 133, 134, 137, 138, 139, 14 0, 143, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, and 195.

[0114] Another preferred embodiment of the present invention relates to a nuclease, preferably an endonuclease, more preferably a non-specific endonuclease, comprising an amino acid sequence having at least 65% identity to SEQ ID NO: 1, wherein the amino acid sequence is substituted at least at one amino acid position selected from the group consisting of E239, T56, K84, D191, T77, and N58 compared to SEQ ID NO: 1, in each case the native amino acid at the selected position is selected from the group consisting of A, G, V, L, I, and P; preferably selected from the group consisting of A, G, V, L, and P; more preferably selected from the group consisting of A, G, L, and P; even more preferably selected from the group consisting of A, G, and P; even more preferably selected from the group consisting of A and G; most preferably replaced by A.

[0115] In a preferred embodiment, the nuclease, preferably the endonuclease, has the following sequence relative to SEQ ID NO: 1: is substituted at position E239, preferably the substitution is selected from the group consisting of E239A, E239G, E239V, E239L, E239I and E239P, preferably E239A, is substituted at position T56, preferably the substitution is selected from the group consisting of T56A, T56G, T56V, T56L, T56I and T56P, preferably T56P and T56G, more preferably T56P; is substituted at position K84, preferably the substitution is selected from the group consisting of K84A, K84G, K84V, K84L, K84I and K84P, preferably K84G; is substituted at position D191, preferably the substitution is selected from the group consisting of D191A, D191G, D191V, D191L, D191I and D191P, preferably D191P, is substituted at position T77, preferably the substitution is selected from the group consisting of T77A, T77G, T77V, T77L, T77I and T77P, preferably T77L; It is substituted at position N58, preferably the substitution comprises an amino acid sequence selected from the group consisting of N58A, N58G, N58V, N58L, N58I and N58P, preferably N58V.

[0116] In a preferred embodiment, the nuclease, preferably the endonuclease, is selected from the group consisting of P51, A52, S53, G54, K55, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, K84, D86, A94, S116, D117, N119, Q120, A124, D128, Q129, K132, S133, D134, S135, D136, S137, D138, S139, D140, S141, D142, S143, D144, S145, D146, S147, D148, S149, D150, S151, D152, S153, D154, S155, D156, S157, S158, S159, D160, S161, D162, S163, D164, S165, D166, S167, D168, S169, D170, S171, D172, S173, D174, S175, D176, S177, D178, S179, D180, S181, D182, D183, D184, D185, D186, D187, D188, D189, D190, D191, D192, D193, D194, D195, D196, D197, D198, D199, D200, D201, D202, D203, D204, D205, D206, D20 , D135, E151, K162, D191, P195, K196, G197, D199, R204, I218, A228, and E239; preferably P51, K55, T56, N58, A74, T77, N80, A81, K84, D86, A94, S116, D117, A124, D128, K132, D135, more preferably selected from the group consisting of P51, K55, T56, N58, A74, T77, N80, K84, D86, A94, S116, D117, A124, D128, K132, D135, E151, K162, D191, R204, I218, A228, and E239; 31, 32, 33, 34, 35, 36, or 37 additional positions selected from the group consisting of A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, A21, A22, A23, A24, A25, A26, A27, A28, A29, A30, A31, A32, A33, A34, A35, A36, A37, A48, A49, A50, A51, A52, A53, A54, A55, A56, A57, A58, A59, A60, A61, A62, A63, A64, A65, A66, A67, A68, A69, A70, A71, A72, A73, A74, A75, A76, A77, A78, A79, A80, A81, A82, A83, A84, A85, A86, A87, A88, A89, A91, A92, A93, A94, A95, A96, A97, A98, A99, A91, A92, A93, A94, A95, A96, A97, A98, A99, A91, A92, A93, A94, A95, A96, A97, A98, A99, A99, A91 ...

[0117] In a preferred embodiment, the nuclease, preferably the endonuclease, comprises a substituted amino acid sequence compared to SEQ ID NO: 1, wherein the amino acid sequence is (i) a substitution at a position selected from the group consisting of E239, T56, K84, D191, T77, and N58 (in each case, a native amino acid of SEQ ID NO: 1 is replaced by an amino acid selected from the group consisting of A, G, V, L, I, and P); and (ii) a second substitution at another position selected from the group consisting of E239, T56, K84, D191, T77, and N58 (in each case, a native amino acid of SEQ ID NO: 1 is replaced by an amino acid selected from the group consisting of G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S, and T).

[0118] In a preferred embodiment, the nuclease, preferably an endonuclease, (ii) the second substitution is selected from the group consisting of: E239A, E239R, E239N, E239D, E239C, E239Q, E239G, E239H, E239I, E239L, E239K, E239M, E239F, E239P, E239T, E239W, E239Y, E239V, and E239S, preferably E239G, E239A, E239V, E 239L, E239I, E239P, E239W, E239F, E239Y, E239C, E239M, E239N, E239Q, E239S, E239T, E239H and E239K, preferably E239A, E239G, E239V, E239L, E239I and E239P, more preferably E239A; T56A, T56R, T56N, T56D, T56C, T56Q, T56E, T56G, T56H, T56I, T56L, T56K, T56M, T56F, T56P, T56W, T56Y, T56V, and T56S, preferably T56P, T56D, T56R, T56K, T56S, T56G, T56A, T56V, T56I, T56F, T56L, and T56M, even more preferably T56P, T56D, T56R, T56K, T56S, and T56G, most preferably T56R, T56K, T56S, and T56G; K84A, K84R, K84N, K84D, K84C, K84Q, K84E, K84G, K84H, K84I, K84L, K84M, K84F, K84P, K84T, K84W, K84Y, K84V, and K84S, preferably K84G, K84A, K84V, K84L, K84I, K84P, K84R, K84H, K84N, K84Q, K84S, K84T, K84C, and K84M, preferably K84G, K84A, K84V, K84L, K84I, and K84P, more preferably K84G; D191G, D191A, D191V, D191L, D191I, D191P, D191W, D191F, D191Y, D191C, D191M, D191S, D191Q, D191T, D191N, D191E, D191H, D191R and D191K, preferably D191G, D191A, D191V, D191L, D191I, D191P, D191C, D191M, D191S, D191Q, and D191T, more preferably D191G, D191A, D191V, D191L, D191I, and D191P, most preferably D191P; T77A, T77R, T77N, T77D, T77C, T77Q, T77E, T77G, T77H, T77I, T77L, T77K, T77M, T77F, T77P, T77W, T77Y, T77V, and T77S, preferably T77R, T77K, T77H, T77G, T77A, T77V, T77L, T77I, T77P, T77W, T77F, T77Y, and T77D, more preferably T77K, T77R, T77H, T77F, and T77L, most preferably T77K, T77R, T77F, and T77L; and N58A, N58R, N58D, N58C, N58Q, N58E, N58G, N58H, N58I, N58L, N58K, N58M, N58F, N58P, N58T, N58W, N58Y, N58V, and N58S, preferably N58R, N58K, N58H, N58G, N58A, N58V, N58L, N58I, N58P, N58W, N58F, N58Y, and N58D, more preferably N58R, N58K, N58H, N58G, N58A, N58V, N58L, N58I, and N58P, more preferably N58K, N58R, and N58V, and most preferably N58K and N58R.

[0119] In a preferred embodiment, the nuclease, preferably the endonuclease, comprises an amino acid sequence, which, compared to SEQ ID NO: 1, is (i) is substituted at position P51, preferably the substitution is selected from the group consisting of P51A, P51R, P51N, P51D, P51C, P51Q, P51E, P51G, P51H, P51I, P51L, P51K, P51M, P51F, P51T, P51W, P51Y, P51V and P51S, preferably P51D, P51E, P51R, P51K, P51H, P51G, P51A, P51V, P51L and P51I, more preferably P51L, P51R, P51G, P51D, P51A, P51V, P51I, even more preferably P51D, P51R, P51G and P51L, most preferably P51R, P51G and P51L; (ii) is substituted at position P51, preferably the substitution is selected from the group consisting of P51A, P51R, P51N, P51D, P51C, P51Q, P51E, P51G, P51H, P51I, P51L, P51K, P51M, P51F, P51T, P51W, P51Y, P51V and P51S, preferably P51D, P51E, P51R, P51K, P51H, P51G, P51A, P51V, P51L and P51I, more preferably P51L, P51R, P51G, P51D, P51A, P51V, P51I, even more preferably P51D, P51R, P51G and P51L, most preferably P51R, P51G and P51L; (iii) is substituted at position A52, preferably the substitution is selected from the group consisting of A52R, A52N, A52D, A52C, A52Q, A52E, A52G, A52H, A52I, A52L, A52K, A52M, A52F, A52P, A52T, A52W, A52Y, A52V, and A52S, preferably A52R, A52K, A52H, more preferably A52R; (iv) substituted at position S53, preferably the substitution is S53A, S53R, S53N, S53D, S53C, S53Q, S53E, S53G, S53H, S53I, S53L, S53K, S53M, S53F, S53P, S53T, S53W, S53Y, and S53V, preferably S53R, S53K, S53H, S53I, S53L, S53K, S53M, S53F, S53P, S53T, S53W, S53Y, and S53V. S53G, S53A, S53V, S53L, S53I, S53P, S53W, S53F, S53Y, and S53D, more preferably S53R, S53K, S53H, S53G, S53A, S53V, S53L, S53I, and S53P, even more preferably S53K, and S53A, most preferably S53K; (v) is substituted at position G54, preferably the substitution is selected from the group consisting of G54A, G54R, G54N, G54D, G54C, G54Q, G54E, G54H, G54I, G54L, G54K, G54M, G54F, G54P, G54T, G54W, G54Y, G54V, and G54S, preferably G54D, G54E, G54R, G54K, G54H, G54A, G54V, G54L, G54I, and G54P, preferably G54K, G54H, and G54R, more preferably G54R; (vi) is substituted at position K55, preferably the substitution is selected from the group consisting of K55A, K55R, K55N, K55D, K55C, K55Q, K55E, K55G, K55H, K55I, K55L, K55M, K55F, K55P, K55T, K55W, K55Y, K55V, and K55S, preferably K55G, K55A, K55V, K55L, K55I, K55P, K55R, K55H, K55N, K55Q, K55S, K55T, K55C, and K55M, more preferably K55H and K55R, most preferably K55R; (vii) is substituted at position A72, preferably the substitution is selected from the group consisting of A72R, A72N, A72D, A72C, A72Q, A72E, A72G, A72H, A72I, A72L, A72K, A72M, A72F, A72P, A72T, A72W, A72Y, A72V, and A72S, preferably A72D, A72E, A72R, A72K, A72H, A72G, A72V, A72L, A72I, and A72P, more preferably A72K, A72H and A72R, most preferably A72K; (viii) is substituted at position P73, preferably the substitution is selected from the group consisting of P73A, P73R, P73N, P73D, P73C, P73Q, P73E, P73G, P73H, P73I, P73L, P73K, P73M, P73F, P73T, P73W, P73Y, P73V, and P73S, preferably P73D, P73E, P73R, P73K, P73H, P73G, P73V, P73L, P73I, and P73A, more preferably P73K, P73H and P73R, most preferably P73K and P73R; (ix) is substituted at position A74, preferably the substitution is selected from the group consisting of A74R, A74N, A74D, A74C, A74Q, A74E, A74G, A74H, A74I, A74L, A74K, A74M, A74F, A74P, A74T, A74W, A74Y, A74V, and A74S, preferably A74D, A74E, A74R, A74K, A74H, A74G, A74V, A74L, A74I, and A74P, more preferably A74K, A74H, and A74R, most preferably A74K and A74R; (x) is substituted at position G78, preferably the substitution is selected from the group consisting of G78A, G78R, G78N, G78D, G78C, G78Q, G78E, G78H, G78I, G78L, G78K, G78M, G78F, G78P, G78T, G78W, G78Y, G78V, and G78S, preferably G78R, G78K, and G78H, more preferably G78R; (xi) is substituted at position N80, preferably the substitution is selected from the group consisting of N80A, N80R, N80D, N80C, N80Q, N80E, N80G, N80H, N80I, N80L, N80K, N80M, N80F, N80P, N80T, N80W, N80Y, N80V, and N80S, preferably N80R, N80K, N80H, N80G, N80A, N80V, N80L, N80I, N80P, N80W, N80F, N80Y, and N80D, preferably N80K, N80R, and N80H, more preferably N80H and N80R, most preferably N80H; (xii) substituted at position A81, preferably the substitution is selected from the group consisting of A81R, A81N, A81D, A81C, A81Q, A81E, A81G, A81H, A81I, A81L, A81K, A81M, A81F, A81P, A81T, A81W, A81Y, A81V, and A81S, preferably A81K, A81H, and A81R, more preferably A81K; (xiii) is substituted at position A82, preferably the substitution is selected from the group consisting of A82R, A82N, A82D, A82C, A82Q, A82E, A82G, A82H, A82I, A82L, A82K, A82M, A82F, A82P, A82T, A82W, A82Y, A82V, and A82S, preferably A82K, A82H, and A82R, more preferably A82R; (xiv) is substituted at position D86, preferably the substitution is selected from the group consisting of D86A, D86R, D86N, D86C, D86Q, D86E, D86G, D86H, D86I, D86L, D86K, D86M, D86F, D86P, D86T, D86W, D86Y, D86V, and D86S; D86G, D86A, D86V, D86L, D86I, D86P, D86C, D86M, D86S, D86T, and D86Q, preferably D86C, D86M, D86S, D86T, and D86Q, more preferably D86S; (xv) substituted at position A94, preferably the substitution is selected from the group consisting of A94R, A94N, A94D, A94C, A94Q, A94E, A94G, A94H, A94I, A94L, A94K, A94M, A94F, A94P, A94T, A94W, A94Y, A94V, and A94S; preferably A94G, A94V, A94L, A94I, and A94P; more preferably A94G, (xvi) is substituted at position S116, preferably the substitution is selected from the group consisting of S116A, S116R, S116N, S116D, S116C, S116Q, S116E, S116G, S116H, S116I, S116L, S116K, S116M, S116F, S116P, S116T, S116W, S116Y and S116V, preferably S116R, S116K, S116H, S116G, S116A, S116V, S116L, S116I, S116P, S116W, S116F, S116Y and S116D, preferably S116K, S116H and S116R, more preferably S116K; (xvii) substituted at position D117, preferably the substitution is D117G, D117A, D117V, D117L, D117I, D117P, D117W, D117F, D117Y, D117C, D117M, D117S, D117Q, D117T, D117N, D117E, D117H, D117R and D117K, preferably D117G, D117A, D117V, D117L, D117I, D117P, D117W, D117F, D117Y, D117C, D117M, D117S, D117Q, D117T, D117N, D117E, D117H, D117R and D117K 7M, D117S, D117Q, D117T, D117N, D117H, D117R and D117K, more preferably D117K, D117A, D117S, D117N, D117R, D117H, and D117Y, even more preferably D117A, D117N, D117R, D117K, and D117S, even more preferably D117A, D117N, D117K, and D117S, most preferably D117A, D117N, and D117S; (xviii) is substituted at position N119, preferably the substitution is selected from the group consisting of N119A, N119R, N119D, N119C, N119Q, N119E, N119G, N119H, N119I, N119L, N119K, N119M, N119F, N119P, N119T, N119W, N119Y, N119V, and N119S, preferably N119R, N119K, and N119H, more preferably N119R; (xix) is substituted at position Q120, preferably the substitution is selected from the group consisting of Q120A, Q120R, Q120N, Q120D, Q120C, Q120E, Q120G, Q120H, Q120I, Q120L, Q120K, Q120M, Q120F, Q120P, Q120T, Q120W, Q120Y, Q120V and Q120S, preferably Q120R, Q120K, Q120H, Q120G, Q120A, Q120V, Q120L, Q120I, Q120P, Q120W, Q120F, Q120Y and Q120D, more preferably Q120K, Q120H and Q120R, most preferably Q120R; (xx) substituted at position A124, preferably the substitution is selected from the group consisting of A124R, A124N, A124D, A124C, A124Q, A124E, A124G, A124H, A124I, A124L, A124K, A124M, A124F, A124P, A124T, A124W, A124Y, A124V, and A124S, preferably A124D, A124E, A124R, A124K, A124H, A124G, A124V, A124L, A124I, and A124P, preferably A124K, A124H and A124R, more preferably A124K and A124R; (xxi) Substitutions at position D128, preferably the substitutions are D128A, D128R, D128N, D128C, D128Q, D128E, D128G, D128H, D128I, D128L, D128K, D128M, D128F, D128P, D128T, D128W, D128Y, D128V, and D128S, preferably D128G, D128A , D128V, D128L, D128I, D128P, D128W, D128F, D128Y, D128C, D128M, D128N, D128Q, D128S, D128T, D128H, D128R and D128K, most preferably D128G, D128Y, D128F, D128M, D128N, D128S, D128R and D128K; (xxii) substituted at position Q129, preferably the substitution is Q129A, Q129R, Q129N, Q129D, Q129C, Q129E, Q129G, Q129H, Q129I, Q129L, Q129K, Q129M, Q129F, Q129P, Q129T, Q129W, Q129Y, Q129V, and Q129S, preferably and wherein the amino acid sequence is selected from the group consisting of: Q129R, Q129K, Q129H, Q129G, Q129A, Q129V, Q129L, Q129I, Q129P, Q129W, Q129F, Q129Y, and Q129D, preferably Q129K, Q129H, and Q129R, more preferably Q129K, Q129R, and even more preferably Q129R; (xxiii) is substituted at position K132, preferably the substitution is selected from the group consisting of K132A, K132R, K132N, K132D, K132C, K132Q, K132E, K132G, K132H, K132I, K132L, K132M, K132F, K132P, K132T, K132W, K132Y, K132V, and K132S, preferably K132R, and K132H, most preferably K132R; (xxiv) substituted at position D135, preferably the substitutions are D135A, D135R, D135N, D135C, D135Q, D135E, D135G, D135H, D135I, D135L, D135K, D135M, D135F, D135P, D135T, D135W, D135Y, D135V, and D135S, preferably D135G, D135A, D135V, D135L, D135I, D135P, D135W, D135F, D135Y, D 135C, D135M, D135N, D135Q, D135S, D135T, D135R, D135H, and D135K, more preferably D135G, D135A, D135V, D135L, D135I, D135P, D135C, D135M, D135N, D135Q, D135S, D135T, D135R, D135H, and D135K, most preferably D135A, D135M, D135G, D135N, D135R, and D135K; (xxv) substituted at position E151, preferably the substitutions are E151A, E151R, E151N, E151D, E151C, E151Q, E151G, E151H, E151I, E151L, E151K, E151M, E151F, E151P, E151T, E151W, E151Y, E151V, and E151S, E151G, E151A , E151V, E151L, E151I, E151P, E151W, E151F, E151Y, E151C, E151M, E151N, E151Q, E151S, E151T, E151H and E151K, preferably E151A, E151G, E151V, E151L, E151I and E151P, more preferably E151L, (xxvi) is substituted at position K162, preferably the substitution is selected from the group consisting of K162A, K162R, K162N, K162D, K162C, K162Q, K162E, K162G, K162H, K162I, K162L, K162M, K162F, K162P, K162T, K162W, K162Y, K162V, and K162S, preferably K162N, K162Q, K162S, K162T, K162C and K162M, most preferably K162N; (xxvii) is substituted at position P195, preferably the substitution is selected from the group consisting of P195A, P195R, P195N, P195D, P195C, P195Q, P195E, P195G, P195H, P195I, P195L, P195K, P195M, P195F, P195T, P195W, P195Y, P195V, and P195S, preferably P195K, P195H and P195R, most preferably P195R; (xxviii) substituted at position K196, preferably the substitution is K196A, K196R, K196N, K196D, K196C, K196Q, K196E, K196G, K196H, K196I, K196L, K196M, K196F, K196P, K196T, K196W, K196Y, K196V, and and K196S, preferably K196G, K196A, K196V, K196L, K196I, K196P, K196R, K196H, K196N, K196Q, K196S, K196T, K196C, and K196M, more preferably K196R, and K196H, most preferably K196R; (xxix) is substituted at position G197, preferably the substitution is selected from the group consisting of G197A, G197R, G197N, G197D, G197C, G197Q, G197E, G197H, G197I, G197L, G197K, G197M, G197F, G197P, G197T, G197W, G197Y, G197V, and G197S, preferably G197D, G197E, G197R, G197K, G197H, G197A, G197V, G197L, G197I, and G197P, preferably G197K, G197H, and G197R, more preferably G197K; (xxx) Substitutions at position D199, preferably the substitutions are D199G, D199A, D199V, D199L, D199I, D199P, D199W, D199F, D199Y, D199C, D199M, D199S, D199Q, D199T, D199N, D199E, D199H, D199R and D199K, preferably D199G, D199A, D199V, D199I D199L, D199I, D199P, D199W, D199F, D199Y, D199C, D199M, D199N, D199S, D199Q, D199T, and D199K, preferably D199N, D199S, D199Q, D199T, D199C, D199M, D199H, and D199K, more preferably D199N, D199S, and D199K; (xxxi) Substitutions at position R204, preferably substitutions are R204A, R204N, R204D, R204C, R204Q, R204E, R204G, R204H, R204I, R204L, R204K, R204M, R204F, R204P, R204T, R204W, R204Y, R204V, and R204S, preferably selected from the group consisting of R204G, R204A, R204V, R204L, R204I, R204P, R204K, R204H, R204N, R204Q, R204S, R204T, R204C, and R204M, more preferably R204Q, R204N, R204S, R204T, R204C and R204M, most preferably R204Q; (xxxii) is substituted at position I218, preferably the substitution is selected from the group consisting of I218R, I218N, I218D, I218C, I218Q, I218E, I218G, I218H, I218A, I218L, I218K, I218M, I218F, I218P, I218T, I218W, I218Y, I218V, and I218S, preferably I218F, I218W and I218Y, more preferably I218F; (xxxiii) is substituted at position A228, preferably the substitution is selected from the group consisting of A228R, A228N, A228D, A228C, A228Q, A228E, A228G, A228H, A228I, A228L, A228K, A228M, A228F, A228P, A228T, A228W, A228Y, A228V, and A228S, preferably A228K, A228H and A228R, more preferably A228R.

