Peptide fragment condensation and cyclization with subtilisin variants having improved synthetic hydrolysis ratio

Abstract

The present invention relates to a method for the enzymatic synthesis of (oligo)peptides comprising coupling (a) a (oligo)peptide C-terminal ester or thioester and (b) a (oligo)peptide nucleophile having an N-terminal unprotected amine, wherein the coupling is performed in a fluid comprising water, and wherein the coupling is catalyzed by a subtilisin BPN' variant or a homologue thereof comprising the following mutations compared to subtilisin BPN' as set forth in SEQUENCE ID NO: 2: deletion of the amino acids corresponding to positions 75-83; a mutation at the amino acid position corresponding to S221 which is S221C or S221 selenocysteine; preferably a mutation at the amino acid position corresponding to P225, wherein the amino acid positions are defined according to the sequence of subtilisin BPN' as set forth in SEQUENCE ID NO: 2. In addition, the present invention relates to enzymes suitable for use as catalysts in the method of the present invention.

Description

[0001] This application is a divisional application of patent application No. 2015800677876, filed on October 9, 2015, entitled "Condensation and Cyclation of Peptide Fragments of Subtilis Protease Variants with Improved Synthetic Hydrolysis Ratio".

[0002] This invention relates to a method for the enzymatic synthesis of (oligo)peptides (i.e., a peptide, especially an oligopeptide), to an enzyme suitable for catalyzing the synthesis, to a host cell capable of functionally expressing the enzyme, and to a method for preparing the enzyme.

[0003] Peptides, especially oligopeptides, have many applications, such as as pharmaceutical, food or feed ingredients, or cosmetic ingredients.

[0004] Methods for synthesizing (oligo)peptides are generally known in the art.

[0005] Oligopeptides can be chemically synthesized in the solid phase via highly optimized processes or in solution in a stepwise manner. However, due to side reactions and the resulting cumbersome purification, it is generally very difficult to synthesize peptides longer than 10–15 amino acids. Therefore, peptides longer than 10 amino acids are typically synthesized by combining solid-phase synthesis of side-chain-protected oligopeptide fragments (which are then chemically condensed in solution, e.g., by 10+10 condensation to prepare 20-amino acid oligopeptides). The main drawback of chemically side-chain-protected oligopeptide fragment condensation is that racemization occurs after activation of the C-terminal C-terminal amino acid side chain of the acyl donor. In contrast, enzyme-catalyzed peptide coupling completely avoids racemization and has several other advantages over chemical peptide synthesis, such as the absence of side reactions on the side chain functional groups. For industrial applications, the concept of enzymatic peptide synthesis based on dynamic methods, i.e., using acyl donor C-terminal esters, is most attractive (see, for example, N. Sewald and H.-D. Jakubke, in “Peptides: Chemistry and Biology”, first reprint, edited by Wiley-VCH Verlag GmbH, Weinheim 2002).

[0006] Chemical-enzymatic peptide synthesis may require the enzymatic coupling of oligopeptide fragments, which have been synthesized individually using chemical synthesis, fermentation, or a combination of chemical and enzymatic coupling steps. Several reports have disclosed the enzymatic condensation of oligopeptide fragments in aqueous solution (Kumaran et al., Protein Science, 2000, 9, 734; Björup et al., Bioorg. Med. Chem. 1998, 6, 891; Homandberg et al., Biochemistry, 1981, 21, 3387; Komoriya et al., Int. J. Pep. Prot. Res. 1980, 16, 433). However, a major drawback of this aqueous-solid-state enzymatic oligopeptide fragment condensation is the simultaneous hydrolysis of peptide bonds and C-terminal ester functional groups within the oligopeptide fragment, leading to low yields and numerous byproducts.

[0007] Proteases have to date been primarily used commercially for hydrolytic applications, such as cleaning, where peptide bonds are hydrolyzed by the protease. A typical example is subtilisin, whose formation is a crucial enzyme type for its application as a detergent. Therefore, subtilisin has been the subject of numerous protein engineering studies. Subtilisin has been used for oligopeptide synthesis; however, it is almost always accompanied by a significant degree of hydrolytic side reaction. Wells et al. (US 5,403,737) found that the condensation of oligopeptides in aqueous solution can be significantly affected by altering the active site of the BPN' of subtilisin, derived from Bacillus amyloliquefaciens (…). B. amyloliquefaciensThe subtilisin (SEQUENCE ID NO:2) was used. When two mutations, S221C and P225A, were introduced, a subtilisin BPN' variant, a subtiligase, was obtained with a 500-fold increase in the hydrolysis ratio (S / H ratio) compared to the wild-type subtilisin BPN'. However, the average ligation yield was only about 66%, and the hydrolysis of the oligopeptidyl donor C-terminal C-terminal ester remained significant (Wells et al., Science, 1994, 266, 243). Most commonly, 10 equivalents of the oligopeptidyl donor C-terminal C-terminal ester are used to obtain good reaction yields. Another disadvantage of subtilisin is its poor stability to organic co-solvents required to dissolve oligopeptide fragments, elevated temperatures, and denaturing agents, all of which are generally necessary for successful oligopeptide condensation. Therefore, Wells et al. added five additional mutations to the subtilisin: M50F, N76D, N109S, K213R, and N218S, to make the enzyme more stable (Proc. Natl. Acad. Sci. USA, 1994, 91, 12544). The new mutant, called a stabilizing ligase, appears to have moderately stronger resistance to sodium dodecyl sulfate and guanidine hydrochloride, but hydrolysis remains a major side reaction. For example, the yield of ligating oligopeptide carboxymethyl ester (Cam-ester) to oligopeptide amine using the stabilizing ligase was 44%. In this example, 10 equivalents of the oligopeptide C-terminal C-terminal ester were used, and therefore 9.56 equivalents of the oligopeptide C-terminal C-terminal ester were hydrolyzed at the C-terminal C-terminal ester functional group, and only 0.44 equivalents were ligated to oligopeptide amine to form the product. Clearly, improved enzymes with high S / H ratios are needed to make oligopeptide condensation reactions an economically viable process. Perhaps for this reason, Bacillus subtilis ligase, or stable ligase, has been industrially applied to the limits of the inventors' knowledge over the past 20 years.