[0120] In a preferred embodiment, the amino acid sequence is selected from the group consisting of the following substitutions compared to SEQ ID NO: 1: P51L, P51G, P51D, K55R, T56A, T56V, T56L, T56I, T56P, T56D, T56S, T56G, T56R, N58A, N58G, N58V, N58L, N58I, N58P, N58R, A74R, T77K, T77F, T77A, T77G, T77V, T77I, T77P, T77L, T77R, N80H, A81K, K84G, K84A, K84V; K84L, K84I, K84P, D86S, A94G, S116K, D117N, A12 4K, D128R, D128M, D128Y, D128F, K132R, D135A, D135G, D135N, D135M, D135R, E151L, K162N, D191A, D191G, D191V, D191L, D191I, D191P, G197K, R204Q, I218F, A228R, E239G, E239V, E239L, E239I, E239P, and E239A, preferably P51L, P51G, P51D, K55R, T56P, T56D, T56S, T56G, T56R, N58V, N58R, A74R, T77K, T77F, T 77L, T77R, N80H, A81K, K84G, D86S, A94G, S116K, D117N, A124K, D128R, D128M, D128Y, D128F, K132R, D135A, D135G, D135N, D135M, D135R, E151L, K162N, D191P, G197K, R204Q, I218F, A228R, and E239A, more preferably P51L, P51G, P51D, K55R, T56P, T56G, N58V, A74R, T77L, N80H, A81K, K84G, D86S, A94G, S116K, D117 at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, or at least 17 selected from the group consisting of N, A124K, D128R, D128M, D128Y, D128F, K132R, D135A, D135G, D135N, D135M, D135R, E151L, K162N, D191P, G197K, R204Q, I218F, A228R, and E239A;At least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 positions are substituted, such as to include at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 substitutions.

[0121] In a preferred embodiment, the nuclease, preferably the endonuclease, has at least 65% identity to SEQ ID NO: 1 and comprises an amino acid sequence comprising: [1] (i) a substitution at position T56, which is substituted with an amino acid selected from the group consisting of A, G, V, L, I, and P, preferably P; and (ii) a substitution at position K84, which is substituted with an amino acid selected from the group consisting of A, G, V, L, I, and P, preferably G; [2] (i) a substitution at position T56, which is substituted with an amino acid selected from the group consisting of A, G, V, L, I, and P, preferably P; (ii) a substitution at position K84, which is substituted with an amino acid selected from the group consisting of A, G, V, L, I, and P, preferably G; and (iii) a substitution at position P51, which is substituted with an amino acid selected from the group consisting of A, G, V, L, and I, preferably L; [3] (i) a substitution at position P51, which is substituted with an amino acid selected from the group consisting of A, G, V, L, and I, preferably L; and (ii) a substitution at position T56, which is substituted with an amino acid selected from the group consisting of A, G, V, L, I, and P, preferably P; [4] (i) a substitution at position P51, which is substituted by an amino acid selected from the group consisting of A, G, V, L, and I, preferably L; (ii) a substitution at position T56, which is substituted by an amino acid selected from the group consisting of A, G, V, L, I, and P, preferably P; and (iii) a substitution at position D135, which is substituted by an amino acid selected from the group consisting of N, T, Q, S, C, M, A, G, V, L, I, and P, preferably N and A; [5] (i) a substitution at position E239, which is substituted by an amino acid selected from the group consisting of A, G, V, L, I, and P, preferably A; (ii) a substitution at position P51, which is substituted by an amino acid selected from the group consisting of A, G, V, L, and I, preferably L; (iii) a substitution at position T56, which is substituted by an amino acid selected from the group consisting of D, E, C, M, N, Q, S, A, G, V, L, I, and P, preferably P, G, D, and S, more preferably D.

[0122] In a preferred embodiment, the nuclease, preferably the endonuclease, comprises an amino acid sequence which has at least 76%, even more preferably at least 78%, even more preferably at least 80%, most preferably at least 84%, especially at least 87% identity to SEQ ID NO: 1.

[0123] Preferably, the amino acid sequence of the nuclease, preferably endonuclease, according to the invention has at least 88%, preferably at least 89%, more preferably at least 90%, even more preferably at least 91%, even more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and especially at least 95% identity with SEQ ID NO: 1.

[0124] In a preferred embodiment, the nuclease, preferably the endonuclease, comprises an amino acid sequence set forth in SEQ ID NOs: 2, 4, 5, 8, 9, 10, 13, 19, 21, 22, 23, 24, 25, 26, 27, 28, 32, 34, 36, 37, 41, 44, 46, 48, 53, 54, 68, 72, 73, 74, 75, 76, 77, 81, 99, 100, 101, 102, 106, 107, 108, 109, 110, 111, 122, 123, 124 , 128, 129, 130, 131, or 137, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, most preferably at least 97%, most preferably at least 98%, and especially at least 99%, or 100% identical to the sequences of ...

[0125] In a preferred embodiment of the nuclease, preferably the endonuclease, the nuclease is characterized by (A) an increased thermostability compared to the wild-type nuclease of SEQ ID NO:1.

[0126] Another preferred embodiment of the present invention relates to a nuclease, preferably an endonuclease, having at least 70% identity to SEQ ID NO: 1, wherein the amino acid sequence has been substituted in at least two amino acid positions selected from the group consisting of P51, T56, K84, and N58 compared to SEQ ID NO: 1, in each case the native amino acid at the selected position has been replaced by an amino acid selected from the group consisting of L, P, A, G, V, and I, preferably selected from the group consisting of L, P, I, G, and V, more preferably selected from the group consisting of L, P, I, and G, even more preferably selected from the group consisting of L, P, and G, even more preferably selected from the group consisting of L and P.

[0127] In a preferred embodiment, the amino acid sequence has a substitution at position P51 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of P51G, P51A, P51V, P51I and P51L, preferably P51G and P51L, more preferably P51L.

[0128] In a preferred embodiment, the amino acid sequence has a substitution at position T56 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of T56G, T56A, T56V, T56L, T56I and T56P, preferably T56G and T56P.

[0129] In a preferred embodiment, the amino acid sequence has a substitution at position K84 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of K84G, K84A, K84V, K84L, K84I and K84P, preferably K84G.

[0130] In a preferred embodiment, the amino acid sequence has a substitution at position N58 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of N58G, N58A, N58V, N58L, N58I and N58P, preferably N58V.

[0131] In a preferred embodiment, the amino acid sequence has the following repeat sequences compared to SEQ ID NO: 1: P51, A52, S53, G54, K55, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, K84, D86, A94, S116, D117, N119, Q120, A124, D128, Q129, K132, D 135, E151, K162, D191, P195, K196, G197, D199, R204, I218, A228, and E239; preferably P51, K55, T56, N58, P73, A74, T77, G78, A81, K84, D86, S116, D117, Q120, A124, D128, Q129, D1 and 35, K196, G197, D199, and E239, more preferably P51, T56, N58, T77, A81, K84, D86, S116, D117, Q120, A124, D128, Q129, D135, K196, and E239, even more preferably P51, T56, N58, T77, K84, D86, S116, and D135.

[0132] In a preferred embodiment, the nuclease, preferably the endonuclease, is [1]: (i) two substitutions at positions selected from the group consisting of P51, T56, K84, and N58 (in each case, a native amino acid of SEQ ID NO: 1 is replaced by an amino acid selected from the group consisting of L, P, A, G, V, and I); and [2]:(ii) a third substitution at another position selected from the group consisting of P51, T56, K84, and N58 (in each case, a naturally occurring amino acid of SEQ ID NO: 1 is replaced with an amino acid selected from the group consisting of G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S, and T).

[0133] In a preferred embodiment, (ii) the third substitution is selected from the group consisting of: P51L, P51D, P51E, P51R, P51K, P51H, P51G, P51A, P51V, P51I, P51F, P51W, P51Y, P51N, P51T, P51S, P51Q, P51C, and P51M, preferably P51G, P51A, P51V, P51I, and P51L, more preferably P51G and P51L, even more preferably P51L; T56P, T56G, T56D, T56E, T56R, T56K, T56H, T56A, T56V, T56L, T56I, T56F, T56W, T56Y, T56N, T56S, T56Q, T56C, and T56M, preferably T56R, T56K, T56H, T56G, T56A, T56V, T56L, T56I, T56P, and T56D, more preferably T56G, T56A, T56V, T56L, T56I, T56P, and T56D, even more preferably T56G, T56P, and T56D; K84G, K84D, K84E, K84R, K84H, K84A, K84V, K84L, K84I, K84P, K84F, K84W, K84Y, K84N, K84T, K84S, K84Q, K84C, and K84M, preferably K84G, K84A, K84V, K84L, K84I, and K84P, more preferably K84G; and N58V, N58D, N58E, N58R, N58K, N58H, N58G, N58A, N58L, N58I, N58P, N58F, N58W, N58Y, N58T, N58S, N58Q, N58C, and N58M; N58R, N58K, N58H, N58G, N58A, N58V, N58L, N58I, and N58P, more preferably N58R, N58K, and N58V, even more preferably N58R, and N58V.

[0134] In a preferred embodiment, the amino acid sequence comprises a substitution at position A52 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of A52R, A52N, A52D, A52C, A52Q, A52E, A52G, A52H, A52I, A52L, A52K, A52M, A52F, A52P, A52T, A52W, A52Y, A52V and A52S, preferably A52R, A52K, A52H, more preferably A52R.

[0135] In a preferred embodiment, the amino acid sequence has a substitution at position S53 compared to SEQ ID NO: 1, preferably the substitution is S53A, S53R, S53N, S53D, S53C, S53Q, S53E, S53G, S53H, S53I, S53L, S53K, S53M, S53F, S53P, S53T, S53W, S53Y, and S53V, preferably S53R, It is preferably selected from the group consisting of S53K, S53H, S53G, S53A, S53V, S53L, S53I, S53P, S53W, S53F, S53Y, and S53D, more preferably S53R, S53K, S53H, S53G, S53A, S53V, S53L, S53I, and S53P, even more preferably S53K, and S53A, and most preferably S53K.

[0136] In a preferred embodiment the amino acid sequence comprises a substitution at position G54 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of G54A, G54R, G54N, G54D, G54C, G54Q, G54E, G54H, G54I, G54L, G54K, G54M, G54F, G54P, G54T, G54W, G54Y, G54V and G54S, preferably G54D, G54E, G54R, G54K, G54H, G54A, G54V, G54L, G54I and G54P, preferably G54K, G54H and G54R, more preferably G54R.

[0137] In a preferred embodiment the amino acid sequence comprises a substitution at position K55 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of K55A, K55R, K55N, K55D, K55C, K55Q, K55E, K55G, K55H, K55I, K55L, K55M, K55F, K55P, K55T, K55W, K55Y, K55V and K55S, preferably K55G, K55A, K55V, K55L, K55I, K55P, K55R, K55H, K55N, K55Q, K55S, K55T, K55C and K55M, more preferably K55H and K55R, most preferably K55R.

[0138] In a preferred embodiment, the amino acid sequence comprises a substitution at position A72 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of A72R, A72N, A72D, A72C, A72Q, A72E, A72G, A72H, A72I, A72L, A72K, A72M, A72F, A72P, A72T, A72W, A72Y, A72V, and A72S, preferably A72D, A72E, A72R, A72K, A72H, A72G, A72V, A72L, A72I, and A72P, more preferably A72K, A72H and A72R, most preferably A72K.

[0139] In a preferred embodiment, the amino acid sequence has a substitution at position P73 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of P73A, P73R, P73N, P73D, P73C, P73Q, P73E, P73G, P73H, P73I, P73L, P73K, P73M, P73F, P73T, P73W, P73Y, P73V and P73S, preferably P73D, P73E, P73R, P73K, P73H, P73G, P73V, P73L, P73I and P73A, more preferably P73K, P73H and P73R, most preferably P73K and P73R.

[0140] In a preferred embodiment, the amino acid sequence comprises a substitution at position A74 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of A74R, A74N, A74D, A74C, A74Q, A74E, A74G, A74H, A74I, A74L, A74K, A74M, A74F, A74P, A74T, A74W, A74Y, A74V, and A74S, preferably A74D, A74E, A74R, A74K, A74H, A74G, A74V, A74L, A74I, and A74P, more preferably A74K, A74H, and A74R, most preferably A74K and A74R.

[0141] In a preferred embodiment the amino acid sequence comprises a substitution at position T77 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of T77R, T77N, T77D, T77C, T77Q, T77E, T77G, T77H, T77I, T77L, T77K, T77M, T77F, T77P, T77A, T77W, T77Y, T77V and T77S, preferably T77R, T77K, T77H, T77G, T77A, T77V, T77L, T77I, T77P, T77W, T77F, T77Y and T77D, preferably T77L, T77F, T77K, T77R and T77H, more preferably T77K, T77R and T77H, even more preferably T77R.

[0142] In a preferred embodiment, the amino acid sequence comprises a substitution at position G78 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of G78A, G78R, G78N, G78D, G78C, G78Q, G78E, G78H, G78I, G78L, G78K, G78M, G78F, G78P, G78T, G78W, G78Y, G78V and G78S, preferably G78R, G78K and G78H, more preferably G78R.

[0143] In a preferred embodiment the amino acid sequence comprises a substitution at position N80 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of N80A, N80R, N80D, N80C, N80Q, N80E, N80G, N80H, N80I, N80L, N80K, N80M, N80F, N80P, N80T, N80W, N80Y, N80V and N80S, preferably N80R, N80K, N80H, N80G, N80A, N80V, N80L, N80I, N80P, N80W, N80F, N80Y and N80D, preferably N80K, N80R and N80H, more preferably N80H and N80R, most preferably N80H.

[0144] In a preferred embodiment, the amino acid sequence has a substitution at position A81 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of A81R, A81N, A81D, A81C, A81Q, A81E, A81G, A81H, A81I, A81L, A81K, A81M, A81F, A81P, A81T, A81W, A81Y, A81V, and A81S, preferably A81K, A81H, and A81R, more preferably A81K.

[0145] In a preferred embodiment, the amino acid sequence comprises a substitution at position A82 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of A82R, A82N, A82D, A82C, A82Q, A82E, A82G, A82H, A82I, A82L, A82K, A82M, A82F, A82P, A82T, A82W, A82Y, A82V, and A82S, preferably A82K, A82H, and A82R, more preferably A82R.

[0146] In a preferred embodiment, the amino acid sequence has a substitution at position D86 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of D86A, D86R, D86N, D86C, D86Q, D86E, D86G, D86H, D86I, D86L, D86K, D86M, D86F, D86P, D86T, D86W, D86Y, D86V and D86S; D86G, D86A, D86V, D86L, D86I, D86P, D86C, D86M, D86S, D86T and D86Q, preferably D86C, D86M, D86S, D86T and D86Q, more preferably D86S.

[0147] In a preferred embodiment, the amino acid sequence comprises a substitution at position A94 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of A94R, A94N, A94D, A94C, A94Q, A94E, A94G, A94H, A94I, A94L, A94K, A94M, A94F, A94P, A94T, A94W, A94Y, A94V, and A94S; preferably A94G, A94V, A94L, A94I, and A94P; more preferably A94G.

[0148] In a preferred embodiment, the amino acid sequence has a substitution at position S116 compared to SEQ ID NO: 1, preferably the substitution is S116A, S116R, S116N, S116D, S116C, S116Q, S116E, S116G, S116H, S116I, S116L, S116K, S116M, S116F, S116P, S116T, S116L, S116K, S116M, S116F, S116P, S116T, S116L, S116K, S116M, S116N, S116D, S116C, S116Q, S116E, S116G, S116H, S116I, S116L, S116K, S116M, S116F, S116P, S116T, S116L, S116K, S116M, S116L, S116K, S116M, S116F, S116P, S116T, S116L, S116K, S116R, S116N, S116D, S116C, S116Q, S116E, S116G, S116H, S116I ... S116W, S116Y, and S116V, preferably S116R, S116K, S116H, S116G, S116A, S116V, S116L, S116I, S116P, S116W, S116F, S116Y, and S116D, preferably S116K, S116H, and S116R, more preferably S116K.

[0149] In a preferred embodiment the amino acid sequence has a substitution at position D117 compared to SEQ ID NO: 1, preferably the substitution is D117G, D117A, D117V, D117L, D117I, D117P, D117W, D117F, D117Y, D117C, D117M, D117S, D117Q, D117T, D117N, D117E, D117H, D117R and D117K, preferably D117G, D117A, D117V, D117L, D117I, D117P, More preferably, D117K, D117R, D117H, D117W, D117F, D117Y, D117C, D117M, D117S, D117Q, D117T, D117N, D117H, D117R and D117K, even more preferably, D117R, D117K, D117H, and D117Y, even more preferably, D117R, D117K, and D117H, and even more preferably, D117K.

[0150] In a preferred embodiment, the amino acid sequence comprises a substitution at position N119 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of N119A, N119R, N119D, N119C, N119Q, N119E, N119G, N119H, N119I, N119L, N119K, N119M, N119F, N119P, N119T, N119W, N119Y, N119V and N119S, preferably N119R, N119K and N119H, more preferably N119R.

[0151] In a preferred embodiment, the amino acid sequence has a substitution at position Q120 compared to SEQ ID NO: 1, preferably the substitution is Q120A, Q120R, Q120N, Q120D, Q120C, Q120E, Q120G, Q120H, Q120I, Q120L, Q120K, Q120M, Q120F, Q120P, Q120T, Q120W, Q120C, Q120D, Q120E, Q120G, Q120H, Q120I, Q120L, Q120K, Q120M, Q120F, Q120P, Q120T, Q120W, Q120F, Q120G, Q120H, Q120I, Q120L, Q120K, Q120M, Q120F, Q120P, Q120T, Q120W, Q120H, Q120I, Q120L, Q120K, Q120M, Q120F, Q120P, Q120T, Q120W, Q120H, Q120I, Q120H, Q120I, Q120L, Q120K, Q120M, Q120F, Q120P, Q120T, Q120W, Q120H, Q120H, Q120I ... Q120Y, Q120V, and Q120S, preferably Q120R, Q120K, Q120H, Q120G, Q120A, Q120V, Q120L, Q120I, Q120P, Q120W, Q120F, Q120Y, and Q120D, more preferably Q120K, Q120H, and Q120R, and most preferably Q120R.

[0152] In a preferred embodiment, the amino acid sequence comprises a substitution at position A124 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of A124R, A124N, A124D, A124C, A124Q, A124E, A124G, A124H, A124I, A124L, A124K, A124M, A124F, A124P, A124T, A124W, A124Y, A124V, and A124S, preferably A124D, A124E, A124R, A124K, A124H, A124G, A124V, A124L, A124I, and A124P, preferably A124K, A124H and A124R, more preferably A124K.

[0153] In a preferred embodiment, the amino acid sequence comprises a substitution at position D128 compared to SEQ ID NO: 1, preferably the substitution is D128A, D128R, D128N, D128C, D128Q, D128E, D128G, D128H, D128I, D128L, D128K, D128M, D128F, D128P, D128T, D128W, D128Y, D128V, and D128S, preferably D128G, D128A, D128V, D128L, D128I, D128P, D128W, D128F, D128Y, D128C, D128M, D128N, D128Q, D128S, D128T, D128H, D128R and D128K, most preferably D128G, D128Y, D128F, D128M, D128N, D128S, D128R and D128K.

[0154] In a preferred embodiment, the amino acid sequence comprises a substitution at position Q129 compared to SEQ ID NO: 1, preferably the substitution is Q129A, Q129R, Q129N, Q129D, Q129C, Q129E, Q129G, Q129H, Q129I, Q129L, Q129K, Q129M, Q129F, Q129P, Q129T, Q129W, Q129Y, Q129V and and Q129S, preferably Q129R, Q129K, Q129H, Q129G, Q129A, Q129V, Q129L, Q129I, Q129P, Q129W, Q129F, Q129Y, and Q129D, preferably Q129K, Q129H, and Q129R, more preferably Q129K, Q129R, even more preferably Q129R.

[0155] In a preferred embodiment, the amino acid sequence comprises a substitution at position K132 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of K132A, K132R, K132N, K132D, K132C, K132Q, K132E, K132G, K132H, K132I, K132L, K132M, K132F, K132P, K132T, K132W, K132Y, K132V, and K132S, preferably K132R, and K132H, most preferably K132R.

[0156] In a preferred embodiment, the amino acid sequence has a substitution at position D135 compared to SEQ ID NO: 1, preferably the substitution is D135A, D135R, D135N, D135C, D135Q, D135E, D135G, D135H, D135I, D135L, D135K, D135M, D135F, D135P, D135T, D135W, D135Y, D135V and D135S, preferably D135G, D135A, D135 D135R, D135H, and D135K, more preferably D135G, D135A, D135V, D135L, D135I, D135P, D135C, D135M, D135N, D135Q, D135S, and D135T, even more preferably D135A, and D135N.

[0157] In a preferred embodiment, the amino acid sequence has a substitution at position E151 compared to SEQ ID NO: 1, preferably the substitution is E151A, E151R, E151N, E151D, E151C, E151Q, E151G, E151H, E151I, E151L, E151K, E151M, E151F, E151P, E151T, E151W, E151Y, E151V, and E151S. , E151G, E151A, E151V, E151L, E151I, E151P, E151W, E151F, E151Y, E151C, E151M, E151N, E151Q, E151S, E151T, E151H and E151K, preferably E151A, E151G, E151V, E151L, E151I, and E151P, more preferably E151L.

[0158] In a preferred embodiment, the amino acid sequence comprises a substitution at position K162 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of K162A, K162R, K162N, K162D, K162C, K162Q, K162E, K162G, K162H, K162I, K162L, K162M, K162F, K162P, K162T, K162W, K162Y, K162V and K162S, preferably K162N, K162Q, K162S, K162T, K162C and K162M, most preferably K162N.

[0159] In a preferred embodiment, the amino acid sequence has a substitution at position D191 compared to SEQ ID NO: 1, the substitution being D191G, D191A, D191V, D191L, D191I, D191P, D191W, D191F, D191Y, D191C, D191M, D191S, D191Q, D191T, D191N, D191E, D191H, ...G, D191H, D191G, D191G, D191G, D191H, D191I, D191I, D191I, D191I, D191I, 1R and D191K, preferably D191G, D191A, D191V, D191L, D191I, D191P, D191C, D191M, D191S, D191Q, and D191T, more preferably D191G, D191A, D191V, D191L, D191I, and D191P, most preferably D191P.

[0160] In a preferred embodiment, the amino acid sequence comprises a substitution at position P195 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of P195A, P195R, P195N, P195D, P195C, P195Q, P195E, P195G, P195H, P195I, P195L, P195K, P195M, P195F, P195T, P195W, P195Y, P195V and P195S, preferably P195K, P195H and P195R, most preferably P195R.

[0161] In a preferred embodiment, the amino acid sequence has a substitution at position K196 compared to SEQ ID NO: 1, preferably the substitution is K196A, K196R, K196N, K196D, K196C, K196Q, K196E, K196G, K196H, K196I, K196L, K196M, K196F, K196P, K196T, K196W, K196H, K196I, K196L, K196M, K196F, K196P, K196T, K196W, K196H, K196I, K196L, K196M, K196F, K196P, K196T, K196W, K196H, K196I, K196L, K196R, K196N, K196D, K196C, K196Q, K196E, K196G, K196H, K196I, K196L, K196M, K196F, K196P, K196T, K196W ...H, K196I, K196L, K196R, K196N, K196D, K196C, K196Q, K196E, K196G, K196H, K196I, K196L, K196M, K196F, K196P, K196T, K196W, K196H, K196H, K196H 6Y, K196V, and K196S, preferably K196G, K196A, K196V, K196L, K196I, K196P, K196R, K196H, K196N, K196Q, K196S, K196T, K196C, and K196M, more preferably K196R, and K196H, most preferably K196R.

[0162] In a preferred embodiment, the amino acid sequence comprises a substitution at position G197 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of G197A, G197R, G197N, G197D, G197C, G197Q, G197E, G197H, G197I, G197L, G197K, G197M, G197F, G197P, G197T, G197W, G197Y, G197V and G197S, preferably G197D, G197E, G197R, G197K, G197H, G197A, G197V, G197L, G197I and G197P, preferably G197K, G197H and G197R, more preferably G197K.

[0163] In a preferred embodiment, the amino acid sequence has a substitution at position D199 compared to SEQ ID NO: 1, preferably the substitution is D199G, D199A, D199V, D199L, D199I, D199P, D199W, D199F, D199Y, D199C, D199M, D199S, D199Q, D199T, D199N, D199E, D199H, D199R and D199K, preferably D199G, D199A, D199V, D199L, D199I, D199P, D199W, D199F, D199Y, D199C, D199M, D199S, D199Q, D199T, D199N, D199E, D199H, D199R and D199K, D199A, D199V, D199L, D199I, D199P, D199W, D199F, D199Y, D199C, D199M, D199N, D199S, D199Q, D199T, and D199K, preferably D199N, D199S, D199Q, D199T, D199C, D199M, D199H, and D199K, more preferably D199N, D199S, and D199K.

[0164] In a preferred embodiment, the amino acid sequence has a substitution at position R204 compared to SEQ ID NO: 1, preferably the substitution is R204A, R204N, R204D, R204C, R204Q, R204E, R204G, R204H, R204I, R204L, R204K, R204M, R204F, R204P, R204T, R204W, R204Y, R204V, and R204S, preferably R204G, R204A, R204V, R204L, R204I, R204P, R204K, R204H, R204N, R204Q, R204S, R204T, R204C, and R204M, more preferably R204Q, R204N, R204S, R204T, R204C and R204M, most preferably R204Q.

[0165] In a preferred embodiment the amino acid sequence comprises a substitution at position I218 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of I218R, I218N, I218D, I218C, I218Q, I218E, I218G, I218H, I218A, I218L, I218K, I218M, I218F, I218P, I218T, I218W, I218Y, I218V and I218S, preferably I218F, I218W and I218Y, more preferably I218F.

[0166] In a preferred embodiment, the amino acid sequence comprises a substitution at position A228 compared to SEQ ID NO: 1, preferably the substitution is selected from the group consisting of A228R, A228N, A228D, A228C, A228Q, A228E, A228G, A228H, A228I, A228L, A228K, A228M, A228F, A228P, A228T, A228W, A228Y, A228V, and A228S, preferably A228K, A228H and A228R, more preferably A228R.

[0167] In a preferred embodiment, the amino acid sequence comprises a substitution at position E239 compared to SEQ ID NO: 1, the substitution being E239A, E239R, E239N, E239D, E239C, E239Q, E239G, E239H, E239I, E239L, E239K, E239M, E239F, E239P, E239T, E239W, E239Y, E239V, and E239S, preferably E239G, E239A, E239V, E239L, E239I, E239P, E239W, E239F, E239Y, E239C, E239M, E239N, E239Q, E239S, E239T, E239H and E239K, preferably E239A, E239G, E239V, E239L, E239I, and E239P, more preferably E239A.

[0168] In a preferred embodiment, the amino acid sequence has the following residues at positions P51, K55, T56, N58, P73, A74, T77, G78, A81, K84, D86, S116, D117, Q120, A124, D128, Q129, D135, K196, G197, D199, and E239, preferably P51, T56, N58, T77, A81, K84, D86, S116, D117, Q120, A124, D128, Q129, D135, K196, and E239, more preferably P51, T56, N58, T77, , K84, D86, S116, and D135, and exhibits improved T activity compared to the T activity of SEQ ID NO: 1; (i) if the amino acid substitution comprises a substitution at amino acid position P51, the amino acid substitution at position P51 is selected from the group consisting of substitutions D, E, R, K, H, G, A, V, L, and I, preferably G, A, V, I, and L, more preferably G and L, even more preferably L; (ii) if the amino acid substitution comprises a substitution at amino acid position K55, the amino acid substitution at position K55 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably H and R, more preferably R; (iii) if the amino acid substitution comprises a substitution at amino acid position T56, the amino acid substitution at position T56 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, N, S, Q, C, M, and D, preferably R, K, H, G, A, V, L, I, P, and D, more preferably G, A, V, L, I, P, and D, even more preferably G, P, and D; (iv) if the amino acid substitution comprises a substitution at amino acid position N58, the amino acid substitution at position N58 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, and P, more preferably R, K, and V, even more preferably R and V; (v) if the amino acid substitution comprises a substitution at amino acid position P73, the amino acid substitution at position P73 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and A, preferably K, H and R, more preferably K and R; (vi) if the amino acid substitution comprises a substitution at amino acid position A74, the amino acid substitution at position A74 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K and R; (vii) if the amino acid substitution comprises a substitution at amino acid position T77, the amino acid substitution at position T77 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably L, F, K, R, and H, more preferably K, R, and H, even more preferably R; (viii) if the amino acid substitution comprises a substitution at amino acid position G78, the amino acid substitution at position G78 is selected from the group consisting of substitutions R, K, H, A, V, L, I, P, W, F, Y, N, Q, S, T, C, M and D, preferably K, R, and H, more preferably R; (ix) if the amino acid substitution comprises a substitution at amino acid position A81, the amino acid substitution at position A81 is selected from the group consisting of substitutions R, K, and H, preferably K; (x) if the amino acid substitution comprises a substitution at amino acid position K84, the amino acid substitution at position K84 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably G, A, V, L, I, and P, more preferably G; (xi) if the amino acid substitution comprises a substitution at amino acid position D86, the amino acid substitution at position D86 is selected from the group consisting of substitutions G, A, V, L, I, P, C, M, S, Q, and T, preferably Q, T, and S, more preferably S; (xii) if the amino acid substitution comprises a substitution at amino acid position S116, the amino acid substitution at position S116 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, H, and R, more preferably K; (xiii) if the amino acid substitution comprises a substitution at amino acid position D117, the amino acid substitution at position D117 is selected from the group consisting of the substitutions G, A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, and K, preferably G, V, L, I, P, W, F, Y, C, M, S, N, Q, H, R, and K, more preferably Y, W, F, K, R, and H, even more preferably Y, K, R, and H, even more preferably K, R, and H, even more preferably K; (xiv) if the amino acid substitution comprises a substitution at amino acid position Q120, the amino acid substitution at position Q120 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, H, and R, more preferably R; (xv) if the amino acid substitution comprises a substitution at amino acid position A124, the amino acid substitution at position A124 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K; (xvi) if the amino acid substitution comprises a substitution at amino acid position D128, the amino acid substitution at position D128 is selected from the group consisting of the substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, preferably G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, more preferably G, Y, F, M, N, S, R, and K; (xvii) if the amino acid substitution comprises a substitution at amino acid position Q129, the amino acid substitution at position Q129 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, H, and R, more preferably R; (xviii) if the amino acid substitution comprises a substitution at amino acid position D135, the amino acid substitution at position D135 is selected from the group consisting of the substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, preferably R, K, G, A, V, L, I, P, C, M, N, Q, S, and T, more preferably G, A, V, L, I, P, C, M, N, Q, S, and T, even more preferably A and N; (xix) if the amino acid substitution comprises a substitution at amino acid position K196, the amino acid substitution at position K196 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably H and R, more preferably R; (xx) if the amino acid substitution includes a substitution at amino acid position G197, the amino acid substitution at position G197 is selected from the group consisting of substitutions D, E, R, K, H, A, V, L, I, and P, preferably K, H, and R, more preferably K; (xxi) if the amino acid substitution comprises a substitution at amino acid position D199, the amino acid substitution at position D199 is selected from the group consisting of the substitutions G, A, V, L, I, P, W, F, Y, C, M, Q, T, N, S, H, R, and K, preferably G, A, V, L, I, P, W, F, Y, C, M, Q, T, N, S, H, and K, more preferably S, Q, T, N, C, M, H, and K, even more preferably S, N, and K; or (xxii) If the amino acid substitution comprises a substitution at amino acid position E239, the amino acid substitution at position E239 is selected from the group consisting of the substitutions G, A, V, L, I, P, W, F, Y, C, M, Q, T, N, S, H, R, K, and D, preferably G, A, V, L, I, and P, more preferably A.

[0169] In a preferred embodiment, the amino acid sequence is selected from the group consisting of the following substitutions compared to SEQ ID NO: 1: P51L, P51D, P51G, P51R, K55R, T56M, T56L, T56S, T56A, T56V, T56I, T56G, T56D, T56P, T56R, T56K, T56F, N58V, N58K, N58R, P73K, P73R, A74K, A74R, T77K, T77L, T77R, T77F, G78R, K84G, D86S, S116K, D117K, D117H, D117Y, D117R, Q120R, A124K, D128Y, D128M, D128Y ... 28N, D128K, D128F, D128R, D128S, D128G, Q129R, D135A, D135K, D135R, D135N, D135M, D135G, K196R, G197K, D199K, D199S, D199N, and E239A, preferably P51L, T56A, T56V, T56I, T56M, T56L, T56F, T56D, T56P, T56G, T56K, T56S, N58V, N58R, T77K, T77R, K84G, D86S, S116K, D117H, D117R, D117Y, Q120R, A124K , D128G, D128N, D128S, Q129R, D135A, D135M, D135N, K196R, and E239A, more preferably at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 selected from the group consisting of P51L, T56D, T56P, T56G, N58V, N58R, T77R, K84G, D86S, S116K, D135A, and D135N. , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 positions to include at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 substitutions.

[0170] In a preferred embodiment, the nuclease, preferably the endonuclease, has at least 70% identity to SEQ ID NO: 1; (i) a substitution at position P51 by an amino acid selected from the group consisting of A, G, V, L, and I, preferably L; (ii) a substitution at position T56 by an amino acid selected from the group consisting of A, G, V, L, I, and P, preferably G, A, L, I, and P, more preferably G and P, even more preferably P.

[0171] In a preferred embodiment, the nuclease, preferably the endonuclease, has at least 70% identity to SEQ ID NO: 1; (i) a substitution at position T56 by an amino acid selected from the group consisting of A, G, V, L, I, and P, preferably G, A, L, I, and P, more preferably G and P, even more preferably P; (ii) a substitution at position K84, which is substituted by an amino acid selected from the group consisting of A, G, V, L, I, and P, preferably G.

[0172] In a preferred embodiment, the nuclease, preferably the endonuclease, has at least 70% identity to SEQ ID NO: 1; (i) a substitution at position T56 by an amino acid selected from the group consisting of A, G, V, L, I, and P, preferably G, A, L, I, and P, more preferably G and P, even more preferably P; (ii) a substitution at position K84 by an amino acid selected from the group consisting of A, G, V, L, I, and P, preferably G; (iii) a substitution at position P51, where the amino acid is substituted with an amino acid selected from the group consisting of A, G, V, L, and I, preferably L.

[0173] In a preferred embodiment, the nuclease, preferably the endonuclease, has at least 70% identity to SEQ ID NO: 1; (i) a substitution at position P51 by an amino acid selected from the group consisting of A, G, V, L, and I, preferably L; (ii) a substitution at position T56 by an amino acid selected from the group consisting of A, G, V, L, I, and P, preferably G, A, L, I, and P, more preferably G and P, even more preferably P; (iii) a substitution at position D135, wherein the amino acid is substituted by an amino acid selected from the group consisting of N, T, Q, S, C, M, A, G, V, L, I, and P, preferably N and A.

[0174] In a preferred embodiment, the nuclease, preferably the endonuclease, has at least 70% identity to SEQ ID NO: 1; (i) a substitution at position P51 by an amino acid selected from the group consisting of A, G, V, L, and I, preferably L; (ii) a substitution at position T56 by an amino acid selected from the group consisting of A, G, V, L, I, and P, preferably G, A, L, I, and P, more preferably G and P, even more preferably G; (iii) a substitution at position A81 by an amino acid selected from the group consisting of R, K, and H, preferably R and K, more preferably K.