[0008] Another aspect of the subtilisin BPN' that has garnered attention is the enzyme's stability at higher temperatures and / or in the presence of metal chelators when used as a detergent (i.e., for peptide bond hydrolysis). A prime example of this research was disclosed by Bryan et al., whose engineered version lacked high affinity Ca2+. 2+ The binding site of the subtilisin BPN' variant (WO02 / 22796). High affinity Ca in the subtilisin BPN'. 2+ The binding site consists of a ring containing amino acids 74-82, as well as amino acids Gln2 (Q2) and Asp41 (D41). Comparison of the 3D structure of subtilisin BPN' with that of its homologous subtilisin reveals high affinity Ca. 2+The binding site is highly conserved. In known subtilisin proteases, this binding site is important for their stability. For example, back-extraction of Ca by metal chelators... 2+ Ions lead to unfolding and consequent inactivation of known subtilisin proteases. When Bryan et al. deleted amino acids 75-83 of the subtilisin BPN'... 75-83) and additionally, mutations were performed on Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, Y217L, N218S, T254A, and Q271E, resulting in the BPN' variant of subtilisin (called BS149, also known as Sbt149), which lacks Ca2+. 2+ It binds to structural domains and exhibits significantly improved stability (1000-fold) against metal chelators. However, this enzyme cannot be used for peptide fragment condensation in aqueous solution because it only possesses hydrolytic activity.

[0009] The object of this invention is to provide an enzymatic method for preparing oligopeptides by condensing first and second oligopeptide fragments or by cyclizing oligopeptides, which can be used as an alternative to known methods for preparing oligopeptides. Alternative methods are generally needed, especially to broaden the range of tools available for preparing specific oligopeptides.

[0010] Specifically, one objective is to provide an enzymatic method for preparing (oligopeptides) by condensing first and second (oligopeptide) fragments or by cyclizing (oligopeptides), wherein an enzyme having an improved S / H ratio and stability compared to BPN' of subtilisin is used at least under certain reaction conditions.

[0011] Another objective is to provide an enzymatic method for preparing (oligopeptides) by condensing first and second (oligopeptide) fragments or by cyclizing (oligopeptides), wherein an enzyme with improved stability and S / H ratio compared to Bacillus subtilis ligase is used, at least under certain reaction conditions.

[0012] Another object of the present invention is to provide a coupling method for (oligo)peptides to proteins. Providing an enzymatic method that allows for the coupling of peptides to proteins is particularly challenging, especially due to the additional complexity of the three-dimensional structure of proteins.

[0013] Another objective is to provide a novel variant of subtilisin BPN' capable of catalyzing the condensation or cyclization of two (oligo)peptides, especially an enzyme with improved properties, such as an improved synthetic hydrolysis ratio and / or improved stability compared to known enzymes suitable for catalyzing such condensation, such as subtilisin BPN' and / or subtilisin ligase, under certain reaction conditions.

[0014] One or more other objectives that may be the purpose of this invention are described below.

[0015] It has now been surprisingly discovered that it is possible to provide a subtilisin BPN' variant in which the calcium-binding domain corresponding to amino acid positions 75-83 has been inactivated by deletion, which is catalytically active for the condensation or cyclization of two (oligo)peptide fragments, and specifically provides a variant with an improved S / H ratio compared to subtilisin BPN' and / or subtilisin ligase, by providing a subtilisin BPN' variant having specific mutations (preferably specific combinations of mutations) other than the deletion of amino acids corresponding to 75-83.

[0016] Therefore, the present invention relates to a method for the enzymatic synthesis of (oligo)peptides, comprising coupling (a) a (oligo)peptide C-terminal C-terminal ester or thioester and (b) a (oligo)peptide nucleophilic group having an N-terminal deprotected amine. The coupling takes place in a fluid containing water, and The coupling is catalyzed by a variant of subtilisin BPN' or its homolog, which contains the following mutations compared to the subtilisin BPN' or its homologous sequence shown in SEQUENCE ID NO:2: i) Delete the amino acid corresponding to positions 75-83; ii) A mutation at the amino acid position corresponding to S221, which is S221C or S221 selenocysteine ​​(S221U). iii) Preferably, a mutation at the amino acid position corresponding to P225; The amino acid positions are defined according to the sequence of BPN', the subtilisin shown in SEQUENCE ID NO: 2.

[0017] Furthermore, the present invention relates to a method for the enzymatic synthesis of cyclic (oligo)peptides of at least 12 amino acids, comprising a cyclization step of a C-terminal C-terminal ester or thioester of an (oligo)peptide having an N-terminal deprotected amine, wherein the cyclization is carried out in a fluid containing water, and The cyclization is catalyzed by a subtilisin BPN' variant or its homolog, which contains the following mutations compared to the subtilisin BPN' or its homologous sequence shown in SEQUENCE ID NO:2: i) Delete the amino acid corresponding to positions 75-83; ii) A mutation at the amino acid position corresponding to S221, which is either S221C or S221 selenocysteine; iii) Preferably, a mutation at the amino acid position corresponding to P225; The amino acid positions are defined according to the sequence of BPN', the subtilisin shown in SEQUENCE ID NO: 2.

[0018] In addition, the present invention relates to an enzyme, which is a variant of subtilisin BPN' or a homolog thereof, containing the following mutations compared to the subtilisin BPN' or its homologous sequence shown in SEQUENCEID NO: 2: i) Delete the amino acid corresponding to positions 75-83; ii) A mutation at the amino acid position corresponding to S221, which is either S221C or S221 selenocysteine; iii) A mutation at the amino acid position corresponding to P225; The amino acid positions are defined according to the sequence of BPN', the subtilisin shown in SEQUENCE ID NO: 2.

[0019] Furthermore, this invention relates to a recombinant method for preparing the enzyme of this invention, the method comprising: a) Provide a recombinant host cell that functionally expresses a gene encoding the enzyme; b) The host cells are cultured under conditions that provide for the expression of functionally active enzymes; and c) Recover the expressed enzymes from the microbial host.

[0020] In addition, the present invention relates to recombinant polynucleotides comprising sequences encoding the enzymes of the present invention.

[0021] Furthermore, this invention relates to host cells containing the polynucleotides of the present invention. These host cells are capable of functionally expressing the enzymes of the present invention.

[0022] Furthermore, this invention relates to the use of the enzymes of the present invention as catalysts. Such uses generally include contacting one or more substrates (reactants) in the presence of an enzyme under conditions where the enzyme catalyzes a chemical reaction involving the substrate. The enzyme has been found to be particularly useful as a catalyst in peptide synthesis. Specifically, the enzymes of the present invention are contemplated for use in catalyzing reactions in which the known subtilisin protease has catalytic activity. In one embodiment, the synthesized peptide is a protein. In one embodiment, the synthesized peptide is an oligopeptide. In another embodiment, the synthesized peptide comprises at least 201 amino acid units.

[0023] This invention provides a useful alternative to known methods for preparing (oligo)peptides, including (oligo)peptide-extended proteins.

[0024] Furthermore, it has been surprisingly discovered that the method of the present invention can enzymatically condense two (oligo)peptide fragments or cyclize (oligo)peptides in a liquid containing water at a high synthetic-to-hydrolysis ratio. The advantage of the method of the present invention lies in its ability to couple various oligopeptide fragments in aqueous solution in high yield without significant hydrolytic side reactions. This remarkable discovery is illustrated in the examples, which show that the method of the present invention is not only suitable for synthesizing (oligo)peptides lacking secondary and tertiary protein structures, but also capable of coupling two peptide fragments, at least one of which is a protein, thereby synthesizing (elongated) proteins with additional sequences of amino acid units. It has also been found that it is possible to synthesize such proteins while maintaining the secondary and tertiary structures of the protein.