[0175] In a preferred embodiment, the nuclease, preferably the endonuclease, has at least 70% identity to SEQ ID NO: 1; (i) a substitution at position P51 by an amino acid selected from the group consisting of A, G, V, L, and I, preferably L; (ii) a substitution at position T56, wherein the amino acid is substituted by an amino acid selected from the group consisting of D, E, C, M, N, Q, S, A, G, V, L, I, and P, preferably P, G, D, and S, more preferably D.

[0176] In a preferred embodiment, the nuclease, preferably the endonuclease, has at least 70% identity to SEQ ID NO: 1; (i) a substitution at position P51 by an amino acid selected from the group consisting of A, G, V, L, and I, preferably L; (ii) a substitution at position T56 by an amino acid selected from the group consisting of D, E, C, M, N, Q, S, A, G, V, L, I, and P, preferably P, G, D, and S, more preferably D; (iii) a substitution at position E239, which is substituted by an amino acid selected from the group consisting of A, G, V, L, I, and P, preferably A.

[0177] In a preferred embodiment, the nuclease, preferably endonuclease, amino acid sequence has at least 76%, even more preferably at least 78%, even more preferably at least 80%, most preferably at least 84%, and especially at least 87% identity to SEQ ID NO:1.

[0178] In a preferred embodiment, the nuclease, preferably endonuclease, amino acid sequence has at least 88%, preferably at least 89%, more preferably at least 90%, even more preferably at least 91%, even more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and especially at least 95% identity to SEQ ID NO:1.

[0179] In a preferred embodiment, the nuclease, preferably endonuclease, amino acid sequence is selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 57, 60, 61, 62, 63, 64, 65, 66, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 102, 112, 116, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 1 20, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, or 144, preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, most preferably at least 97%, most preferably at least 98%, and especially at least 99%, or 100% identical to

[0180] In a preferred embodiment, the nuclease, preferably endonuclease, amino acid sequence is set forth in SEQ ID NOs: 2, 3, 4, 5, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 57, 60, 61, 62, 63, 64, 65, 66, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 7, 112, 116, or 119, preferably at least 90%, preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, most preferably at least 97%, most preferably at least 98%, and especially at least 99%, or 100% identical to the amino acid sequence of ...

[0181] In a preferred embodiment, the nuclease, preferably the endonuclease, has an amino acid sequence (i) SEQ ID NOs: 64, 65, 66, 73, 74, 75, 76, 81, 83, 88, 89, 90, 94, 120, 122, 123, 128, 131, and 135, or (ii) has at least 90%, preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, most preferably at least 97%, most preferably at least 98%, and especially at least 99%, or 100% identity to SEQ ID NOs: 64, 65, 66, 73, 74, 75, 76.

[0182] In a preferred embodiment, the nuclease, preferably the endonuclease, is (A) improved T low activity compared to the T low activity of SEQ ID NO: 1; and / or (B) Characterized by an improved relative low-temperature activity compared to the relative low-temperature activity of SEQ ID NO:1.

[0183] In a preferred embodiment, the nuclease, preferably the endonuclease, has the following sequence relative to SEQ ID NO: 1: (i) an improvement of at least 1.05-fold, preferably at least 1.5-fold, more preferably at least 2.0-fold, and even more preferably at least 3.0-fold; or (ii) have a relative low temperature activity that is improved by 1.05 to 5 times, preferably 1.5 to 5 times, more preferably 2 to 5 times, and even more preferably about 3 times;

[0184] In a preferred embodiment, the nuclease, preferably the endonuclease, has the following sequence relative to SEQ ID NO: 1: (i) an improvement of at least 1.01-fold, preferably at least 1.1-fold, more preferably at least 1.3-fold, even more preferably at least 1.5-fold, and even more preferably at least 1.55-fold; or (ii) have a 1.01-fold to 2-fold, preferably 1.1-fold to 2-fold, more preferably 1.3-fold to 1.6-fold, even more preferably 1.5-fold to 1.6-fold, and even more preferably about 1.55-fold improved T activity;

[0185] In a preferred embodiment, nuclease, preferably endonuclease, low temperature activity and relative low temperature activity are measured at a Tlow of 5°C and a Thigh of 30°C.

[0186] In a preferred embodiment of the nuclease according to the present invention, the amino acid sequence is selected from the group consisting of P51, A52, S53, G54, K55, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, K84, D86, A94, S116, D117, N119, Q120, A124, D128, Q129, K132, D135, E151, K162, D191, P195, K196, G197, D199, R204, I218, A228, and E239, compared to SEQ ID NO: 1. at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 amino acid positions selected from the group consisting of:

[0187] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence is P51, S53, K55, T56, N58, A72, P73, A74, T77, N80, K84, D86, S116, D117, Q120, A124, D128, Q129, K132, D135, D191, K196, G197, D199, R204, and E239, preferably P51, T56, N58, T77, K84, Q120, A124, D128, Q129, D135, K196, and G197, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 amino acid positions selected from the group consisting of:

[0188] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence is P51, S53, K55, T56, N58, A72, P73, A74, T77, N80, K84, D86, S116, D117, Q120, A124, D128, Q129, K132, D135, D191, K196, G197, D199, R204, and E239, preferably P51, T56, N58, T77, K84, Q120, A124, D128, Q129, D135, K196, and G197, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 amino acid positions selected from the group consisting of: G197, If the amino acid substitution comprises a substitution at amino acid position P51, the amino acid substitution at position P51 is selected from the group consisting of substitutions D, E, R, K, H, G, A, V, L, and I, preferably D, R, G, and L; If the amino acid substitution comprises a substitution at amino acid position S53, the amino acid substitution at position S53 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, and P, more preferably K, and A; If the amino acid substitution comprises a substitution at amino acid position K55, the amino acid substitution at position K55 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position T56, the amino acid substitution at position T56 is selected from the group consisting of substitutions S, M, R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably S, M, R, K, G, A, V, L, I, F, P, and D; If the amino acid substitution comprises a substitution at amino acid position N58, the amino acid substitution at position N58 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, and P, more preferably K, R, and V; If the amino acid substitution comprises a substitution at amino acid position A72, the amino acid substitution at position A72 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K; If the amino acid substitution comprises a substitution at amino acid position P73, the amino acid substitution at position P73 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and A, preferably K, H, and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position A74, the amino acid substitution at position A74 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position T77, the amino acid substitution at position T77 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, R, F, and L; If the amino acid substitution comprises a substitution at amino acid position N80, the amino acid substitution at position N80 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, R, and H, more preferably H; If the amino acid substitution comprises a substitution at amino acid position K84, the amino acid substitution at position K84 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably G, A, V, L, I, and P, more preferably G; If the amino acid substitution comprises a substitution at amino acid position D86, the amino acid substitution at position D86 is selected from the group consisting of substitutions G, A, V, L, I, P, C, M, S, Q, and T, preferably Q, T, and S, more preferably S; If the amino acid substitution comprises a substitution at amino acid position S116, the amino acid substitution at position S116 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, H, and R, more preferably K; If the amino acid substitution comprises a substitution at amino acid position D117, the amino acid substitution at position D117 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, and K, preferably A, G, V, L, I, P, C, M, N, T, Q, K, H, R, and S, more preferably A, N, Y, K, H, R, and S; If the amino acid substitution comprises a substitution at amino acid position Q120, the amino acid substitution at position Q120 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, H, and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position A124, the amino acid substitution at position A124 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position D128, the amino acid substitution at position D128 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, preferably G, Y, F, M, N, S, R, and K; If the amino acid substitution comprises a substitution at amino acid position Q129, the amino acid substitution at position Q129 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, H, and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position K132, the amino acid substitution at position K132 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position D135, the amino acid substitution at position D135 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, preferably G, A, V, L, I, P, C, M, N, Q, S, T, H, R, and K, more preferably A, M, G, N, R, and K; If the amino acid substitution comprises a substitution at amino acid position D191, the amino acid substitution at position D191 is selected from the group consisting of substitutions G, A, V, L, I, P, C, M, S, Q, and T, preferably G, A, V, L, I, and P, more preferably P; If the amino acid substitution comprises a substitution at amino acid position K196, the amino acid substitution at position K196 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably H, and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position G197, the amino acid substitution at position G197 is selected from the group consisting of substitutions D, E, R, K, H, A, V, L, I, and P, preferably K, H, and R, more preferably K; If the amino acid substitution comprises a substitution at amino acid position D199, the amino acid substitution at position D199 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, Q, T, N, S, H, R, and K, preferably S, Q, T, N, C, M, H, R, and K, more preferably N, S, and K; If the amino acid substitution comprises a substitution at amino acid position R204, the amino acid substitution at position R204 is selected from the group consisting of substitutions G, A, V, L, I, P, K, H, N, Q, S, T, C, and M, preferably Q, N, S, T, C, and M, more preferably Q; or If the amino acid substitution comprises a substitution at amino acid position E239, the amino acid substitution at position E239 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, preferably A, G, V, L, I, and P, more preferably A.

[0189] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence is selected from the group consisting of P51D, P51R, P51G, P51L, S53K, S53A, K55H, K55R, T56S, T56M, T56R, T56K, T56G, T56A, T56V, T56L, T56I, T56F, T56P, T56D, N58K, N58R, N58V, A72K, A72H, A72R, P73K, P73R, A74K, A74H, A74R, T77K, T77R, T77F, T77L, N80K, N80R, N81K, N81R, N82K, N82R, N83K, N83R, N84K, N84R, N85K, N85R, N86K, N86R, N87K, N87K, N88K, N88K, N89K, N89K, N90K, N91K, N92K, N93K, N94K, N94K, N95K, N96K, N97K, N97K, N98K, N99K, N99K, N99K, N91K, N91K, N92K, N92K, N93K, N94K, N94K, N95K, N95K, N96K, N97K, N98K, N9 ... 80H, K84G, D86S, S116K, D117A, D117N, D117Y, D117K, D117H, D117R, D117S, Q120R, A124K, A124R, D128G, D128Y, D128F, D128M, D128N, D128S, D128R, D128K, Q129R, K132R, D135A, D135M, D135G, D135N, D135R, D135K, D191P, K196R, G197K, D199N, D199S, D199K, R204Q, and E239A, are preferred. or P51R, P51G, P51L, T56S, T56M, T56R, T56K, T56G, T56A, T56V, T56L, T56I, T56F, T56P, T56D, N58K, N58R, N58V, T77K, T77R, T77F, T77L, K 84G, Q120R, A124K, A124R, D128G, D128Y, D128F, D128M, D128N, D128S, D128R, D128K, Q129R, D135A, D135M, D135G, D135N, D135R, D135K, K1 at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 amino acid positions selected from the group consisting of G196R, G197K ...

[0190] In a preferred embodiment of the nuclease according to the present invention, the amino acid sequence comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 1 ...9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25, or at least 26, or at least 27, or at least 28 amino acid positions, and the amino acid sequence further comprises at least one additional substitution, wherein the at least one additional substitution position is selected from the group consisting of P51, S53, K55, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, K84, D86, S116, D117, Q120, A124, D128, Q129, K132, D135, D191, K196, G197, D199, R204, and E239.

[0191] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence is selected from the group consisting of P51D, P51R, P51G, P51L, S53K, S53A, K55H, K55R, T56S, T56M, T56R, T56K, T56G, T56A, T56V, T56L, T56I, T56F, T56P, T56D, N58K, N58R, N58V, A72K, A72H, A72R, P73K, P73R, A74K, A74H, A74R, T77K, T77R, T77F, T77L, N80K, N80R, N80H, K84G, D86S, S116K, D86R ... at least two, at least three, at least four, at least five, at least six, or at least seven selected from the group consisting of 117A, D117N, D117Y, D117K, D117H, D117R, D117S, Q120R, A124K, A124R, D128G, D128Y, D128F, D128M, D128N, D128S, D128R, D128K, Q129R, K132R, D135A, D135M, D135G, D135N, D135R, D135K, D191P, K196R, G197K, D199N, D199S, D199K, R204Q, and E239A; at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25, or at least 26, or at least 27, or at least 28 amino acid positions, and the amino acid sequence further comprises at least one additional substitution, wherein the at least one additional substitution position is selected from the group consisting of P51D, P51R, P51G, P51L, S53K, S53A, K55H, K55R, T56S, T56M, T56R, T56K, T56G, T56A, T56V, T56L, T56I, T56F, T56P, T56D, N58K, N58R, N58V, A72K, A72 H, A72R, P73K, P73R, A74K, A74H, A74R, T77K, T77R, T77F, T77L, N80K, N80R, N80H, K84G, D86S, S116K, D117A, D117N, D117Y, D117K, D117H, D117R, D117S,Q120R, A124K, A124R, D128G, D128Y, D128F, D128M, D128N, D128S, D128R, D128K, Q129R, K132R, D135A, D135M, D135G, D135N, D135R, D135K, D191P, K196R, G197K, D199N, D199S, D199K, R204Q, and E239A.

[0192] <Functional characteristics of the nuclease according to the present invention> In a preferred embodiment of a nuclease according to the present invention, the nuclease is capable of hydrolyzing the phosphodiester of a polynucleotide substrate into a cleaved polynucleotide or oligonucleotide.

[0193] In a preferred embodiment of a nuclease according to the present invention, the nuclease catalyzes the hydrolysis of phosphodiesters of polynucleotide substrates into cleaved polynucleotides or oligonucleotides.

[0194] In a preferred embodiment of the nuclease according to the invention, the polynucleotide substrate is selected from the group consisting of single-stranded RNA, single-stranded DNA, double-stranded DNA, double-stranded RNA and hybrid-stranded DNA / RNA, preferably double-stranded DNA.

[0195] In preferred embodiments of the nuclease according to the invention, the cleaved polynucleotide substrate is an oligonucleotide, preferably the oligonucleotide is at least 200 nucleotides in length, more preferably at least 150 nucleotides in length, even more preferably at least 100 nucleotides in length, even more preferably at least 50 nucleotides in length, even more preferably at least 25 nucleotides in length, most preferably at least 13 nucleotides in length, most preferably at least 5 nucleotides in length, and especially 2 nucleotides in length, optionally the cleaved polynucleotide is a mononucleotide.

[0196] In a preferred embodiment, the nuclease according to the invention comprises: (A) improved thermostability compared to the wild-type nuclease of SEQ ID NO: 1; and / or (B) an improved relative salt activity compared to the relative salt activity of the wild-type nuclease of SEQ ID NO: 1; and / or (C) SEQ ID NO: 1 wild-type nuclease 高 Improved activity compared to S 高 activity; and / or (D) an improved relative low-temperature activity compared to the wild-type nuclease relative low-temperature activity of SEQ ID NO: 1; and / or (E) T of the wild-type nuclease of SEQ ID NO: 1 低 Improved T compared to activity 低 The compounds are characterized by either:

[0197] For purposes herein, the enzymatic activity of a nuclease according to the present invention is detected by the nuclease activity assay described in Example 2. The nuclease activity assay is based on the release of short, acid-soluble, cleaved polynucleotides or oligonucleotides from a DNA polynucleotide substrate, leading to an increase in absorbance at 260 nm. The term A260 refers to the absorbance of a sample in a nuclease activity assay and corresponds to the difference between the absorbance at 260 nm of such an enzyme sample and that of a control sample containing no nuclease.

[0198] In a preferred embodiment, the nuclease according to the invention has improved thermostability compared to SEQ ID NO: 1, said thermostability being at a temperature T 安定性 The ATP-binding protein is characterized by a higher residual activity after 15 min of incubation at RT.

[0199] In a preferred embodiment of the nucleases according to the invention, the residual activity of the individual nucleases is: (i) Two vessels were incubated at 23°C or 37°C with a specific amount of nuclease in a buffer containing 50 mM Tris·HCl, pH 8.0, 5 mM MgCl2, and 0.1 mg / ml BSA. 安定性 Incubate separately for 15 min in either (ii) then cooling the two vessels by incubation on ice for 15 minutes, centrifuging the vessels to remove denatured proteins, and isolating the supernatant containing the nuclease; (iii) transferring a specific proportional amount of either nuclease into a new separate container and mixing with salmon testis-derived DNA sodium salt to a final concentration of 0.8 mg / ml, incubating each new container at 30°C for 1 hour, then terminating the reaction by adding 100 μl of 4% perchloric acid, incubating the two resulting compositions on ice for 45 minutes, subsequently centrifuging the resulting compositions at 3,800 × g and 4°C for 30 minutes, and measuring the residual activity of either nuclease from step (ii) by measuring the absorbance at 260 nm of each resulting supernatant, which correlates with the enzymatic activity of the nuclease in the corresponding container; (iv) T expressed as a percentage 安定性 and the ratio of absorbance measured for the sample at 23°C (A260[T 安定性 ]:A260[T 23℃ ]) and calculate the residual activity as For any nuclease variant, the specific ratio of the amount of nuclease transferred to the two vessels in step (ii) is the required temperature at 23°C or T required to obtain a detectable linear signal when measuring the residual activity in step (iii). 安定性 The amount of nuclease of SEQ ID NO: 1 after incubation with either

[0200] In a preferred embodiment of the nuclease according to the invention, the residual activity is at a temperature T of 40°C to 60°C, preferably 45°C to 55°C, more preferably 49°C to 53°C, even more preferably 51°C. 安定性 It is measured in

[0201] In a preferred embodiment of the nuclease according to the present invention, the thermostability of the nuclease is such that the residual activity is at a temperature T 安定性, is improved by at least 16%, preferably at least 23%, more preferably at least 29%, even more preferably at least 59% compared to SEQ ID NO: 1.

[0202] In a preferred embodiment of the nuclease according to the invention, the thermostability of the nuclease is improved by at least 16%, preferably at least 23%, more preferably at least 29%, even more preferably at least 59% compared to SEQ ID NO:1.

[0203] In preferred embodiments, the nuclease according to the present invention has improved thermal stability by at least 1.051-fold, at least 1.5-fold, or at least 2-fold, more preferably at least 3-fold, at least 4-fold, or at least 5-fold, even more preferably at least 6-fold, at least 6.5-fold, or at least 7-fold compared to SEQ ID NO:1.

[0204] In a preferred embodiment, the nuclease according to the present invention has a thermal stability that is improved by 1.051 to 100 times, preferably 1.5 to 50 times, more preferably 2 to 40 times, even more preferably 3 to 30 times, even more preferably 4 to 23 times, even more preferably 5 to 21 times, and most preferably 6 to 15 times, compared to SEQ ID NO: 1.

[0205] In a preferred embodiment, the nuclease according to the invention comprises: (i) Salt concentration S 高 (S 高 activity) than SEQ ID NO: 1; and / or (ii) has a higher relative salt activity compared to SEQ ID NO: 1, and the relative salt activity is 低 Salt concentration S compared to 高 is the ratio of enzyme activity in

[0206] In a preferred embodiment of the nuclease according to the invention, S 高 activity, S 低 activity, and relative salt activity, (i) A specific ratio of amounts of such nuclease was transferred to two separate containers and mixed with a buffer containing 50 mM Tris·HCl, pH 8.0, 5.0 mM MgCl , 0.1 mg / ml BSA, and 0.8 mg / ml of DNA sodium salt from salmon testis, and S 低 or S 高 The final salt concentration was adjusted to 100 μl, and the mixture was incubated at 30° C. for 1 hour. Then, 100 μl of 4% perchloric acid was added to terminate the reaction. The two resulting compositions were incubated on ice for 45 minutes. The resulting compositions were then centrifuged at 3,800×g and 4° C. for 30 minutes. The absorbance at 260 nm of each resulting supernatant was measured (the detected absorbance was the salt concentration S). 低 (S 低 activity) and S 高 (S 高 The enzyme activity of the nuclease is correlated with the salt concentration (S) by measuring 低 and S 高 separately detecting the enzymatic activity of the nuclease in (ii) S expressed as a percentage 高 (S 高 (activity) and S 低 (S 低 Relative salt activity (A260[S 高 ]:A260[S 低 ]) and For any nuclease variant, the specific ratio of the amount of nuclease transferred to the two vessels in step (i) is S 低 Activity or S 高 It is defined by the amount of nuclease of SEQ ID NO: 1 required to obtain a detectable linear signal when measuring activity.

[0207] In a preferred embodiment of the nuclease according to the invention, the salt is NaCl or KCl, preferably NaCl.

[0208] In a preferred embodiment of the nuclease according to the invention, S高 activity, S 低 When measuring the activity and relative salt activity, S 低 is in the range of 0.00M to 0.15M, preferably 0.00M; S 高 is at a concentration of 0.01M to 1.00M, preferably 0.15M to 0.85M, more preferably 0.20M to 0.70M, even more preferably 0.25M to 0.60M, even more preferably 0.30M to 0.60M, even more preferably 0.35M to 0.60M, most preferably 0.40M to 0.60M, and especially 0.50M.

[0209] In a preferred embodiment, the nuclease according to the present invention has a relative salt activity that is improved by at least 1.05-fold, preferably at least 7-fold, more preferably at least 9-fold, even more preferably at least 13-fold, even more preferably at least 60-fold, even more preferably at least 110-fold compared to SEQ ID NO: 1.

[0210] In a preferred embodiment, the nuclease according to the present invention has a relative salt activity that is improved by 1.05 to 300 times, preferably 7 to 250 times, more preferably 9 to 200 times, even more preferably 13 to 150 times, and even more preferably about 115 times, compared to SEQ ID NO: 1.

[0211] In preferred embodiments of the nuclease according to the invention, the relative salt activity is improved by at least 3%, preferably at least 25%, more preferably at least 50%, even more preferably at least 100%, even more preferably at least 130%, even more preferably at least 200%, and most preferably at least 800% compared to SEQ ID NO: 1.

[0212] In a preferred embodiment, the nuclease according to the invention is SEQ ID NO: 1 高activity is improved by at least 1.01-fold, preferably at least 1.5-fold, more preferably at least 2-fold, even more preferably at least 5-fold, even more preferably at least 12-fold, even more preferably at least 19-fold compared to 高 It has activity.

[0213] In a preferred embodiment, the nuclease according to the present invention has an improved SEQ ID NO: 1 of 1.01 to 200 fold, preferably 1.5 to 150 fold, more preferably 2 to 100 fold, even more preferably 5 to 75 fold, even more preferably 12 to 50 fold, even more preferably about 20 fold. 高 It has activity.

[0214] In a preferred embodiment of the nuclease according to the invention, the salt activity and relative salt activity are 低 and 500 mM S 高 It is measured in

[0215] In a preferred embodiment, the nuclease according to the invention comprises: (i) a temperature T30 ° C. higher than SEQ ID NO: 1 低 Enzyme activity in (T 低 activity); and / or (ii) has a higher relative low-temperature activity than SEQ ID NO: 1, and the relative low-temperature activity is at a temperature T 高 Enzyme activity at temperatures below 30°C (T 低 The ratio of enzyme activity in (T 低 Activity:T 高 activity).

[0216] In a preferred embodiment of the nuclease according to the invention, T 低 activity, T 高 activity, and relative low-temperature activity, (i) Transfer specific ratio amounts of such nuclease into two separate containers and mix with buffer to a final concentration of 50 mM Tris·HCl, pH 8.0, 5.0 mM MgCl2, 0.1 mg / ml BSA, and 0.8 mg / ml of DNA sodium salt from salmon testis, each at temperature T低 and T 高 After incubation at 4°C for 1.0 hour, 100 μl of 4% perchloric acid was added to terminate the reaction, and the two resulting compositions were incubated on ice for 45 minutes. Subsequently, the resulting compositions were centrifuged at 3,800 × g and 4°C for 30 minutes, and the absorbance at 260 nm of each resulting supernatant was measured (the detected absorbance was T 低 (T 低 activity) and T 高 (T 高 The activity of the nuclease is correlated with the enzymatic activity of the nuclease at temperature T 低 and T 高 separately detecting the enzymatic activity of the nuclease in (ii) T expressed as a percentage 低 (T 低 Samples incubated with T 高 (T 高 Relative cold activity (A260[T activity) was measured as the ratio of absorbance measured for a sample incubated at 200°C to that of a sample incubated at 200°C. 低 ]:A260[T 高 ]) and For any nuclease variant, the specific ratio of the amount of nuclease transferred to the two vessels in step (i) is T 低 Activity or T 高 It is defined by the amount of nuclease of SEQ ID NO: 1 required to obtain a detectable linear signal when measuring activity.

[0217] In a preferred embodiment of the nuclease according to the invention, T 低 is in the range of 4°C to 25°C, preferably 4.0°C to 15°C, more preferably 4.0°C to 10°C, even more preferably 4.0°C to 6.0°C, and even more preferably 5.0°C; T 高 is in the range of 30°C to 60°C, preferably 30°C to 50°C, more preferably 30°C to 40°C, and even more preferably 30°C.

[0218] In a preferred embodiment of the nuclease according to the invention, T 低 is 4.0℃ to 15℃, and T 高 is 25°C to 60°C; preferably T 低 is 4.0℃ to 10℃, and T 高 is 25°C to 50°C; more preferably T 低 is 4.0 to 6.0°C, and T 高 is 25°C to 40°C; even more preferably T 低 is 5.0°C, and T 高 It's 30 degrees Celsius.

[0219] In a preferred embodiment, the nuclease according to the present invention has a relative low temperature activity that is improved by at least 1.05-fold, preferably at least 1.5-fold, more preferably at least 2.0-fold, and even more preferably at least 3.0-fold compared to SEQ ID NO:1.

[0220] In a preferred embodiment, the nuclease according to the present invention has a relative low-temperature activity that is improved by 1.05 to 5 times, preferably 1.5 to 5 times, more preferably 2 to 5 times, and even more preferably about 3 times, compared to SEQ ID NO: 1.

[0221] In a preferred embodiment, the nuclease according to the invention has an improved T activity of at least 1.01-fold, preferably at least 1.1-fold, more preferably at least 1.3-fold, even more preferably at least 1.5-fold, even more preferably at least 1.55-fold compared to SEQ ID NO: 1. 低 It has activity.

[0222] In a preferred embodiment, the nuclease according to the present invention has a T activity that is improved by 1.01 to 2 fold, preferably 1.1 to 2 fold, more preferably 1.3 to 1.6 fold, even more preferably 1.5 to 1.6 fold, and even more preferably about 1.55 fold compared to SEQ ID NO: 1. 低 It has activity.

[0223] In a preferred embodiment of the nuclease according to the invention, the low temperature activity and relative low temperature activity are低 and T of 30°C 高 It is measured in

[0224] Combination of structural and functional features of the nuclease according to the present invention In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 amino acid positions selected from the group consisting of positions P51, K55, T56, N58, A74, T77, N80, K84, D86, A94, S116, D117, A124, D128, K132, D135, E151, K162, D191, R204, I218, A228, and E239 compared to SEQ ID NO: 1, and exhibits improved thermostability compared to SEQ ID NO: 1.

[0225] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 14, at least 16, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least and have substitutions at at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 positions, and exhibit improved thermal stability of at least 1.051-fold, at least 1.5-fold, or at least 2-fold; more preferably at least 3-fold, at least 4-fold, or at least 5-fold; even more preferably at least 6-fold, at least 6.5-fold, or at least 7-fold compared to the thermal stability of SEQ ID NO:1.

[0226] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 positions selected from the group consisting of positions P51, K55, T56, N58, T77, A74, N80, K84, D86, A94, S116, D117, A124, D128, K132, D135, E151, K162, D191, R204, I218, A228, and E239 compared to SEQ ID NO: 1, and exhibits improved thermostability compared to SEQ ID NO: 1, If the amino acid substitution comprises a substitution at amino acid position P51, the amino acid substitution at position P51 is selected from the group consisting of substitutions D, E, R, K, H, G, A, V, L, and I, preferably D, E, G, A, V, L, and I, more preferably G, L, and D; If the amino acid substitution comprises a substitution at amino acid position K55, the amino acid substitution at position K55 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position T56, the amino acid substitution at position T56 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, P, and D, more preferably G, A, V, L, I, P, and D, even more preferably P and D; If the amino acid substitution comprises a substitution at amino acid position N58, the amino acid substitution at position N58 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, and P, more preferably G, A, V, L, I, and P, even more preferably V; If the amino acid substitution comprises a substitution at amino acid position A74, the amino acid substitution at position A74 is selected from the group consisting of substitutions G, I, V, L, P, W, F, Y, C, M, N, Q, S, T, D, E, H, R, and K, preferably R, Q, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position T77, the amino acid substitution at position T77 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, R, F, and L; If the amino acid substitution comprises a substitution at amino acid position N80, the amino acid substitution at position N80 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, R, and H, more preferably H; If the amino acid substitution comprises a substitution at amino acid position K84, the amino acid substitution at position K84 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably G, A, V, L, I, and P, more preferably G; If the amino acid substitution comprises a substitution at amino acid position D86, the amino acid substitution at position D86 is selected from the group consisting of substitutions G, A, V, L, I, P, C, M, S, Q, and T, preferably Q, T, and S, more preferably S; If the amino acid substitution comprises a substitution at amino acid position A94, the amino acid substitution at position A94 is selected from the group consisting of substitutions G, I, V, L, P, W, F, Y, C, M, N, Q, S, T, D, E, H, R, and K, preferably G, I, V, L, and P, more preferably G; If the amino acid substitution comprises a substitution at amino acid position S116, the amino acid substitution at position S116 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, H, and R, more preferably K; If the amino acid substitution comprises a substitution at amino acid position D117, the amino acid substitution at position D117 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, C, M, S, Q, T, and N, preferably C, M, S, Q, T, and N, more preferably C, M, S, Q, and N, even more preferably N; If the amino acid substitution comprises a substitution at amino acid position A124, the amino acid substitution at position A124 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K; If the amino acid substitution comprises a substitution at amino acid position D128, the amino acid substitution at position D128 is selected from the group consisting of the substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, preferably G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, and K, more preferably W, F, Y, C, M, N, Q, S, T, even more preferably M, F, and Y; If the amino acid substitution comprises a substitution at amino acid position K132, the amino acid substitution at position K132 is selected from the group consisting of the substitutions R, H, G, A, V, L, I, P, W, F, Y, C, M, S, Q, T, and N, preferably R, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position D135, the amino acid substitution at position D135 is selected from the group consisting of the substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R and K, preferably G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H and K, more preferably G, A, V, L, I, P, C, M, N, Q, S, T, H and K, even more preferably G, A, V, L, I, P, C, M, Q, S, T and N, even more preferably G and N; If the amino acid substitution comprises a substitution at amino acid position E151, the amino acid substitution at position E151 is selected from the group consisting of substitutions D, R, K, H, G, A, V, L, I, P, W, F, Y, C, M, S, Q, T, and N, preferably G, A, V, L, I, and P, more preferably L; If the amino acid substitution comprises a substitution at amino acid position K162, the amino acid substitution at position K162 is selected from the group consisting of substitutions R, H, G, A, V, L, I, P, W, F, Y, C, M, S, Q, T, and N, preferably C, M, S, Q, T, and N, more preferably N; If the amino acid substitution comprises a substitution at amino acid position D191, the amino acid substitution at position D191 is selected from the group consisting of substitutions G, A, V, L, I, P, C, M, S, Q, and T, preferably G, A, V, L, I, and P, more preferably P; If the amino acid substitution comprises a substitution at amino acid position R204, the amino acid substitution at position R204 is selected from the group consisting of substitutions G, A, V, L, I, P, K, H, N, Q, S, T, C, and M, preferably Q, N, S, T, C, and M, more preferably Q; If the amino acid substitution comprises a substitution at amino acid position 1218, the amino acid substitution at position 1218 is selected from the group consisting of substitutions G, A, V, L, P, W, F, Y, C, M, N, Q, S, T, D, E, H, R, and K, preferably W, Y, and F, more preferably F; If the amino acid substitution comprises a substitution at amino acid position A228, the amino acid substitution at position A228 is selected from the group consisting of substitutions G, I, V, L, P, W, F, Y, C, M, N, Q, S, T, D, E, H, R, and K, preferably H, R, and K, more preferably R; or If the amino acid substitution comprises a substitution at amino acid position E239, the amino acid substitution at position E239 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, preferably A, G, V, L, I, and P, more preferably A.

[0227] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence is such that, compared to SEQ ID NO: 1, it has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 2 or at least 3 substitutions selected from the group consisting of P51L, P51G, P51D, K55R, T56P, T56D, N58V, A74R, T77K, T77F, T77L, T77R, N80H, K84G, D86S, A94G, S116K, D117N, A124K, D128M, D128Y, D128F, K132R, D135G, D135N, E151L, K162N, D191P, R204Q, I218F, A228R, and E239A. at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 substitutions at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23, and exhibit improved thermostability compared to SEQ ID NO: 1.

[0228] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, or at least 27 positions compared to SEQ ID NO: 1, and the substitutions are , positions P51, A52, S53, G54, K55, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, D86, S116, D117, N119, Q120, A124, D128, D135, P195, K196, G197, and D199, preferably P51, S53, K55, T56, N58, A72, P73, A74, T77, A81, A82, D86, S116, D117, A124, D128, D135, K196, G197, and D199, wherein the nuclease exhibits improved relative salt activity compared to the relative salt activity of SEQ ID NO: 1.

[0229] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, or at least 27 positions compared to SEQ ID NO: 1, wherein the substitutions are at positions P51, A52, S53, G54, K55, T56, N58, A72, P73, A74, T77, G 78, N80, A81, A82, D86, S116, D117, N119, Q120, A124, D128, D135, P195, K196, G197, and D199, preferably P51, S53, K55, T56, N58, A72, P73, A74, T77, A81, A82, D86, S116, D117, A124, D128, D135, K196, G197, and D199, and wherein the nuclease exhibits an improved relative salt activity of at least 1.05-fold, preferably at least 7-fold, more preferably at least 9-fold, even more preferably at least 13-fold, even more preferably at least 60-fold, and even more preferably at least 110-fold compared to the relative salt activity of SEQ ID NO: 1.

[0230] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence is from positions P51, A52, S53, G54, K55, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, D86, S116, D117, N119, Q120, A124, D128, D135, P195, K196, G197 and D199, preferably from P51, S53, K55, T56, N58, A72, P73, A74, T77, A81, A82, D86, S116, D117, A124, D128, D135, K196, G197 and D199, compared to SEQ ID NO: 1. at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, or at least 27 positions selected from the group consisting of: If the amino acid substitution comprises a substitution at amino acid position P51, the amino acid substitution at position P51 is selected from the group consisting of substitutions D, E, R, K, H, G, A, V, L, and I, preferably R, G, and L; If the amino acid substitution comprises a substitution at amino acid position A52, the amino acid substitution at position A52 is selected from the group consisting of substitutions G, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably K, H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position S53, the amino acid substitution at position S53 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, and P, more preferably K, and A; If the amino acid substitution comprises a substitution at amino acid position G54, the amino acid substitution at position G54 is selected from the group consisting of substitutions A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably K, H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position K55, the amino acid substitution at position K55 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position T56, the amino acid substitution at position T56 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, P, C, M, S, N, T, Q and D, more preferably K, R, S and G; If the amino acid substitution comprises a substitution at amino acid position N58, the amino acid substitution at position N58 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, and P, more preferably K, R, and H, even more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position A72, the amino acid substitution at position A72 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K; If the amino acid substitution comprises a substitution at amino acid position P73, the amino acid substitution at position P73 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and A, preferably K, H and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position A74, the amino acid substitution at position A74 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position T77, the amino acid substitution at position T77 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, R, H, W, F, and Y, more preferably K, R, and F; If the amino acid substitution comprises a substitution at amino acid position G78, the amino acid substitution at position G78 is selected from the group consisting of substitutions A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably K, H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position N80, the amino acid substitution at position N80 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, S, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position A81, the amino acid substitution at position A81 is selected from the group consisting of substitutions R, K, and H, preferably K; If the amino acid substitution comprises a substitution at amino acid position A82, the amino acid substitution at position A82 is selected from the group consisting of the substitutions R, K, and H, preferably R; If the amino acid substitution comprises a substitution at amino acid position D86, the amino acid substitution at position D86 is selected from the group consisting of substitutions N, Q, S, T, C, and M, preferably S; If the amino acid substitution comprises a substitution at amino acid position S116, the amino acid substitution at position S116 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, H, and R, more preferably K; If the amino acid substitution comprises a substitution at amino acid position D117, the amino acid substitution at position D117 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, and K, preferably A, G, V, L, I, P, C, M, N, T, Q, K, H, R, and S, more preferably A, K, R, N, and S; If the amino acid substitution comprises a substitution at amino acid position N119, the amino acid substitution at position N119 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, S, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position Q120, the amino acid substitution at position Q120 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, N, T, S, H, R, K, D and E, preferably R, K, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position A124, the amino acid substitution at position A124 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position D128, the amino acid substitution at position D128 is selected from the group consisting of the substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, preferably G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, more preferably G, Y, F, M, N, S, R, and K; If the amino acid substitution comprises a substitution at amino acid position D135, the amino acid substitution at position D135 is selected from the group consisting of the substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, preferably G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, and K, more preferably G, A, V, L, I, P, C, M, N, Q, S, T, H, and K, even more preferably G, A, N, and K; If the amino acid substitution comprises a substitution at amino acid position P195, the amino acid substitution at position P195 is selected from the group consisting of substitutions A, V, L, I, G, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably K, H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position K196, the amino acid substitution at position K196 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position G197, the amino acid substitution at position G197 is selected from the group consisting of substitutions D, E, R, K, H, A, V, L, I, and P, preferably K, H, and R, more preferably K; or If the amino acid substitution comprises a substitution at amino acid position D199, the amino acid substitution at position D199 is selected from the group consisting of the substitutions G, A, V, L, I, P, W, F, Y, C, M, Q, T, N, S, H, R, and K, preferably G, A, V, L, I, P, W, F, Y, C, M, Q, T, N, S, H, and K, more preferably S, Q, T, N, C, M, H, and K, even more preferably S, N, and K.

[0231] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence is such that, compared to SEQ ID NO: 1, it contains the substitutions P51R, P51G, P51L, A52R, S53K, S53A, G54R, K55R, T56G, T56S, T56R, T56K, N58R, N58K, A72K, P73K, P73R, A74R, A74K, T77F, T77K, T77R, G78R, N80R, A81K, A82R, D86S, S116K, D117A, D117S, D117K, D117R, D117N, N119R, Q120R, A124K, A124R, D128K, D 128R, D128Y, D128S, D128M, D128N, D128F, D128G, D135A, D135G, D135N, D135K, P195R, K196R, G197K, D199K, D199N, and D199S, preferably P51R, P51G, P51L, S53K, S53A, K55R, T56G, T56S, T56R, T56K, N58R, N58K, A72K, P73K, P73R, A74R, A74K, T77F, T77K, T77R, A81K, A82R, D86S, S116K, D117A, D117S, D117K, D1 at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or a few selected from the group consisting of 17R, D117N, A124K, A124R, D128K, D128R, D128Y, D128S, D128M, D128N, D128F, D128G, D135A, D135G, D135N, D135K, K196R, G197K, D199K, D199N, and D199S at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, or at least 27 substitutions.Substitutions at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, or at least 27 positions are present, and the relative salt activity is improved compared to the relative salt activity of SEQ ID NO: 1.