[0025] For the purposes of this invention, the “synthesis-hydrolysis ratio” (S / H ratio) represents the amount of enzymatically synthesized (oligo)peptide product divided by the amount of (oligo)peptide C-terminal C-terminal ester or thioester that has been hydrolyzed by the ester or thioester group.

[0026] The S / H ratio of the enzymes of the present invention depends on a variety of factors, such as substrate properties (amino acid sequence of the nucleophilic group of the (oligopeptide) and the amino acid sequence of the C-terminal C-terminal ester or thioester of the (oligopeptide)) and reaction conditions (e.g., temperature, pH, peptide fragment concentration, enzyme concentration). As shown in the examples, it has been found that the S / H ratio is higher than that of known subtilis proteases, such as subtilis ligase and subtilisin BPN', under various reaction conditions and with a variety of substrates. Therefore, it is expected that the enzymes of the present invention will generally have a significantly higher S / H ratio than subtilis ligase and subtilisin BPN' when tested under the same reaction conditions and using the same substrates, and specifically, it is expected that the enzymes of the present invention will have a significantly higher S / H ratio under the conditions used in Example 1 or one or more other examples (100 mM phosphate buffer, pH 8.0, temperature about 20°C, concentration of C-terminal C-terminal ester of (oligopeptide) 0.83 mM, concentration of nucleophilic group of (oligopeptide) 3.33 mM, enzyme concentration 5.5 mg / L). Therefore, specifically, the present invention relates to a variant of the subtilisin BPN' or a homolog thereof, wherein, at least under the conditions described in Example 1 or one or more other examples, the S / H ratio of the subtilisin BPN' variant or a homolog thereof divided by the S / H ratio of the subtilisin ligase is greater than 1, preferably 2 or greater, especially 5 or greater. The upper limit of this quotient is not critical; in practice, it can be, for example, 100 or less, especially 20 or less.

[0027] At least under the conditions described in Example 1 or one or more other examples, the S / H ratio of the subtilisin BPN' variant or its homolog divided by the S / H ratio of the subtilisin BPN' is generally greater than 100, preferably 250 or greater, more preferably 500 or greater, and especially 1000 or greater. The upper limit of this quotient is not critical; under the reaction conditions at least shown herein, the S / H ratio of the subtilisin BPN' is generally very low, and can even be 0 (no detectable synthesis). Therefore, the S / H ratio of the subtilisin BPN' variant or its homolog of the present invention divided by the S / H ratio of the subtilisin BPN' may be approximately infinite. In the potential case where the subtilisin BPN' has significant ligase or cyclase activity, the inventors believe that the S / H ratio of the subtilisin BPN' variant or its homolog of the present invention divided by the S / H ratio of the subtilisin BPN' is also very high, for example, up to 100,000, especially up to 25,000, and even more especially up to 10,000.

[0028] In addition, the method of the present invention makes it very easy to purify (oligo)peptide products from the reaction mixture because very few hydrolysis byproducts are formed.

[0029] Another advantage of the present invention is that, due to the improved S / H ratio, a small or no excess of the (oligo)peptide C-terminal C-terminal ester or thioester or (oligo)peptide nucleophilic group is required to achieve a high yield (over 80%) in the condensation reaction. Therefore, in a preferred embodiment, the (oligo)peptide C-terminal C-terminal ester or thioester and the (oligo)peptide nucleophilic group are contacted with the (oligo)peptide fragment in a small excess or approximately stoichiometric ratio, although a larger excess of one over the other may also be used, as described below.

[0030] As illustrated in the examples, the enzyme of the present invention also has the advantage of being able to synthesize cyclic (oligo)peptides in significantly higher yields than Bacillus subtilis ligase (78% compared to 61% for Bacillus subtilis ligase). Cyclic (oligo)peptides are a class of peptides of particular interest because they are generally more potent due to their more restricted three-dimensional structure and greater resistance to proteolysis. Attached Figure Description

[0031] Figure 1A and 1BThe S / H ratios of the enzyme activities of the different enzymes of the present invention compared to Bacillus subtilis ligase are shown respectively; for BS149-DM, all the mutations shown at M222, Y104, I107 and / or L135 are additional. The name "BS149-DM" is used herein for the Bacillus subtilis protease BPN' variant, which has the following mutations compared to Bacillus subtilis protease BPN' (SEQUENCE ID NO 2): deletion of amino acids 75-83 (Δ75-83), S221C, P225A, Y217L, Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, N218S, T254A and Q271E.

[0032] Figure 2A and 2B The activity S / H ratios of different BS149-DM + M222P + L217 mutants are shown separately; for BS149-DM + M222P, all the mutations shown on L217 are additional.

[0033] Figure 3A P4 bag specificity of BS149-DM and BS149-DM + Y104 mutants.

[0034] Figure 3B P4 bag specificity of BS149-DM and BS149-DM + I107 mutants.

[0035] Figure 3C P4 bag specificity of BS149-DM and BS149-DM+L135 mutants.

[0036] Figure 4A P1' bag specificity of BS149-DM and BS149-DM+M222A, M222E and M222Q mutants.

[0037] Figure 4B P2' bag specificity of BS149-DM and BS149-DM+M222A, M222E and M222Q mutants.

[0038] Figure 4C P1' bag specificity of BS149-DM and BS149-DM+M222G, M222N and M222P mutants.

[0039] Figure 4D P2' bag specificity of BS149-DM and BS149-DM+M222G, M222N and M222P mutants.

[0040] Figure 5A P1' bag specificity of BS149-DM + M222P + L217N, L217T and L217E mutants.

[0041] Figure 5B P2' bag specificity of BS149-DM + M222P + L217N, L217T and L217E mutants.

[0042] Figure 5C P1' bag specificity of BS149-DM + M222P + L217I, L217V and L217A mutants.

[0043] Figure 5D P2' bag specificity of BS149-DM + M222P + L217I, L217V and L217A mutants.

[0044] Figure 5E P1' bag specificity of BS149-DM + M222P + L217M, L217K and L217Q mutants.

[0045] Figure 5F P2' bag specificity of BS149-DM + M222P + L217M, L217K and L217Q mutants.

[0046] Figure 5G P1' bag specificity of BS149-DM + M222P + L217S, L217G and L217Y mutants.

[0047] Figure 5H P2' bag specificity of BS149-DM + M222P + L217S, L217G and L217Y mutants.

[0048] Figure 5I P1' bag specificity of BS149-DM + M222P + L217F, L217H and L217W mutants.

[0049] Figure 5J P2' bag specificity of BS149-DM + M222P + L217F, L217H and L217W mutants.

[0050] Figure 5K P1' bag specificity of BS149-DM + M222P + L217R, L217C, L217D and L217P mutants.

[0051] Figure 5L P2' bag specificity of BS149-DM + M222P + L217R, L217C, L217D and L217P mutants.

[0052] Figure 6A P1' bag substrate specificity of BS149-DM + M222G + L217N, L217T and L217E mutants.

[0053] Figure 6B P1' bag substrate specificity of BS149-DM + M222G + L217I, L217V and L217A mutants.

[0054] Figure 6C P1' bag substrate specificity of BS149-DM + M222G + L217M, L217K and L217Q mutants.

[0055] Figure 6D P1' bag substrate specificity of BS149-DM + M222G + L217S, L217G and L217Y mutants.

[0056] Figure 6E P1' bag substrate specificity of BS149-DM + M222G + L217F, L217H and L217R mutants.

[0057] Figure 6F P1' bag substrate specificity of BS149-DM + M222G + L217C, L217D and L217P mutants.

[0058] Figure 7A P1' bag specificity of BS149-DM and BS149-DM+M222G and BS149-DM+I107V+M222G mutants.

[0059] Figure 7B P2' bag specificity of BS149-DM and BS149-DM+M222G and BS149-DM+I107V+M222G mutants.

[0060] Figure 7C P4 bag specificity of BS149-DM, BS149-DM + I107V and BS149-DM + I107V + M222G mutants.

[0061] Figure 8 S / H ratio of BS149-DM + M222G mutant at different pH values.

[0062] Figure 9A S / H ratio of BS149-DM+M222G mutants using different concentrations of acyl donors and H-Glu-Leu-Arg-NH2 nucleophiles.

[0063] Figure 9B S / H ratio of BS149-DM+M222G mutants using different concentrations of acyl donors and H-Ala-Leu-Arg-NH2 nucleophiles.

[0064] Figure 10 The S / H ratio of the different enzymes of the present invention used for (oligo)peptide cyclization compared to Bacillus subtilis ligase.

[0065] Figure 11 S / H ratio of BS149-DM + M222G mutant used for (oligo)peptide cyclization at different pH values.

[0066] Figure 12 Bacillus subtilis with the BS149-DM gene ( B. subtilis ) / E. coli ( E. coli ) Shuttle vector pBE-S (pBES DNA-BS149-DM HIStag).

[0067] Figure 13 PBS42-S5, a Bacillus subtilis / Escherichia coli shuttle vector containing the Bacillus subtilis ligase gene.

[0068] Figure 14 List of subtilisin proteases that can be used as templates for providing homologs of the subtilisin BPN' variant of the present invention and comparison of sequence fragments containing Ca2+ binding loops with the corresponding loops (SEQ ID NO 2) in subtilisin BPN' and the loops deleted in BS149-DM (SEQ ID NO 5).

[0069] The polynucleotides of this invention can be in single-stranded or double-stranded form, and unless otherwise limited, include known analogs having the basic properties of natural nucleotides that hybridize with single-stranded nucleic acids in a manner similar to naturally occurring nucleotides. Polynucleotides can be full-length or subsequences of natural or hybridized structures or regulatory sequences. Unless otherwise stated, this data includes specific sequences and their complementary sequences. Thus, a main strand of DNA or RNA modified for stability or other reasons is a "polynucleotide," as the term refers herein. The term polynucleotide as used herein includes such chemically, enzymatically, or metabolically modified forms of polynucleotides, as well as chemical forms characteristic of the DNA and RNA of viruses and cells, including single or complex cells.

[0070] The recombinant polynucleotides of this invention are generally synthetic. This invention is specifically extended to DNA or RNA isolated from any organism. In a particular embodiment, this invention is extended to host cells containing the recombinant DNA of this invention. The host cells are generally transgenic.

[0071] As used herein, the term "recombinant" refers to a polynucleotide or a cell containing that polynucleotide, resulting from one or more genetic modifications using recombinant DNA technology and / or other mutagenesis techniques. Specifically, a recombinant cell may contain a polynucleotide not present in the corresponding wild-type cell, which has been introduced into the cell (transgenic cell) using recombinant DNA technology, or the absence of that polynucleotide in the wild-type cell may be the result of one or more mutations—for example, using recombinant DNA technology or other mutagenesis techniques such as UV radiation—that modify the polynucleotide sequence present in the wild-type cell (such as a gene encoding a wild-type polypeptide, like an enzyme) or the polynucleotide sequence of a gene therein to target the polypeptide product (encoding it) to another cell compartment. Furthermore, the term "recombinant (cell)" specifically refers to a line (cell) from which a DNA sequence has been extracted using recombinant DNA technology.

[0072] Specifically, mutations can be introduced into polynucleotide sequences to exchange one polynucleotide for another through site-directed mutagenesis using any method known in the art. Alternatively, mutated genes can be obtained through gene synthesis, which, in addition to introducing changes at the amino acid level, can be used to optimize coding sequences to improve transcription and translation (R. Carlson, Nature Biotechnology, 2009, 27, 1091; E. Angov et al., PLoS ONE 2008, 3(5):e2189).

[0073] The term "transgenic cell" as used in this article refers to a line (cell) containing a polynucleotide that is not naturally present in the line (cell) and that has been introduced into the line (cell) using recombinant DNA technology, i.e., a recombinant cell.

[0074] The term “or” as used herein is defined as “and / or”, unless otherwise stated or followed by meaning “...or...either of the two”.

[0075] The term “a” or “an” as used herein is defined as “at least one”, unless otherwise stated or if what follows should refer only to the singular.

[0076] When referring to a noun in the singular form (e.g., compound, additive, etc.), the plural form is often included, unless the content following it should refer only to the singular.

[0077] For the purposes of this invention, "peptide" refers to any chain containing two or more amino acids. Thus, a peptide is generally an amide that at least conceptually contains two or more aminocarboxylic acid molecules (i.e., amino acids) through the covalent bonding (formal loss of water) of the carbonyl carbon of one amino group to the nitrogen atom of another. The term is generally applied to structures formed from α-amino acids. Peptides can be linear, branched, or cyclic. A peptide may have a single chain containing two or more amino acids, or it may have multiple chains. In the case where a peptide contains two or more chains, each chain generally contains two or more amino acid molecules. The amino acid sequence of a peptide is referred to as its primary structure.

[0078] In one implementation, the peptide has virtually no secondary structure and virtually no tertiary structure.

[0079] In another embodiment, the peptide has a secondary structure. The secondary structure is generally a highly regular local substructure, such as an α-helix and a β-sheet (or β-chain), formed by interactions between individual amino acids and the peptide backbone.