[0232] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has the following amino acids at positions P51, A52, S53, G54, K55, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, D86, S116, D117, N119, Q120, A124, D128, D135, P195, K196, G197, and D199, preferably P51, A52, S53, G54, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, D117, N119, Q120, A124, D128, D135, P195, K196, and G197, more preferably P51, S53, T56, N5 at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, or at least 27 positions selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, 高 Improved activity compared to S 高 Shows activity.

[0233] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has the following amino acids at positions P51, A52, S53, G54, K55, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, D86, S116, D117, N119, Q120, A124, D128, D135, P195, K196, G197, and D199, preferably P51, A52, S53, G54, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, D117, N119, Q120, A124, D128, D135, P195, K196, and G197, more preferably P51, S53, T56, N5 at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, or at least 27 positions selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, 高 activity is improved by at least 1.01-fold, preferably at least 1.5-fold, more preferably at least 2-fold, even more preferably at least 5-fold, even more preferably at least 12-fold, even more preferably at least 19-fold compared to 高 Shows activity.

[0234] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has the following amino acids at positions P51, A52, S53, G54, K55, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, D86, S116, D117, N119, Q120, A124, D128, D135, P195, K196, G197, and D199, preferably P51, A52, S53, G54, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, D117, N119, Q120, A124, D128, D135, P195, K196, and G197, more preferably P51, S53, T56, N5 at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, or at least 27 positions selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, 高 Improved activity compared to S 高 Shows activity, If the amino acid substitution comprises a substitution at amino acid position P51, the amino acid substitution at position P51 is selected from the group consisting of substitutions D, E, R, K, H, G, A, V, L, and I, preferably R, G, and L; If the amino acid substitution comprises a substitution at amino acid position A52, the amino acid substitution at position A52 is selected from the group consisting of substitutions G, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably K, H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position S53, the amino acid substitution at position S53 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, and P, more preferably K, R, and H, even more preferably K; If the amino acid substitution comprises a substitution at amino acid position G54, the amino acid substitution at position G54 is selected from the group consisting of substitutions A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably K, H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position K55, the amino acid substitution at position K55 is selected from the group consisting of substitutions D, E, R, H, G, V, L, I, P, C, M, S, N, T, Q, W, F, and Y, preferably R, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position T56, the amino acid substitution at position T56 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, C, M, S, N, T, Q and D, preferably R, K, H, G, A, V, L, I, P, C, M, S, N, T, Q and D, more preferably K, R, G and S; If the amino acid substitution comprises a substitution at amino acid position N58, the amino acid substitution at position N58 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, and P, more preferably K, R, and H, even more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position A72, the amino acid substitution at position A72 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K; If the amino acid substitution comprises a substitution at amino acid position P73, the amino acid substitution at position P73 is selected from the group consisting of substitutions G, V, L, I, A, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably R and K; If the amino acid substitution comprises a substitution at amino acid position A74, the amino acid substitution at position A74 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position T77, the amino acid substitution at position T77 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably W, F, Y, K, R, and H, more preferably K, R, and H, even more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position G78, the amino acid substitution at position G78 is selected from the group consisting of substitutions A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably K, H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position N80, the amino acid substitution at position N80 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, S, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position A81, the amino acid substitution at position A81 is selected from the group consisting of substitutions G, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably K; If the amino acid substitution comprises a substitution at amino acid position A82, the amino acid substitution at position A82 is selected from the group consisting of substitutions G, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position D86, the amino acid substitution at position D86 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, and E, preferably C, M, S, N, T, and Q, more preferably S; If the amino acid substitution comprises a substitution at amino acid position S116, the amino acid substitution at position S116 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, N, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably K; If the amino acid substitution comprises a substitution at amino acid position D117, the amino acid substitution at position D117 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, and K, preferably A, G, V, L, I, P, C, M, N, T, Q, R, K, H, and S, more preferably A, G, V, L, I, P, C, M, N, T, Q, and S, even more preferably A, N, and S; If the amino acid substitution comprises a substitution at amino acid position N119, the amino acid substitution at position N119 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, S, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position Q120, the amino acid substitution at position Q120 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, N, T, S, H, R, K, D and E, preferably R, K, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position A124, the amino acid substitution at position A124 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position D128, the amino acid substitution at position D128 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, preferably G, Y, F, M, N, S, R, and K; If the amino acid substitution comprises a substitution at amino acid position D135, the amino acid substitution at position D135 is selected from the group consisting of the substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, preferably G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, and K, more preferably G, A, V, L, I, P, C, M, N, Q, S, T, H, and K, even more preferably A, G, N, and K; If the amino acid substitution comprises a substitution at amino acid position P195, the amino acid substitution at position P195 is selected from the group consisting of substitutions A, V, L, I, G, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably K, H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position K196, the amino acid substitution at position K196 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position G197, the amino acid substitution at position G197 is selected from the group consisting of substitutions D, E, R, K, H, A, V, L, I, and P, preferably K, H, and R, more preferably K; or If the amino acid substitution comprises a substitution at amino acid position D199, the amino acid substitution at position D199 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, and E, preferably C, M, S, N, T, Q, K, H, and R, more preferably K, S, and N.

[0235] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence is such that, compared to SEQ ID NO: 1, it contains the substitutions P51L, P51R, P51G, A52R, S53A, S53K, K55R, G54R, T56G, T56S, T56R, T56K, N58R, N58K, A72K, P73K, P73R, A74K, A74R, T77R, T77K, T77F, A78R, N80R, A81K, A82R, D86S, S116K, D117A, D117S, D117N, D117R, N119R, Q120R, A124K, A124R, D128S, D128N, D 128K, D128R, D128G, D128M, D128Y, D128F, D135A, D135K, D135G, D135N, P195R, K196R, G197K, D199K, D199S, and D199N, preferably P51R, P51G, A52R, S53K, G54R, T56G, T56S, T56R, T56K, N58R, N58K, A72K, P73R, A74K, A74R, T77R, T77K, G78R, N80R, A81K, A82R, D117A, D117S, D117N, N119R, Q120R, A124K, A124R, D128S, D128N, D128K, D128R, D128G, D128M, D128Y, D128F, D135A, D135K, D135G, D135N, P195R, K196R, and G197K, more preferably P51R, P51G, S53K, T56G, T56S, T56R, T56K, N58R, N58K, A72K, A74K, A74R, T77R, T77K, A81K, A82R, D117A, D117S, D117N, A124K, A124R, D128S, D128N, D128K, D128R, D128G, D128M, D12 at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 selected from the group consisting of 8Y, D128F, D135A, D135K, D135G, D135N, K196R, and G197K;at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, or at least 27 positions, such that there are at least 26, or at least 27 substitutions; 高 Improved activity compared to S 高 Shows activity.

[0236] In a preferred embodiment of the nuclease according to the present invention, the amino acid sequence comprises substitutions at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 positions selected from the group consisting of positions P51, K55, T56, N58, P73, A74, T77, G78, K84, D86, S116, D117, D128, D135, G197, and D199 compared to SEQ ID NO: 1, and exhibits improved relative low-temperature activity compared to the relative low-temperature activity of SEQ ID NO: 1.

[0237] In a preferred embodiment of the nuclease according to the present invention, the amino acid sequence comprises substitutions at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 positions selected from the group consisting of positions P51, K55, T56, N58, P73, A74, T77, G78, K84, D86, S116, D117, D128, D135, G197, and D199 compared to SEQ ID NO: 1, and exhibits a relative low-temperature activity that is improved by at least 1.05-fold, preferably at least 1.5-fold, more preferably at least 2.0-fold, and even more preferably at least 3.0-fold compared to the relative low-temperature activity of SEQ ID NO: 1.

[0238] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises substitutions at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 positions selected from the group consisting of positions P51, K55, T56, N58, P73, A74, T77, G78, K84, D86, S116, D117, D128, D135, G197, and D199 compared to SEQ ID NO: 1, and exhibits an improved relative low-temperature activity compared to the relative low-temperature activity of SEQ ID NO: 1; If the amino acid substitution comprises a substitution at amino acid position P51, the amino acid substitution at position P51 is selected from the group consisting of substitutions D, E, R, K, H, G, A, V, L, and I, preferably D, R, G, and L; If the amino acid substitution comprises a substitution at amino acid position K55, the amino acid substitution at position K55 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position T56, the amino acid substitution at position T56 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, P, and D, more preferably K, R, P, and D; If the amino acid substitution comprises a substitution at amino acid position N58, the amino acid substitution at position N58 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, and P, more preferably K, R, and V; If the amino acid substitution comprises a substitution at amino acid position P73, the amino acid substitution at position P73 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and A, preferably K, H and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position A74, the amino acid substitution at position A74 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position T77, the amino acid substitution at position T77 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, R, F, and L; If the amino acid substitution comprises a substitution at amino acid position G78, the amino acid substitution at position G78 is selected from the group consisting of substitutions R, K, H, A, V, L, I, P, W, F, Y, N, Q, S, T, C, M and D, preferably K, R, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position K84, the amino acid substitution at position K84 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably G, A, V, L, I, and P, more preferably G; If the amino acid substitution comprises a substitution at amino acid position D86, the amino acid substitution at position D86 is selected from the group consisting of substitutions G, A, V, L, I, P, C, M, S, Q, and T, preferably Q, T, and S, more preferably S; If the amino acid substitution comprises a substitution at amino acid position S116, the amino acid substitution at position S116 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, H, and R, more preferably K; If the amino acid substitution comprises a substitution at amino acid position D117, the amino acid substitution at position D117 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R and K, preferably G, A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H and K, more preferably A, G, V, L, I, P, C, M, N, T, Q, K, H and S, even more preferably H and K, most preferably K; If the amino acid substitution comprises a substitution at amino acid position D128, the amino acid substitution at position D128 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R and K, preferably G, Y, F, M, N, S, R and K; If the amino acid substitution comprises a substitution at amino acid position D135, the amino acid substitution at position D135 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R and K, preferably G, A, V, L, I, P, C, M, N, Q, S, T, H, R and K, more preferably G, A, N, R and K, even more preferably G, A, N and K; If the amino acid substitution comprises a substitution at amino acid position G197, the amino acid substitution at position G197 is selected from the group consisting of substitutions D, E, R, K, H, A, V, L, I, and P, preferably K, H, and R, more preferably K; or If the amino acid substitution comprises a substitution at amino acid position D199, the amino acid substitution at position D199 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, Q, T, N, S, H, and K, preferably S, Q, T, N, C, M, H, and K, more preferably N, S, and K.

[0239] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence is such that, compared to SEQ ID NO: 1, it contains the substitutions P51L, P51D, P51G, P51R, K55R, T56D, T56R, T56P, T56K, N58V, N58K, N58R, P73R, P73K, A74K, A74R, T77K, T77L, T77R, T77F, G78R, K84G, D86S, S116K, D117K, D128Y, D128M, D128N, D128K, D128R, D128F, D128S, D128G, D135A, D135K, D135R, D135N, D135G, G197K, D199K, D199N, and D199S, preferably P51L, P51D, P51G, P51R, K55R, T56D, T56R, T56P, T56K, N58V, N58K, N58R, P73R, P73K, A74K, A74R, T77K, T77L, T77R, T77F, G78R, K84G, D86S, S116K, D117 at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 positions to include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 substitutions selected from the group consisting of K, D128Y, D128M, D128N, D128K, D128R, D128F, D128S, D128G, D135A, D135K, D135N, D135G, G197K, D199K, D199N, and D199S, and exhibit improved relative low-temperature activity compared to the relative low-temperature activity of SEQ ID NO: 1.

[0240] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 positions selected from the group consisting of positions P51, K55, T56, N58, P73, A74, T77, G78, K84, D86, S116, D117, D128, D135, G197, and D199, preferably P51, T56, N58, K84, D86, S116, and D135, compared to SEQ ID NO: 1, and 低 Improved T compared to activity 低 Shows activity.

[0241] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 positions selected from the group consisting of positions P51, K55, T56, N58, P73, A74, T77, G78, K84, D86, S116, D117, D128, D135, G197, and D199, preferably P51, T56, N58, K84, D86, S116, and D135, compared to SEQ ID NO: 1, and 低 activity is improved by at least 1.01-fold, preferably at least 1.1-fold, more preferably at least 1.3-fold, even more preferably at least 1.5-fold, and even more preferably at least 1.55-fold. 低 Shows activity.

[0242] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 positions selected from the group consisting of positions P51, K55, T56, N58, P73, A74, T77, G78, K84, D86, S116, D117, D128, D135, G197, and D199, preferably P51, T56, N58, K84, D86, S116, and D135, compared to SEQ ID NO: 1, and 低 Improved T compared to activity 低 Shows activity, If the amino acid substitution comprises a substitution at amino acid position P51, the amino acid substitution at position P51 is selected from the group consisting of substitutions D, E, R, K, H, G, A, V, L, and I, preferably G, A, V, I, and L, more preferably L; If the amino acid substitution comprises a substitution at amino acid position K55, the amino acid substitution at position K55 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position T56, the amino acid substitution at position T56 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, P, and D, more preferably G, A, V, L, I, P, and D, even more preferably G, P, and D; If the amino acid substitution comprises a substitution at amino acid position N58, the amino acid substitution at position N58 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, and P, more preferably G, A, L, I, P, and V, even more preferably V; If the amino acid substitution comprises a substitution at amino acid position P73, the amino acid substitution at position P73 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and A, preferably K, H and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position A74, the amino acid substitution at position A74 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position T77, the amino acid substitution at position T77 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, R, F, and L; if the amino acid substitution comprises a substitution at amino acid position G78, the amino acid substitution at position G78 is selected from the group consisting of substitutions R, K, H, A, V, L, I, P, W, F, Y, N, Q, S, T, C, M, and D, preferably K, R, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position K84, the amino acid substitution at position K84 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably G, A, V, L, I, and P, more preferably G; If the amino acid substitution comprises a substitution at amino acid position D86, the amino acid substitution at position D86 is selected from the group consisting of substitutions G, A, V, L, I, P, C, M, S, Q, and T, preferably Q, T, and S, more preferably S; If the amino acid substitution comprises a substitution at amino acid position S116, the amino acid substitution at position S116 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, H, and R, more preferably K; If the amino acid substitution comprises a substitution at amino acid position D117, the amino acid substitution at position D117 is selected from the group consisting of the substitutions G, A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, and K, preferably G, V, L, I, P, W, F, Y, C, M, S, N, Q, H, R, and K, more preferably K, R, and H, even more preferably K; If the amino acid substitution comprises a substitution at amino acid position D128, the amino acid substitution at position D128 is selected from the group consisting of the substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, preferably G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, more preferably G, Y, F, M, N, S, R, and K; If the amino acid substitution comprises a substitution at amino acid position D135, the amino acid substitution at position D135 is selected from the group consisting of the substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, preferably G, A, V, L, I, P, C, M, N, Q, S, T, H, R, and K, more preferably G, A, V, L, I, P, C, M, N, Q, S, T, H, and K, even more preferably G, A, V, L, I, and P, even more preferably A; If the amino acid substitution comprises a substitution at amino acid position G197, the amino acid substitution at position G197 is selected from the group consisting of substitutions D, E, R, K, H, A, V, L, I, and P, preferably K, H, and R, more preferably K; or If the amino acid substitution comprises a substitution at amino acid position D199, the amino acid substitution at position D199 is selected from the group consisting of the substitutions G, A, V, L, I, P, W, F, Y, C, M, Q, T, N, S, H, R, and K, preferably G, A, V, L, I, P, W, F, Y, C, M, Q, T, N, S, H, and K, more preferably S, Q, T, N, C, M, H, and K, even more preferably S, N, and K.

[0243] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence is such that, compared to SEQ ID NO: 1, it contains the substitutions P51L, P51D, P51G, P51R, K55R, T56G, T56D, T56P, T56R, T56K, N58V, N58K, N58R, P73K, P73R, A74K, A74R, T77K, T77L, T77R, T77F, G78R , K84G, D86S, S116K, D117K, D128Y, D128M, D128N, D128K, D128F, D128R, D128S, D128G, D135A, D135K, D135R, D135N, D135G, G197K, D199K, D199S, and D199N, preferably P51L, T56D, T56P, T56G, N58 at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 substitutions selected from the group consisting of V, K84G, D86S, S116K, and D135A; 低 Improved T activity compared to 低 Shows activity.

[0244] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises substitutions at at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 amino acid positions selected from the group consisting of positions P51, K55, T56, N58, A74, T77, N80, A81, K84, D86, A94, S116, D117, A124, D128, K132, D135, E151, K162, D191, G197, R204, 1218, A228, and E239 compared to SEQ ID NO: 1, and exhibits improved thermostability compared to SEQ ID NO: 1.

[0245] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 selected from the group consisting of positions P51, K55, T56, N58, A74, T77, N80, A81, K84, D86, A94, S116, D117, A124, D128, K132, D135, E151, K162, D191, G197, R204, I218, A228, and E239 compared to SEQ ID NO: 1. , at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 positions, and exhibiting improved thermal stability of at least 1.051-fold, at least 1.5-fold, or at least 2-fold, more preferably at least 3-fold, at least 4-fold, or at least 5-fold, and even more preferably at least 6-fold, at least 6.5-fold, or at least 7-fold compared to the thermal stability of SEQ ID NO:1.