[0080] In one embodiment, the peptide (or peptides) has a tertiary structure. Tertiary structures are generally formed through multiple interactions, including hydrogen bonds, hydrophobic interactions, van der Waals forces, ionic interactions, and disulfide bonds. Secondary structures can also constitute tertiary structures. Tertiary structures provide a three-dimensional shape (which is essentially fixed in a stable environment, such as in the absence of temperature changes and changes in the medium in which the peptide resides). Those skilled in the art will recognize that a tertiary structure differs from a random helical peptide chain lacking any fixed three-dimensional structure. Proteins are (oligo)peptides having a tertiary structure. A well-known example of a tertiary structure is the globular structure of a globular protein. In one embodiment, the protein is a protein intended for targeted delivery of a pharmaceutically active (oligo)peptide to a specific site, such as a tumor or organ tissue. Known examples of proteins suitable for this purpose are immunoglobulins or portions thereof, such as antigen-binding fragments (Fab) of immunoglobulins. Thus, immunoglobulins conjugated to pharmaceutically active (oligo)peptides can be used to more efficiently deliver the pharmaceutically active (oligo)peptide to a target containing an antigen against the immunoglobulin, such as tumor tissue or organ tissue. In one embodiment, the protein is a protein adapted to increase the half-life of the (oligo)peptide in a living organism, particularly the plasma half-life. Albumin is an example of a protein that can be coupled to the (oligo)peptide to increase its half-life.

[0081] A disulfide bond (disulfide bridge) is generally a bond between two cysteine ​​units (formed by oxidation). Therefore, two amino acids in the same peptide chain (amino acid sequence) can also be covalently bonded if they are not adjacent amino acids in the amino acid sequence. Similarly, disulfide bonds can form between the first cysteine ​​of the first peptide chain and the second cysteine ​​of the second peptide chain to form a peptide, and the first and second peptide chains can have the same or different amino acid sequences. Such a peptide may contain more than one peptide chain. An example of a peptide containing more than one peptide chain, where different chains are linked by disulfide bonds, is insulin. Other bonds linking different peptide chains are known in the art.

[0082] In one embodiment, the (oligo)peptide consists essentially of amino acid units. In another embodiment, the (oligo)peptide consists essentially of amino acid units and protecting groups. In one embodiment, a peptide is a conjugate of two or more amino acid chains and another molecule, particularly a sugar or lipid. These peptides are referred to as glycoproteins and lipoproteins, respectively. In another embodiment, a peptide conjugate is a conjugate of two or more amino acids and an imaging agent such as a fluorescent, phosphorescent, chromogenic, or radioactive group. Peptide conjugates may also contain chelating agents or toxins.

[0083] Generally, peptides—the term includes oligopeptides, proteins, and peptide conjugates—contain up to 35,000 amino acid units, particularly 3-20,000 amino acid units, more especially 4-5,000 amino acid units, and preferably 5-1,000 amino acid units. In a particularly preferred embodiment, the peptide contains 500 amino acid units or less, particularly 200 or less, and more especially 100 or less. In a particularly preferred embodiment, the peptide contains at least 10 amino acid units, more specifically at least 15 amino acids, at least 25 amino acids, or at least 40 amino acids.

[0084] In this invention, "oligopeptide" refers to a peptide containing 2-200 amino acid units, particularly 5-100 amino acid units, and even more particularly 10-50 amino acid units.

[0085] The term “(oligo)peptide” as used in this article is an abbreviation of the phrase “peptide, especially oligopeptide”.

[0086] Synthesized (oligo)peptides can be linear, branched, or cyclic. The synthesis of linear or cyclic oligopeptides has yielded good results. Further good results have been achieved in the synthesis of peptides with more than 200 amino acid units, for example, about 800 amino acid units. Therefore, peptides can have at least 250 or at least 400 amino acid units. Additionally, good results have been achieved by coupling peptide fragments to proteins, such as insulin, while maintaining both secondary and tertiary protein structures. Proteins can have 200 or fewer amino acid units, or more than 201 amino acid units.

[0087] Acyclic oligopeptides are synthesized from a first and a second oligopeptide smaller than the synthesized oligopeptide. The first oligopeptide is a C-terminal C-terminal ester or thioester of the oligopeptide, and the second oligopeptide contains an N-terminal deprotected amine. The C-terminal C-terminal ester or thioester of the oligopeptide is also referred to as the oligopeptide acyl donor. The second oligopeptide is also referred to as the oligopeptide nucleophile. These oligopeptides from which the synthetic oligopeptide is formed are referred to as "oligopeptide fragments". These oligopeptide fragments can themselves be enzymatically synthesized from smaller oligopeptide acyl donors and oligopeptide nucleophiles, or synthesized using conventional chemical solutions or solid-phase peptides known to those skilled in the art.

[0088] For the purposes of this invention, "peptide bond" refers to an amide bond between (i) the α-amino terminus of one α-amino acid or the β-amino terminus of one β-amino acid and (ii) the α-carboxyl terminus of another α-amino acid or the β-carboxyl terminus of another β-amino acid. Preferably, the peptide bond is between the α-amino terminus of one amino acid and the α-carboxyl terminus of another amino acid.

[0089] For the purposes of this invention, "cyclic peptide" refers to an oligopeptide chain in which the α-amino and α-carboxyl terms of a branched or branched oligopeptide are linked by peptide bonds to form a ring structure of at least 12 amino acid units. Specifically, a cyclic peptide comprises 12-200 amino acid units, more particularly 12-100 amino acid units, and preferably 12-50 amino acid units.

[0090] For the purposes of this invention, "condensation" means the formation of a new peptide bond between the C-terminal C-terminal carboxyl functional group of an oligopeptide and the N-terminal amine functional group of the same oligopeptide or another oligopeptide.

[0091] In the context of this application, the term “about” specifically means a deviation from a given value of 10% or less, more specifically 5% or less, or even more specifically 3% or less.

[0092] As defined by Schechter and Berger, the active site residues of proteases, including subtilisin, contain a series of subpositions called pockets. Each pocket binds to a corresponding residue in the peptide substrate sequence, referred to here as a sequence position. According to this definition, the amino acid residues in the substrate sequence are numbered sequentially outward from the cleavage site as follows: ...-P4-P3-P2-P1-P1'-P2'-P3'-P4'-... (the easily cleaved bond lies between the P1 and P1' positions), while the subpositions in the active site are correspondingly labeled as ...-S4-S3-S2-S1-S1'-S2'-S3'-S4'-. (Schechter and Berger, Biochem Biophys ResCommun. April 20, 1967; 27(2): 157-62.) For the purposes of this invention, "S1, S2, S3, and S4 bags" represent the amino acids of a protease that interact with the amino acids of the (oligo)peptide acyl donor. The C-terminal amino acid of the acyl donor (oligo)peptide (first amino acid; P1) interacts with the amino acids in the S1 bag of the protease. The penultimate amino acid of the acyl donor (oligo)peptide (second amino acid; P2) interacts with the amino acids in the S2 bag of the protease, the third amino acid (P3) interacts with P3, and the fourth amino acid (P4) interacts with the S4 bag. The S1-S4 binding bags of the protease are defined by several amino acids that may be far apart in the primary structure of the protease but close together in three-dimensional space. For the purposes of this invention, S1' and S2' bags represent the amino acids of a protease that interact with the N-terminal amino acid of the (oligo)peptide nucleophilic group. The N-terminal amino acid of the (oligo)peptide nucleophilic group interacts with the amino acids in the S1' bag of the protease. The penultimate amino acid of the (oligo)peptide nucleophilic group interacts with the amino acids in the S2' bag of the protease. The S1' and S2' binding pockets of a protease are defined by a few amino acids, which may be far apart in the primary structure of the protease but close together in three-dimensional space.

[0093] For the purposes of this invention, "denaturant" refers to an additive that can potentially disrupt the three-dimensional structure of a protease and thus potentially inactivate the protease.

[0094] In the context of this invention, "amino acid side chain" refers to any protein or non-protein amino acid side chain.

[0095] Protein amino acids are amino acids encoded by specific codons. Protein amino acids include: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), serine (Ser), threonine (Thr), methionine (Met), cysteine ​​(Cys), asparagine (Asn), tyrosine (Tyr), tryptophan (Trp), glycine (Gly), aspartic acid (Asp), glutamic acid (Glu), histidine (His), lysine (Lys), arginine (Arg), proline (Pro), and phenylalanine (Phe). Selenocysteine ​​(Sec, U) is an amino acid whose structure corresponds to cysteine, provided that the selenium atom replaces the sulfur atom.

[0096] Non-protein amino acids may be specifically selected from D-amino acids, L- or D-phenylglycine, DOPA (3,4-dihydroxy-L-phenylalanine), β-amino acids, 4-fluoro-phenylalanine, or C-amino acids. α - Alkylated amino acids.

[0097] Regarding proteins or polypeptides, especially enzymes, the term "mutated" or "mutated" as used herein means that at least one amino acid in the sequence of a wild-type or naturally occurring protein or polypeptide has been replaced, inserted, or deleted from the sequence by mutagenesis of the nucleic acid encoding that amino acid. Mutagenesis is a method well known in the art and includes, for example, site-directed mutagenesis by PCR or oligonucleotide-mediated mutagenesis as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Volumes 1-3 (1989). Regarding genes, the term "mutated" or "mutated" as used herein means that at least one nucleotide in the nucleic acid sequence of the gene or its regulatory sequence has been replaced, inserted, appended, or deleted by mutagenesis, resulting in transcription of a protein sequence having a qualitative or quantitative alteration of function or resulting in knockout of the gene.

[0098] In this specification, the abbreviation for amino acid substitution uses the single-letter amino acid code of the substituted amino acid, followed by a number indicating the position in the protein's amino acid sequence where the substitution occurred. This number represents the amino acid position in the wild-type amino acid sequence (generally BPN' of subtilisin, unless otherwise stated). Therefore, for a mutated amino acid sequence, it corresponds to the amino acid position at that number in the wild-type enzyme. The actual position may not need to be the same due to one or more other mutations (additions, insertions, deletions, etc.) at lower positions. Those skilled in the art will be able to determine the corresponding position using generally known alignment techniques, such as NEEDLE. This number is followed by the single-letter code of the amino acid that substitutes for the wild-type amino acid described herein. For example, G116S indicates that glycine is substituted for serine at position 116. X is used to indicate any other protein amino acid besides the substituted amino acid. For example, G166X indicates that glycine 166 is substituted for any other protein amino acid.

[0099] When referring to a compound that contains stereoisomers, the compound can be any such isomer or a mixture thereof. Therefore, when referring to, for example, an amino acid that contains stereoisomers, the amino acid can be an L-enantiomer, a D-enantiomer, or a mixture thereof. In the case of the presence of native stereoisomers, the compound is preferably a native stereoisomer.

[0100] The term "pH" as used in this article refers to apparent pH, which is the pH measured using a standard calibrated pH electrode.

[0101] When referring to an enzyme in parentheses as an enzyme category (EC), the enzyme category is the class in which the enzyme is classified or may be classified, based on the enzyme nomenclature provided by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), whose nomenclature can be found at http: / / www.chem.qmul.ac.uk / iubmb / enzyme / . It often includes other suitable enzymes that have not yet been classified into a specific category but may have been.

[0102] Homologous sequences generally share the expected function with the corresponding polynucleotide of their homologous polypeptide (enzyme), such as encoding the same peptide that catalyzes the same reaction. The term homologous sequence also refers to an amino acid sequence (polynucleotide sequence) that differs from another amino acid sequence due to the degeneracy of the genetic code but encodes the same polypeptide sequence.

[0103] Amino acid or nucleotide sequences are said to be homologous when they show a certain degree of similarity. Homologous sequences indicate a common evolutionary origin. Whether two homologous sequences are closely related or more distantly related is indicated by a high or low "identity percentage" or "similarity percentage," respectively.

[0104] The terms “homology,” “percentage of homology,” “percentage of similarity,” or “percentage of identical sequence” are used interchangeably herein. For the purposes of this invention, they are defined herein as determining the percentage of identical sequence between two amino acid sequences or two nucleic acid sequences, by aligning complete sequences for optimal comparison purposes. To optimize the alignment between two sequences, gaps may be introduced in either of the two sequences being compared. This alignment is performed over the full length of the sequences being compared. Alternatively, alignment may be performed over a shorter length, for example, over about 20, about 50, about 100, or more nucleic acids or amino acids. The percentage of identical sequence is the percentage of identical matches between two sequences in the reported alignment region.

[0105] Sequence comparison and determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms. Those skilled in the art will recognize that several different computer programs can be used to align two sequences and determine their homology (Kruskal, JB (1983), An overview of sequence comparison; D. Sankoff and JB Kruskal, ed., Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44, Addison Wesley). The Needleman and Wunsch algorithms used for aligning two sequences can be used to determine the percentage of identity between two amino acid sequences (Needleman, SB and Wunsch, CD (1970)). J. Mol. Biol. 48, 443-453). This algorithm compares amino acid sequences with nucleotide sequences. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purposes of this invention, the NEEDLE program from the EMBOSS package (version 2.8.0 or later) is used. EMBOSS: European Molecular Biology Open Software Suite(2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp. 276–277, http: / / emboss.bioinformatics.nl / . For protein sequences, EBLOSUM62 is used for the substitution matrix. For nucleotide sequences, EDNAFULL is used. Other matrices may be specialized. The optimal parameters for aligning amino acid sequences are a 10 nick opening penalty and a 0.5 nick extension penalty. Those skilled in the art will understand that all these different parameters will produce slightly different results, but will not significantly change the overall percentage of similarity between the two sequences when different algorithms are used.