[0246] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises substitutions at at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 positions selected from the group consisting of positions P51, K55, T56, N58, A74, T77, N80, A81, K84, D86, A94, S116, D117, A124, D128, K132, D135, E151, K162, D191, G197, R204, I218, A228 and E239 compared to SEQ ID NO: 1, and exhibits improved thermostability compared to SEQ ID NO: 1, If the amino acid substitution comprises a substitution at amino acid position P51, the amino acid substitution at position P51 is selected from the group consisting of substitutions D, E, R, K, H, G, A, V, L, and I, preferably D, E, G, A, V, L, and I, more preferably G, L, and D; If the amino acid substitution comprises a substitution at amino acid position K55, the amino acid substitution at position K55 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position T56, the amino acid substitution at position T56 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, N, Q, S, C, M, and D, preferably R, K, H, G, A, V, L, I, P, N, Q, S, C, M, and D, more preferably R, G, S, P, and D; If the amino acid substitution comprises a substitution at amino acid position N58, the amino acid substitution at position N58 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, and P, more preferably R and V; If the amino acid substitution comprises a substitution at amino acid position A74, the amino acid substitution at position A74 is selected from the group consisting of substitutions G, I, V, L, P, W, F, Y, C, M, N, Q, S, T, D, E, H, R, and K, preferably R, Q, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position T77, the amino acid substitution at position T77 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, R, F, and L; If the amino acid substitution comprises a substitution at amino acid position N80, the amino acid substitution at position N80 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, R, and H, more preferably H; If the amino acid substitution comprises a substitution at amino acid position A81, the amino acid substitution at position A81 is selected from the group consisting of substitutions G, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably K; If the amino acid substitution comprises a substitution at amino acid position K84, the amino acid substitution at position K84 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably G, A, V, L, I, and P, more preferably G; If the amino acid substitution comprises a substitution at amino acid position D86, the amino acid substitution at position D86 is selected from the group consisting of substitutions G, A, V, L, I, P, C, M, S, Q, and T, preferably Q, T, and S, more preferably S; If the amino acid substitution comprises a substitution at amino acid position A94, the amino acid substitution at position A94 is selected from the group consisting of substitutions G, I, V, L, P, W, F, Y, C, M, N, Q, S, T, D, E, H, R, and K, preferably G, I, V, L, and P, more preferably G; If the amino acid substitution comprises a substitution at amino acid position S116, the amino acid substitution at position S116 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, H, and R, more preferably K; If the amino acid substitution comprises a substitution at amino acid position D117, the amino acid substitution at position D117 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, C, M, S, Q, T, and N, preferably C, M, S, Q, T, and N, more preferably C, M, S, Q, and N, even more preferably N; If the amino acid substitution comprises a substitution at amino acid position A124, the amino acid substitution at position A124 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K; If the amino acid substitution comprises a substitution at amino acid position D128, the amino acid substitution at position D128 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, preferably W, F, Y, C, M, N, Q, S, T, and R, more preferably R, M, F, and Y; If the amino acid substitution comprises a substitution at amino acid position K132, the amino acid substitution at position K132 is selected from the group consisting of the substitutions R, H, G, A, V, L, I, P, W, F, Y, C, M, S, Q, T, and N, preferably R, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position D135, the amino acid substitution at position D135 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, preferably G, A, V, L, I, P, C, M, N, Q, S, T, H, R, and K, more preferably G, A, M, R, and N; If the amino acid substitution comprises a substitution at amino acid position E151, the amino acid substitution at position E151 is selected from the group consisting of substitutions D, R, K, H, G, A, V, L, I, P, W, F, Y, C, M, S, Q, T, and N, preferably G, A, V, L, I, and P, more preferably L; If the amino acid substitution comprises a substitution at amino acid position K162, the amino acid substitution at position K162 is selected from the group consisting of substitutions R, H, G, A, V, L, I, P, W, F, Y, C, M, S, Q, T, and N, preferably C, M, S, Q, T, and N, more preferably N; If the amino acid substitution comprises a substitution at amino acid position D191, the amino acid substitution at position D191 is selected from the group consisting of substitutions G, A, V, L, I, P, C, M, S, Q, and T, preferably G, A, V, L, I, and P, more preferably P; If the amino acid substitution comprises a substitution at amino acid position G197, the amino acid substitution at position G197 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, N, T, S, Q, C, M, Y, F, W and P, preferably R, K, and H, more preferably K; If the amino acid substitution comprises a substitution at amino acid position R204, the amino acid substitution at position R204 is selected from the group consisting of substitutions G, A, V, L, I, P, K, H, N, Q, S, T, C, and M, preferably Q, N, S, T, C, and M, more preferably Q; If the amino acid substitution comprises a substitution at amino acid position 1218, the amino acid substitution at position 1218 is selected from the group consisting of substitutions G, A, V, L, P, W, F, Y, C, M, N, Q, S, T, D, E, H, R, and K, preferably W, Y, and F, more preferably F; If the amino acid substitution comprises a substitution at amino acid position A228, the amino acid substitution at position A228 is selected from the group consisting of substitutions G, I, V, L, P, W, F, Y, C, M, N, Q, S, T, D, E, H, R, and K, preferably H, R, and K, more preferably R; or If the amino acid substitution comprises a substitution at amino acid position E239, the amino acid substitution at position E239 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, preferably A, G, V, L, I, and P, more preferably A.

[0247] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence is such that, compared to SEQ ID NO: 1, it contains the substitutions P51L, P51G, P51D, K55R, T56P, T56D, T56S, T56G, T56R, N58V, N58R, A74R, T77K, T77F, T77L, T77R, N80H, A81K, K84G, D86S, A94G, S116K, D117N, A124K, D128R, At least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 selected from the group consisting of D128M, D128Y, D128F, K132R, D135A, D135G, D135N, D135M, D135R, E151L, K162N, D191P, G197K, R204Q, I218F, A228R, and E239A at least 10, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 substitutions at positions 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, and exhibit improved thermostability compared to SEQ ID NO: 1.

[0248] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 positions compared to SEQ ID NO: 1, wherein the substitutions are at positions P51, A52, A53, A54, A55, A56, A57, A58, A59, A60, A61, A62, A63, A64, A65, A66, A67, A68, A69 ... 52, S53, G54, K55, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, K84, D86, S116, D117, N119, Q120, A124, D128, Q129, D135, P195, K196, G197, D199, and E239, preferably P51, S53, K55 , T56, N58, A72, P73, A74, T77, A81, A82, K84, D86, S116, D117, Q120, A124, D128, Q129, D135, K196, G197, D199, and E239, and the nuclease exhibits improved relative salt activity compared to the relative salt activity of SEQ ID NO:1.

[0249] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence comprises a substitution at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 positions compared to SEQ ID NO: 1, wherein the substitutions are at positions P51, A52, S53, G54, K55, T56, N58, A72, P73, A74, T77, G78, N80, A 81, A82, K84, D86, S116, D117, N119, Q120, A124, D128, Q129, D135, P195, K196, G197, D199, and E239, preferably P51, S53, K55, T56, N58, A72, P73, A74, T77, A81, A82, K84, D86, S116, D117, Q120, A124, D128, Q129, D 135, K196, G197, D199, and E239, and the nuclease exhibits a relative salt activity that is improved by at least 1.05-fold, preferably at least 7-fold, more preferably at least 9-fold, even more preferably at least 13-fold, even more preferably at least 60-fold, and even more preferably at least 110-fold compared to the relative salt activity of SEQ ID NO:1.

[0250] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has the following residues compared to SEQ ID NO: 1 at positions P51, A52, S53, G54, K55, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, K84, D86, S116, D117, N119, Q120, A124, D128, Q1 29, D135, P195, K196, G197, D199, and E239, preferably P51, S53, K55, T56, N58, A72, P73, A74, T77, A81, A82, K84, D86, S116, D117, Q120, A124, D128, Q129, D135, K196, G197, D199, and and E239, and at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 positions selected from the group consisting of E239, E240, E241, E242, E243, E244, E245, E246, E247, E248, E249, E250, E251, E252, E253, E254, E255, E256, E257, E258, E259, E260, E261, E262, E263, E264, E265, E266, E267, E268, E269, E270, E271, E272, E273, E274, E275, E276, E277, E278, E279, E280, E281, E282, E283, E284, E285, E286, E287, E288, E289, E290, E291, E292, E293, E294, E295, E296, E297, E298, E299, E300, E301, E302, E303, E304, E If the amino acid substitution comprises a substitution at amino acid position P51, the amino acid substitution at position P51 is selected from the group consisting of substitutions D, E, R, K, H, G, A, V, L, and I, preferably G, A, V, L, I, and R, more preferably R, G, and L; If the amino acid substitution comprises a substitution at amino acid position A52, the amino acid substitution at position A52 is selected from the group consisting of substitutions G, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position S53, the amino acid substitution at position S53 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, and P, more preferably K, and A; If the amino acid substitution comprises a substitution at amino acid position G54, the amino acid substitution at position G54 is selected from the group consisting of substitutions A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position K55, the amino acid substitution at position K55 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position T56, the amino acid substitution at position T56 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, N, S, Q, C, M, and D, preferably R, K, G, A, V, L, I, P, F, S, M, and D; If the amino acid substitution comprises a substitution at amino acid position N58, the amino acid substitution at position N58 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, and P, more preferably K, R, and H, even more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position A72, the amino acid substitution at position A72 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K; If the amino acid substitution comprises a substitution at amino acid position P73, the amino acid substitution at position P73 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and A, preferably K, H and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position A74, the amino acid substitution at position A74 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position T77, the amino acid substitution at position T77 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, R, H, W, F, and Y, more preferably H, K, R, and F; If the amino acid substitution comprises a substitution at amino acid position G78, the amino acid substitution at position G78 is selected from the group consisting of substitutions A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position N80, the amino acid substitution at position N80 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, C, M, T, Q, S, and D, preferably K, H, and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position A81, the amino acid substitution at position A81 is selected from the group consisting of substitutions G, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably K; If the amino acid substitution comprises a substitution at amino acid position A82, the amino acid substitution at position A82 is selected from the group consisting of substitutions G, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position K84, the amino acid substitution at position K84 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, D and E, preferably G, A, V, L, I, P, more preferably G; If the amino acid substitution comprises a substitution at amino acid position D86, the amino acid substitution at position D86 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, and E, preferably C, M, S, N, T, and Q, more preferably S; if the amino acid substitution comprises a substitution at amino acid position S116, the amino acid substitution at position S116 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, H, and R, more preferably K; If the amino acid substitution comprises a substitution at amino acid position D117, the amino acid substitution at position D117 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, and K, preferably N, Y, A, K, H, R, and S; If the amino acid substitution comprises a substitution at amino acid position N119, the amino acid substitution at position N119 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, C, M, T, Q, S, and D, preferably K, H, and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position Q120, the amino acid substitution at position Q120 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, H, and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position A124, the amino acid substitution at position A124 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position D128, the amino acid substitution at position D128 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, preferably G, Y, F, M, N, S, R, and K; If the amino acid substitution comprises a substitution at amino acid position Q129, the amino acid substitution at position Q129 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, H, and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position D135, the amino acid substitution at position D135 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, preferably G, A, V, L, I, P, C, M, N, Q, S, T, H, R, and K, more preferably G, A, N, R, and K; If the amino acid substitution comprises a substitution at amino acid position P195, the amino acid substitution at position P195 is selected from the group consisting of substitutions G, V, L, I, A, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position K196, the amino acid substitution at position K196 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position G197, the amino acid substitution at position G197 is selected from the group consisting of substitutions D, E, R, K, H, A, V, L, I, and P, preferably K, H, and R, more preferably K; If the amino acid substitution comprises a substitution at amino acid position D199, the amino acid substitution at position D199 is selected from the group consisting of the substitutions G, A, V, L, I, P, W, F, Y, C, M, Q, T, N, S, H, R, and K, preferably S, Q, T, N, C, M, H, R, and K, more preferably N, S, and K; or If the amino acid substitution comprises a substitution at amino acid position E239, the amino acid substitution at position E239 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, Q, T, N, S, H, R, K, and D, preferably G, A, V, L, I, and P, more preferably A.

[0251] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence is such that, compared to SEQ ID NO: 1, it contains the substitutions P51R, P51G, P51L, P51A, P51V, P51I, A52R, S53K, S53A, G54R, K55R, T56R, T56K, T56M, T56L, T56F, T56I, T56V, T56A, T56G, T56P, T56S, T56D, N58R, N58K, A72K, P73K, P73R, A74R, A74K, T77F, T77H, T77K, T77R, G78R, N80R, A81K, A82R, K84G, D86S, S 116K, D117A, D117S, D117K, D117N, D117Y, D117H, D117R, N119R, Q120R, A124K, A124R, A124H, D128R, D128K, D128Y, D128S, D128M, D128N, D128F, D128G, Q129R, D135A, D135R, D135G, D135N, D135K, P195R, K196R, G197K, D199N, D199K, D199S, and E239A, preferably P51R, P51G, P51L, S53K, S53A, K55R, T56R, T56 K, T56M, T56L, T56F, T56I, T56V, T56A, T56G, T56P, T56S, T56D, N58R, N58K, A72K, P73K, P73R, A74R, A74K, T77F, T77K, T77R, A81K, A82R, K84G, D86S, S1 16K, D117A, D117S, D117K, D117N, D117Y, D117H, D117R, Q120R, A124K, A124 R, D128R, D128K, D128Y, D128S, D128M, D128N, D128F, D128G, Q129R, D135A, D at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24 selected from the group consisting of 135R, D135G, D135N, D135K, K196R, G197K, D199N, D199K, D199S, and E239A;At least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 substitutions to contain at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 substitutions, and exhibit improved relative salt activity compared to the relative salt activity of SEQ ID NO: 1.

[0252] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has the following amino acids compared to SEQ ID NO: 1 at positions P51, A52, S53, G54, K55, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, K84, D86, S116, D117, N119, Q120, A124, D128, Q129, D135, P195, K196, G1 97, D199, and E239, preferably P51, A52, S53, G54, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, K84, D117, N119, Q120, A124, D128, Q129, D135, P195, K196, G197, and E239, more preferably P51, S53, T56, N58, A7 at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 positions selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, A74, T77, A81, A82, K84, D117, Q120, A124, D128, Q129, D135, K196, G197, and E239; 高 Improved activity compared to S 高 Shows activity.

[0253] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has the following amino acids compared to SEQ ID NO: 1 at positions P51, A52, S53, G54, K55, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, K84, D86, S116, D117, N119, Q120, A124, D128, Q129, D135, P195, K196, G1 97, D199, and E239, preferably P51, A52, S53, G54, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, K84, D117, N119, Q120, A124, D128, Q129, D135, P195, K196, G197, and E239, more preferably P51, S53, T56, N58, A7 at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 positions selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, A74, T77, A81, A82, K84, D117, Q120, A124, D128, Q129, D135, K196, G197, and E239; 高 activity is improved by at least 1.01-fold, preferably at least 1.5-fold, more preferably at least 2-fold, even more preferably at least 5-fold, even more preferably at least 12-fold, even more preferably at least 19-fold compared to 高 Shows activity.

[0254] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has the following amino acids compared to SEQ ID NO: 1 at positions P51, A52, S53, G54, K55, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, K84, D86, S116, D117, N119, Q120, A124, D128, Q129, D135, P195, K196, G1 97, D199, and E239, preferably P51, A52, S53, G54, T56, N58, A72, P73, A74, T77, G78, N80, A81, A82, K84, D117, N119, Q120, A124, D128, Q129, D135, P195, K196, G197, and E239, more preferably P51, S53, T56, N58, A7 at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 positions selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, A74, T77, A81, A82, K84, D117, Q120, A124, D128, Q129, D135, K196, G197, and E239; 高 Improved activity compared to S 高 Shows activity, If the amino acid substitution comprises a substitution at amino acid position P51, the amino acid substitution at position P51 is selected from the group consisting of substitutions D, E, R, K, H, G, A, V, L, and I, preferably G, A, V, L, I, and R, more preferably R, G, and L; If the amino acid substitution comprises a substitution at amino acid position A52, the amino acid substitution at position A52 is selected from the group consisting of substitutions G, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position S53, the amino acid substitution at position S53 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, and P, more preferably K, R, and H, even more preferably K; If the amino acid substitution comprises a substitution at amino acid position K55, the amino acid substitution at position K55 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position G54, the amino acid substitution at position G54 is selected from the group consisting of substitutions A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position T56, the amino acid substitution at position T56 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, N, S, Q, C, M, and D, preferably G, A, V, L, I, P, F, S, M, D, K, and R; If the amino acid substitution comprises a substitution at amino acid position N58, the amino acid substitution at position N58 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, and P, more preferably K, R, and H, even more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position A72, the amino acid substitution at position A72 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K; If the amino acid substitution comprises a substitution at amino acid position P73, the amino acid substitution at position P73 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and A, preferably K, H and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position A74, the amino acid substitution at position A74 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position T77, the amino acid substitution at position T77 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably W, F, Y, K, R, and H, more preferably K, H, and R, even more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position G78, the amino acid substitution at position G78 is selected from the group consisting of substitutions A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position N80, the amino acid substitution at position N80 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, C, M, T, Q, S, and D, preferably K, H, and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position A81, the amino acid substitution at position A81 is selected from the group consisting of substitutions G, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably K; If the amino acid substitution comprises a substitution at amino acid position A82, the amino acid substitution at position A82 is selected from the group consisting of substitutions G, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position K84, the amino acid substitution at position K84 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, D and E, preferably G, A, V, L, I, P, more preferably G; If the amino acid substitution comprises a substitution at amino acid position D86, the amino acid substitution at position D86 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, K, and E, preferably C, M, S, N, T, and Q, more preferably S; If the amino acid substitution comprises a substitution at amino acid position S116, the amino acid substitution at position S116 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, H, and R, more preferably K; If the amino acid substitution comprises a substitution at amino acid position D117, the amino acid substitution at position D117 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, and K, preferably Y, N, H, R, A, and S; If the amino acid substitution comprises a substitution at amino acid position N119, the amino acid substitution at position N119 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, C, M, T, Q, S, and D, preferably K, H, and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position Q120, the amino acid substitution at position Q120 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, H, and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position A124, the amino acid substitution at position A124 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position D128, the amino acid substitution at position D128 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, preferably G, Y, F, M, N, S, R, and K; If the amino acid substitution comprises a substitution at amino acid position Q129, the amino acid substitution at position Q129 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, H, and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position D135, the amino acid substitution at position D135 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R, and K, preferably G, A, V, L, I, P, C, M, N, Q, S, T, H, R, and K, more preferably A, G, N, R, and K; If the amino acid substitution comprises a substitution at amino acid position P195, the amino acid substitution at position P195 is selected from the group consisting of substitutions G, V, L, I, A, W, F, Y, C, M, S, N, T, Q, H, R, K, D and E, preferably R, K, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position K196, the amino acid substitution at position K196 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position G197, the amino acid substitution at position G197 is selected from the group consisting of substitutions D, E, R, K, H, A, V, L, I, and P, preferably K, H, and R, more preferably K; If the amino acid substitution comprises a substitution at amino acid position D199, the amino acid substitution at position D199 is selected from the group consisting of the substitutions G, A, V, L, I, P, W, F, Y, C, M, Q, T, N, S, H, R, and K, preferably S, Q, T, N, C, M, H, R, and K, more preferably N, S, and K; or If the amino acid substitution comprises a substitution at amino acid position E239, the amino acid substitution at position E239 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, Q, T, N, S, H, R, K, and D, preferably G, A, V, L, I, and P, more preferably A.

[0255] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence is such that, compared to SEQ ID NO: 1, it contains the substitutions P51L, P51R, P51G, P51A, P51V, P51I, A52R, S53A, S53K, G54R, K55R, T56R, T56K, T56M, T56L, T56F, T56I, T56V, T56A, T56G, T56P, T56S, T56D, N58R, N58K, A72K, P73K, P73R, A74K, A74R, T77R, T77K, T77H, T77F, G78R, N80R, A81K, A82R, K84G, D86S, S1 16K, D117A, D117S, D117N, D117Y, D117H, D117R, N119R, Q120R, A124K, A124R, A124H, D128R, D128S, D128N, D128K, D128G, D128M, D128Y, D128F, Q129R, D135A, D135K, D135G, D135N, D135R, P195R, K196R, G197K, D199K, D199S, D199N, and E239A, preferably P51L, P51R, P51G, P51A, P51V, P51I, A52R, S53K, G54R, T56R, T56K, T56M, T56L, T56F, T56I, T56V, T56A, T56G, T56P, T56S, T56D, N5 8R, N58K, A72K, P73R, A74K, A74R, T77R, T77K, T77H, G78R, N80R, A81K, A82R , K84G, D117A, D117S, D117N, D117Y, D117H, D117R, N119R, Q120R, A124K, A1 24R, A124H, D128R, D128S, D128N, D128K, D128G, D128M, D128Y, D128F, Q129R , D135A, D135K, D135G, D135N, D135R, P195R, K196R, G197K, and E239A, more preferably P51L, P51R, P51G, S53K, T56R, T56K, T56M, T56L, T56F, T56I, T56V, T56A, T56G, T56P, T56S, T56D, N58R, N58K, A72K, A74K, A74R, T77R, T77K, A81K, A82R, K84G, D117A, D117S, D117N, D117Y, D117H, D117R, Q120R, A124K, A124R,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 selected from the group consisting of D128R, D128S, D128N, D128K, D128G, D128M, D128Y, D128F, Q129R, D135A, D135K, D135G, D135N, D135R, K196R, G197K, and E239A; at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 substitutions in S of SEQ ID NO: 1, 高 Improved activity compared to S 高 Shows activity.