[0106] Homology or identity between two aligned sequences is calculated as follows: the number of corresponding positions in the alignment showing the same amino acid in both sequences divided by the total alignment length minus the total number of gaps in the alignment. The identity defined herein can be obtained from NEEDLE using the NOBRIEF option and is marked as "Longest Identity" in the program's output. For the purposes of this invention, the level of identity (homology) between two sequences (amino acids or nucleotides) is calculated according to the definition of "Longest Identity" that can be performed using the NEEDLE program.

[0107] The polypeptide sequence representing the enzyme of this invention can also be used as a "reference sequence" for searching sequence databases, for example, to identify other family members or related sequences. Such searches can be performed using a BLAST program. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (http: / / ncbi.nlm.nih.gov / ). BLASTP is used for amino acid sequences and BLASTN is used for nucleotide sequences. The BLAST program uses the following default parameters: - Open gap cost: default = 5 for nucleotides / 11 for proteins - Extended gap cost: default = 2 for nucleotides / 1 for proteins - Nucleotide mismatch penalty: default=-3 - Nucleotide mismatch reward: default=1 - Expected value: default=10 -Word length: default=11 for nucleotides / 28 for megablasts / 3 for proteins Typically, the BLAST procedure determines the degree of local similarity (homology) between an amino acid sequence reference or a nucleic acid sequence reference and a recovered homologous sequence. However, only those sequence segments that give a match above a certain threshold are compared. Therefore, the procedure only calculates the similarity of these matching segments. Thus, the similarity calculated in this way is called local similarity.

[0108] The term "homology," as used herein, specifically refers to polypeptides (enzymes) having at least 50%, preferably at least 60%, more preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence similarity to a homologous peptide. Obviously, sequence similarity will be less than 100%. The percentage of sequence similarity will depend on the number of mutations and the length of the polypeptide being compared to the homolog. Specifically, for the subtilisin BPN' variant, the enzyme of the present invention will generally have at least 11 mutations, of which at least 9 mutations are deletions and at least 2 mutations are substitutions of another amino acid. Deletions are not considered in the "longest similarity" alignment. This means that the enzyme of the present invention generally has 99.25% similarity (2 substitutions in a polypeptide having 266 amino acids) or less compared to subtilisin BPN'. Preferably, the sequence identity of the enzyme of the present invention with SEQUENCE ID NO 2 is 98% or less, more preferably 96% or less, especially 94% or less, even more especially 92% or less, or 90% or less.

[0109] "Expression" refers to the transcription of genes into structural RNA (rRNA, tRNA) or messenger RNA (mRNA), which is then translated into proteins.

[0110] As used herein, “heterologous” in reference to nucleic acids or proteins means a nucleic acid or protein derived from a foreign species, or, if derived from the same substance, significantly modified from its natural form by deliberate human intervention in its composition and / or genetic locus. For example, a promoter operatively linked to a heterologous structural gene may originate from a different species from which that structural gene is derived, or, if derived from the same substance, one or both may be significantly modified from their original form. Heterologous proteins may originate from a foreign species, or, if derived from the same substance, be significantly modified from their original form by deliberate human intervention.

[0111] The term "heterologous expression" refers to the expression of heterologous nucleic acids in host cells. Heterologous protein expression in suitable host cell systems is well known to those skilled in the art. Based on common knowledge and the information disclosed herein, those skilled in the art will be able to provide suitable host cells for the production of the enzymes of this invention from various organisms without undue burden. The term "promoter" as used in this article refers to the DNA sequence that guides the transcription of (structural) genes. Generally, promoters are located in the 5' region of a gene, close to the transcription start site of the (structural) gene. Promoter sequences can be constitutive, inducible, or repressive. If the promoter is inducible, the transcription rate increases in response to the inducer.

[0112] The term "vector" as used in this article includes both autosomal expression vectors and integration vectors used for integration into chromosomes.

[0113] The term "expression vector" refers to a linear or circular DNA molecule containing a polypeptide-coding region (enzyme) of interest, which is controlled (i.e., operatively linked) by other nucleic acid segments that provide transcription. These other segments may include promoter and terminator sequences and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both.

[0114] "Plasmid" refers to autonomously replicating extrachromosomal DNA that is not integrated into the genome of a microorganism and is usually circular in nature.

[0115] An "integrating vector" is a linear or circular DNA molecule that can be integrated into the genome of a microorganism to provide stable inheritance of a gene encoding a polypeptide of interest. An integrating vector typically comprises one or more segments containing the gene sequence encoding the polypeptide of interest, controlled (i.e., operably linked) by other nucleic acid segments that provide transcription. These other segments may include promoter and terminator sequences, and one or more segments that typically drive the integration of the gene of interest into the genome of the target cell via homologous recombination. Generally, the integrating vector will be a vector that can be transferred to the target cell but has a replicon that is not functional in that organism. Integration of the segment containing the gene of interest can be selective if a suitable marker is included in the segment.

[0116] As used herein, the term "operably ligated" refers to a juxtaposition where the relationship between the components described thus allows them to function in a desired manner. "Operably ligated" means ligating to another control sequence and / or the control sequence of a coding sequence in a manner compatible with the control sequence to complete transcription and / or expression of the coding sequence. Generally, operably ligated means that the ligated nucleic acid sequences are contiguous and, when it is necessary to join two protein-coding regions, are contiguous and within the same reading frame.

[0117] "Host cell" refers to a cell that contains a vector and supports the replication or expression of that vector. Host cells can be prokaryotic cells such as bacterial cells, or eukaryotic cells such as yeast, plant, insect, amphibian, or mammalian cells.

[0118] As used herein, “transformation” and “transformed” refer to the insertion of exogenous polynucleotides into host cells, regardless of the method used for insertion, such as direct uptake, transduction, f-matching, or electroporation. The exogenous polynucleotides may remain as non-integrating vectors, such as plasmids, or may be integrated into the host cell genome.

[0119] For the purposes of clarity and brevity, each feature is described in this invention as part of the same or separate implementation; however, it should be understood that the scope of the invention may include implementations having all or some of the described features.

[0120] As used herein, the term "C-terminal C-terminal protection" refers to providing a protective group to the C-terminal carboxyl group of an oligopeptide, generally significantly protecting the carboxyl group from coupling to the N-terminal amine group of another oligopeptide or the same oligopeptide molecule. The C-terminal protecting group can be a tertiary alkyl ester group, such as a tert-butyl ester group, which is a commonly used protecting group. The C-terminal protecting group can also be a C-terminal carboxamide. Primary carboxamides are commonly used protecting groups.

[0121] The term “N-terminal protection” as used in this article refers to providing a protective group to the N-terminal amino group of an oligopeptide, generally at least significantly protecting the N-terminal amino group from coupling to the C-terminal carboxyl group of another oligopeptide or the same oligopeptide molecule.