[0256] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has positions P51, K55, T56, N58, P73, A74, T77, G78, A81, K84, D86, S116, D117, Q120, A124, D128, Q129, D135, G197, D199 and E239 compared to SEQ ID NO: 1, preferably P51, K55, T56, N58, P73, A74, T77, G78, K84, D86, S116, D117, A124, D128, D135, G197 and and D199, and exhibits improved relative low-temperature activity compared to the relative low-temperature activity of SEQ ID NO: 1.

[0257] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has at least two, at least three, selected from the group consisting of positions P51, K55, T56, N58, P73, A74, T77, G78, A81, K84, D86, S116, D117, Q120, A124, D128, Q129, D135, G197, D199, and E239, preferably P51, K55, T56, N58, P73, A74, T77, G78, K84, D86, S116, D117, A124, D128, D135, G197, and D199, compared to SEQ ID NO: 1. , at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 positions, and exhibits a relative low-temperature activity that is improved by at least 1.05-fold, preferably at least 1.5-fold, more preferably at least 2.0-fold, and even more preferably at least 3.0-fold compared to the relative low-temperature activity of SEQ ID NO: 1.

[0258] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence has positions P51, K55, T56, N58, P73, A74, T77, G78, A81, K84, D86, S116, D117, Q120, A124, D128, Q129, D135, G197, D199 and E239 compared to SEQ ID NO: 1, preferably P51, K55, T56, N58, P73, A74, T77, G78, K84, D86, S116, D117, A124, D128, D135, G197 and and D199, wherein the amino acid sequence is substituted at at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 positions selected from the group consisting of D199, D209, D300, D310, D311, D312, D313, D314, D315, D316, D317, D318, D319, D320, D321, D322, D323, D324, D325, D326, D327, D328, D329, D330, D331, D332, D333, D334, D335, D336, D337, D338, D339, D340, D341, D342, D343, D344, D345, D346, D347, D348, D349, D350, D351, D352, D353, D354, D355, D356, D357, D358, D359 ... If the amino acid substitution comprises a substitution at amino acid position P51, the amino acid substitution at position P51 is selected from the group consisting of substitutions D, E, R, K, H, G, A, V, L, and I, preferably D, R, G, and L; If the amino acid substitution comprises a substitution at amino acid position K55, the amino acid substitution at position K55 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position T56, the amino acid substitution at position T56 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, N, Q, S, C, M, and D, preferably R, K, H, G, A, V, L, I, P, and D, more preferably K, R, P, and D; If the amino acid substitution comprises a substitution at amino acid position N58, the amino acid substitution at position N58 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, and P, more preferably K, R, and V; If the amino acid substitution comprises a substitution at amino acid position P73, the amino acid substitution at position P73 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and A, preferably K, H and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position A74, the amino acid substitution at position A74 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position T77, the amino acid substitution at position T77 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, R, F, and L; If the amino acid substitution comprises a substitution at amino acid position G78, the amino acid substitution at position G78 is selected from the group consisting of substitutions R, K, H, A, V, L, I, P, W, F, Y, N, Q, S, T, C, M and D, preferably K, R, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position A81, the amino acid substitution at position A81 is selected from the group consisting of substitutions R, K, and H, preferably K; If the amino acid substitution comprises a substitution at amino acid position K84, the amino acid substitution at position K84 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably G, A, V, L, I, and P, more preferably G; If the amino acid substitution comprises a substitution at amino acid position D86, the amino acid substitution at position D86 is selected from the group consisting of substitutions G, A, V, L, I, P, C, M, S, Q, and T, preferably Q, T, and S, more preferably S; If the amino acid substitution comprises a substitution at amino acid position S116, the amino acid substitution at position S116 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, H, and R, more preferably K; If the amino acid substitution comprises a substitution at amino acid position D117, the amino acid substitution at position D117 is selected from the group consisting of the substitutions G, A, V, L, I, P, W, F, Y, C, M, S, N, T, Q, H, R, and K, preferably A, G, V, L, I, P, R, K, H, W, F, and Y, more preferably Y, H, R, and K, even more preferably K, H, and R, even more preferably K; If the amino acid substitution comprises a substitution at amino acid position Q120, the amino acid substitution at position Q120 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, H, and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position A124, the amino acid substitution at position A124 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K; If the amino acid substitution comprises a substitution at amino acid position D128, the amino acid substitution at position D128 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R and K, preferably G, Y, F, M, N, S, R and K; If the amino acid substitution comprises a substitution at amino acid position Q129, the amino acid substitution at position Q129 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably K, H, and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position D135, the amino acid substitution at position D135 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, N, Q, S, T, H, R and K, preferably G, A, V, L, I, P, C, M, N, Q, S, T, H, R and K, more preferably M, G, A, N, R and K; If the amino acid substitution comprises a substitution at amino acid position G197, the amino acid substitution at position G197 is selected from the group consisting of substitutions D, E, R, K, H, A, V, L, I, and P, preferably K, H, and R, more preferably K; or If the amino acid substitution comprises a substitution at amino acid position D199, the amino acid substitution at position D199 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, Q, T, N, S, H, R, and K, preferably S, Q, T, N, C, M, H, R, and K, more preferably N, S, and K; If the amino acid substitution comprises a substitution at amino acid position E239, the amino acid substitution at position E239 is selected from the group consisting of substitutions G, A, V, L, I, P, W, F, Y, C, M, Q, T, N, S, H, R, K, and D, preferably G, A, V, L, I, and P, more preferably A.

[0259] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence is such that, compared to SEQ ID NO: 1, it contains the substitutions P51L, P51D, P51G, P51R, K55R, T56D, T56R, T56P, T56K, T56S, T56G, T56A, T56M, T56F, T56V, T56L, T56I, N58V, N58K, N58R, P73R, P73K, A74K, A74R, T77K, T77L, T77R, T77F, G78R, A81K, K84G, D86S, S116K, D117K, D117H, D117Y, Q120R, A124K, D125R, Q126R, Q127R, Q128R, Q129R, Q130R, Q131R, Q132R, Q133R, Q134R, Q135R, Q136R, Q137R, Q138R, Q139R, Q140R, Q141R, Q142R, Q143R, Q144R, Q145R, Q146R, Q147R, Q148R, Q149R, Q150R, Q151R, Q152R, Q153R, Q154R, Q155R, Q156R, Q157R, Q158R, Q159R, Q160R, Q161R, Q162R, Q163R, Q164R, Q165R, Q166R, Q167R, Q168R, Q169R, Q170R, Q 28Y, D128M, D128N, D128K, D128F, D128S, D128G, D128R, Q129R, D135A, D135K, D135R, D135N, D135G, D135M, G197K, D199N, D199K, D199S, E239A, preferably P51L, P51D, P51G, P51R, K55R, T56D, T56R, T56P, T56K, N58V, N58K, N58R, P73R, P73K, A74K, A74R, T77K, T77L, T77R, T77F, G78R, K84G, D86S, S116K, D at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 substitutions to contain 7, at least 18, at least 19, at least 20, or at least 21 substitutions, and exhibit improved relative low-temperature activity compared to the relative low-temperature activity of SEQ ID NO: 1.

[0260] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence is at positions P51, K55, T56, N58, P73, A74, T77, G78, A81, K84, D86, S116, D117, Q120, A124, D128, Q129, D135, K196, G197, D199 and E239, preferably P51, T56, N58, T77, A81, K84, D86, S116, D117, Q120, A124, D128, Q129, D135, K196 and E239, more preferably is substituted at at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 positions selected from the group consisting of P51, T56, N58, T77, K84, D86, S116, and D135; 低 Improved T compared to activity 低 Shows activity.

[0261] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence is at positions P51, K55, T56, N58, P73, A74, T77, G78, A81, K84, D86, S116, D117, Q120, A124, D128, Q129, D135, K196, G197, D199 and E239, preferably P51, T56, N58, T77, A81, K84, D86, S116, D117, Q120, A124, D128, Q129, D135, K196 and E239, more preferably is substituted at at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 positions selected from the group consisting of P51, T56, N58, T77, K84, D86, S116, and D135; 低 activity is improved by at least 1.01-fold, preferably at least 1.1-fold, more preferably at least 1.3-fold, even more preferably at least 1.5-fold, and even more preferably at least 1.55-fold. 低 Shows activity.

[0262] In a preferred embodiment of the nuclease according to the invention, the amino acid sequence is at positions P51, K55, T56, N58, P73, A74, T77, G78, A81, K84, D86, S116, D117, Q120, A124, D128, Q129, D135, K196, G197, D199 and E239, preferably P51, T56, N58, T77, A81, K84, D86, S116, D117, Q120, A124, D128, Q129, D135, K196 and E239, more preferably is substituted at at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 positions selected from the group consisting of P51, T56, N58, T77, K84, D86, S116, and D135; 低 Improved T compared to activity 低 Shows activity, If the amino acid substitution comprises a substitution at amino acid position P51, the amino acid substitution at position P51 is selected from the group consisting of substitutions D, E, R, K, H, G, A, V, L, and I, preferably G, A, V, I, and L, more preferably G and L, even more preferably L; If the amino acid substitution comprises a substitution at amino acid position K55, the amino acid substitution at position K55 is selected from the group consisting of substitutions G, A, V, L, I, P, R, H, N, Q, S, T, C, and M, preferably H and R, more preferably R; If the amino acid substitution comprises a substitution at amino acid position T56, the amino acid substitution at position T56 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, N, S, Q, C, M, and D, preferably R, K, H, G, A, V, L, I, P, and D, more preferably G, A, V, L, I, P, and D, even more preferably G, P, and D; If the amino acid substitution comprises a substitution at amino acid position N58, the amino acid substitution at position N58 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably R, K, H, G, A, V, L, I, and P, more preferably R, K, and V, even more preferably R, and V; If the amino acid substitution comprises a substitution at amino acid position P73, the amino acid substitution at position P73 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and A, preferably K, H and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position A74, the amino acid substitution at position A74 is selected from the group consisting of substitutions D, E, R, K, H, G, V, L, I, and P, preferably K, H, and R, more preferably K and R; If the amino acid substitution comprises a substitution at amino acid position T77, the amino acid substitution at position T77 is selected from the group consisting of substitutions R, K, H, G, A, V, L, I, P, W, F, Y, and D, preferably L, F, K, R, and H, more preferably K, R, and H, even more preferably R; If the amino acid substitution comprises a substitution at amino acid position G78, the amino acid substitution at position G78 is selected from the group consisting of substitutions R, K, H, A, V, L, I, P, W, F, Y, N, Q, S, T, C, M and D, preferably K, R, and H, more preferably R; If the amino acid substitution comprises a substitution at amino acid position A81, the amino acid substitution at position A81 is selected from the group consisting of substitutions R, K, and H, preferably K; If the amino acid substitution comprises a substitution at amino acid position K84, the amino acid substitution at position K84 is selected from the gro...

Claims

1. comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 1; (i) a substitution at position P51, where P is replaced by an amino acid selected from the group consisting of L, G, A, V, and I; (ii) a substitution at position T77, where T is replaced by an amino acid selected from the group consisting of R, H, and K; (iii) (a) A124, A52, G54, P73, A74, G78, A81, and P195; or (b) T56, S53, N58, N80, N119, and Q120 and wherein in each case of (a) or (b), an amino acid of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of R, H, and K. A nuclease, preferably an endonuclease, comprising:

2. said nuclease, preferably said endonuclease, (i) a substitution at position P51, where P is replaced by an amino acid selected from the group consisting of L, G, A, V, and I, preferably L; (ii) a substitution at position T77, where T is replaced by an amino acid selected from the group consisting of R, H, and K, preferably R; (iii) a substitution at position A124, in which A is replaced by an amino acid selected from the group consisting of R, H, and K, preferably K; 2. A nuclease, preferably an endonuclease, according to claim 1, comprising:

3. said nuclease, preferably said endonuclease, (i) a substitution at position P51, where P is replaced by an amino acid selected from the group consisting of L, G, A, V, and I, preferably L; (ii) a substitution at position T77, where T is replaced by an amino acid selected from the group consisting of R, H, and K, preferably R; (iii) a substitution at position A81, in which A is replaced by an amino acid selected from the group consisting of R, H, and K, preferably K; 2. A nuclease, preferably an endonuclease, according to claim 1, comprising:

4. The amino acid sequence is selected from the group consisting of P51, T77, A52, S53, G54, K55, T56, N58, A72, P73, A74, G78, N80, A81, A82, K84, D86, A94, S116, D117, N119, Q120, A124, D128, Q129, K132, D135, E151, K162, D191, P195, K196, G197, D199, R204, I218, A228, and E239, compared with SEQ ID NO: 1, preferably P51 , T77, S53, K55, T56, N58, A72, P73, A74, G78, N80, A81, A82, K84, D86, A94, S116, D117, Q120, A124, D128, Q129, K132, D135, E151, K162, D191, K196, G197, D199, R204, I218, A228, and E239. The nuclease, preferably endonuclease, of claim 1, wherein the nuclease, preferably endonuclease, is substituted at at least four positions selected from the group consisting of:

5. The amino acid sequence is selected from the group consisting of A52, S53, G54, K55, T56, N58, A72, P73, A74, G78, N80, A81, A82, K84, D86, A94, S116, D117, N119, Q120, A124, D128, Q129, K132, D135, E151, K162, D191, P195, K196, G197, D199, R204, I218, A228, and E239, compared with SEQ ID NO: 1, preferably S53, K5 5, T56, N58, A72, P73, A74, G78, N80, A81, A82, K84, D86, A94, S116, D117, Q120, A124, D128, Q129, K132, D135, E151, K162, D191, K196, G197, D199, R204, I218, A228, and E239.

6. 2. The nuclease, preferably endonuclease, of claim 1, wherein the amino acid sequence has at least 76%, even more preferably at least 78%, even more preferably at least 81%, most preferably at least 84%, and especially at least 87% identity with SEQ ID NO:

1.

7. 2. The nuclease, preferably endonuclease, of claim 1, wherein the amino acid sequence has at least 88%, preferably at least 89%, more preferably at least 90%, even more preferably at least 91%, even more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and especially at least 95% identity with SEQ ID NO:

1.

8. The amino acid sequence is selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, and 72. ,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,1 37, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, or 195, having at least 90%, preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, most preferably at least 97%, most preferably at least 98%, and especially at least 99% identity.

9. The amino acid sequence is SEQ ID NO: 2, 3, 4, 6, 7, 8, 11, 12, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 107, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 145, 146, 147, 148 8, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, or 195 and at least 90 %, preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, most preferably at least 97%, most preferably at least 98%, and especially at least 99%, or 100% identity with the nuclease, preferably endonuclease, of claim 1.

10. The amino acid sequences are selected from the group consisting of SEQ ID NOs: 55, 57, 58, 59, 61, 64, 66, 69, 86, 128, 131, 132, 133, 134, 137, 138, 139, 140, 143, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186 187, 188, 189, 190, 191, 192, 193, 194, or 195, having at least 90%, preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, most preferably at least 97%, most preferably at least 98%, and especially at least 99%, or 100% identity.

11. (A) an improved relative salt activity compared to the relative salt activity of the wild-type nuclease of SEQ ID NO: 1; and / or (B) the S of the wild-type nuclease of SEQ ID NO: 1 高 Improved activity compared to S 高 A nuclease, preferably an endonuclease, according to claim 1, characterized by any of the following activities:

12. SEQ ID NO: 1 of the wild-type nuclease 高 Improved activity compared to S 高 activity, and 高 The activity is (i) at least 1.01-fold, preferably at least 1.5-fold, more preferably at least 2-fold, even more preferably at least 5-fold, even more preferably at least 12-fold, even more preferably at least 19-fold improvement; (ii) The nuclease, preferably endonuclease, of claim 1, which is improved by 1.01 to 200 times, preferably 1.5 to 150 times, more preferably 2 to 100 times, even more preferably 5 to 75 times, even more preferably 12 to 50 times, even more preferably about 20 times compared to SEQ ID NO:

1.

13. Compared to SEQ ID NO: 1, (i) at least a 1.05-fold to 300-fold improvement, preferably a 7-fold to 250-fold improvement, more preferably a 9-fold to 200-fold improvement, even more preferably a 13-fold to 150-fold improvement, and even more preferably about a 115-fold improvement; (ii) A nuclease, preferably an endonuclease, according to claim 1, having a relative salt activity that is improved by at least 3%, preferably at least 25%, more preferably at least 50%, even more preferably at least 100%, even more preferably at least 130%, even more preferably at least 200%, and most preferably at least 800%.

14. 2. The nuclease, preferably the endonuclease, of claim 1, wherein the nuclease, preferably the endonuclease, is capable of hydrolyzing the phosphodiester of a polynucleotide substrate and hydrolyzing the polynucleotide substrate into cleaved polynucleotides or oligonucleotides.

15. 1. A method for hydrolyzing a polynucleotide substrate, preferably a polynucleotide substrate contained in a sample, comprising: (a) providing a nuclease according to claim 1; (b) providing a composition comprising a polynucleotide substrate; (c) contacting the polynucleotide substrate in the composition provided in step (b) with the nuclease provided in step (a) and allowing the nuclease to hydrolyze the polynucleotide substrate, thereby obtaining a composition comprising cleaved polynucleotides. The method comprising:

16. step (c) is carried out in the presence of a salt, said salt being NaCl or KCl, preferably NaCl; and / or 16. The method of claim 15, wherein step (c) is carried out at a salt concentration of 0.00M to 1.00M, or 0.01M to 1.00M, preferably 0.15M to 0.85M, more preferably 0.20M to 0.70M, even more preferably 0.25M to 0.60M, even more preferably 0.30M to 0.60M, even more preferably 0.35M to 0.60M, most preferably 0.40M to 0.60M, and especially 0.50M.

17. (d) including the additional step of inactivating said nuclease after step (c), thereby obtaining a composition comprising cleaved polynucleotides and an inactivated nuclease; and / or (e) separating said nuclease, preferably said endonuclease, from said composition comprising the cleaved polynucleotides obtained in step (c) and optionally the inactivated nuclease obtained in step (d).

16. The method of claim 15, comprising:

18. 10. Use of the nuclease of claim 1 for hydrolyzing a polynucleotide substrate, preferably a polynucleotide substrate contained in a composition.

19. 19. The use according to claim 18, carried out at a salt concentration of 0.00M to 1.00M, or 0.01M to 1.00M, preferably 0.15M to 0.85M, more preferably 0.20M to 0.70M, even more preferably 0.25M to 0.60M, even more preferably 0.30M to 0.60M, even more preferably 0.35M to 0.60M, most preferably 0.40M to 0.60M, and especially 0.50M.

20. 1. A kit for cleaving a polynucleotide, comprising: a. the nuclease of claim 1; b. a buffer for hydrolyzing the polynucleotide substrate; c. Optionally, agents and / or devices for inactivation and / or removal of said nucleases. and preferably for use in the method according to claim 15 or the use according to claim 18.

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