[0122] C-terminal esters or thioesters of oligopeptides are generally activated thioesters, meaning they contain a carboxyl ester or carboxythioester group that can participate in enzymatic coupling reactions. In principle, any (substituted or unsubstituted) alkyl or (substituted or unsubstituted) aryl (thio) ester can be used. Common forms of (thio) esters that can participate in enzymatic coupling reactions are methyl, ethyl, propyl, isopropyl, phenyl, benzyl, 2,2,2-trichloroethyl-, 2,2,2-trifluoroethyl-, cyanomethyl-, and carboxyamidomethyl- (thio) esters.

[0123] Particularly good results have been obtained with carboxyamide methyl esters represented by the formula peptide-(C=O)-O-CX1X2-C(=O)N-R1R2. Again, each X1 and X2 independently represents a hydrogen atom or an alkyl group. Good results have been achieved when both X1 and X2 are hydrogen atoms (peptide-(C=O)-O-CH2-C(=O)N-R1R2). Here, R1 represents a hydrogen atom or an alkyl group and R2 represents a peptide residue or hydrogen atom or alkyl group or amino acid having a C-terminal carboxyamide or carboxylic acid functional group, optionally protected on a side chain functional group of an amino acid or on one or more of the side chain functional groups of multiple amino acids. Here, each alkyl group can independently represent (substituted or unsubstituted) C1-C7 alkyl, preferably (substituted or unsubstituted) linear C1-C6 alkyl, more preferably (substituted or unsubstituted) linear C1-C3 alkyl, and most preferably methyl. Good results have been achieved in the method of the present invention, wherein R1 and R2 both represent hydrogen atoms or wherein R1 represents a hydrogen atom and R2 represents a hydrogen atom having a hydrogen atom. C-terminus The peptide residue or amino acid with a carboxamide or carboxylic acid functional group is optionally protected on a side chain functional group of the amino acid or on one or more of the side chain functional groups of multiple amino acids. Particularly good results are achieved using Cam-esters when X1, X2, R1, and R2 are hydrogen atoms.

[0124] The C-terminal (thio)ester of the oligopeptide can be N-terminally deprotected or N-terminally protected. In one embodiment, protecting groups are provided to one or more side-chain functional groups (especially carboxyl, amino groups), for example, all side-chain functional groups; in another embodiment, all side-chain functional groups are deprotected. In a preferred embodiment, protecting groups are provided only to the side-chain functional groups (especially hydroxyl, carboxyl, or amino groups) of amino acids at the P4 and P1 positions of the (oligopeptide) acyl donor or at the P1' or P2' positions of the (oligopeptide) nucleophile. Suitable protecting groups are well known to those skilled in the art. For example, cyclohexyl, benzyl, or allyl can be used to protect the carboxylic acid group; for example, allyl carbonyl or trifluoroacetyl can be used to protect the amine functional group.

[0125] Without racemization, activated C-terminal (thio)ester groups of (oligo)peptides can be synthesized in high yield and purity using solid-phase synthesis. Another advantage of using (thio)esters is that their activated C-terminal ester or thioester groups can be synthesized using inexpensive and industrially available 2-chlorotriphenylmethyl chloride resins, where R1 represents a hydrogen atom and R2 represents a peptide residue or amino acid having a C-terminal carboxylic acid functional group, optionally protected on a side chain functional group of the amino acid or on one or more of the side chain functional groups of multiple amino acids.

[0126] Activated C-terminal thioester groups of (oligo)peptides can also be synthesized by microbial fermentation. A reliable method for obtaining (oligo)peptide (thio)esters using fermentation is through so-called integrin expression (see, for example, EKLee, Journal of Chemical Technology and Biotechnology, 2010, 9, 11-18). Various integrin expression system kits are commercially available (e.g., IMPACT™ kits). Other methods for producing (oligo)peptide (thio)esters by fermentation are known in the art.

[0127] The C-terminal amino acid of the C-terminal (thio)ester of the oligopeptide and other amino acids of the C-terminal (thio)ester can, in principle, be any protein or non-protein amino acid. If the amino acid sequence of the C-terminal portion of the (thio)ester is poorly recognized or inaccessible by the coupling enzyme due to amino acid priority and / or due to the secondary or tertiary structure of the (oligopeptide), the primary structure (amino acid sequence) can be extended at the C-terminus. Essentially, the C-terminus of the (oligo)ester is extended by a certain number of amino acids to ensure good recognition or accessibility by the enzyme for enzymatic coupling reactions. Those skilled in the art will recognize how to extend the (oligo)ester C-terminal (thio)ester based on the information disclosed herein and common knowledge. Typically, the number of extended amino acids ranges from 1 to 10, although in principle it can be higher. Good results have been obtained by extending the (oligo)ester C-terminal (thio)ester with 4 amino acid residues, for example, -Phe-Ser-Lys-Leu-(thio)ester.

[0128] Specifically, the (optionally N-terminally protected) (oligo)peptide C-terminal (thio)ester can be represented by a compound of formula I.

[0129] Formula I In this article, Q represents the OR or SR part. R can represent (substituted or unsubstituted) alkyl or (substituted or unsubstituted) aryl.

[0130] In this article, P 1 This represents a hydrogen or N-terminal protecting group. Suitable N-terminal protecting groups are those that can be used to synthesize (oligo)peptides. These types of groups are well known to those skilled in the art.

[0131] Examples of suitable N-protecting groups include urethane or acyl protecting groups, such as "Cbz" (benzyloxycarbonyl), "Boc" (tert-butoxycarbonyl), "For" (formyl), "Fmoc" (9-fluorenylmethoxycarbonyl), "PhAc" (phenylacetyl), and "Ac" (acetyl). The groups For, PhAc, and Ac can be introduced and enzymatically cleaved using peptide deformylase, PenG acyltransferase, or acyltransferase, respectively. Chemical cleavage methods are generally known in the art.

[0132] In this document, n is an integer of at least 2. n can specifically be 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. n can specifically be 100 or less, 75 or less, 50 or less, 25 or less, 20 or less, 15 or less, for example, 10 or less.

[0133] In this article, each R A and each R B The hydrogen atom or organic moiety is represented independently, preferably an amino acid side chain. Therefore, R is not required. A They are identical in all n amino acid units. Similarly, R is not required. B They are identical in all n amino acid units. Optionally, one or more of the side chain functional groups may contain protecting groups.

[0134] The amino acid unit of the nucleophilic group of (oligo)peptides can, in principle, be selected from any protein or non-protein amino acid.

[0135] Specifically, the nucleophilic group of the (oligo)peptide can be represented by a compound of formula II.

[0136] Formula II Here, n, R A and R B As defined above.

[0137] Here, P 2 This indicates the amine moiety or the OR moiety.

[0138] In P 2 In cases where the amine moiety is represented, it can be represented by the formula NR3R4, where R3 and R4 can each independently represent any (substituted or unsubstituted) alkyl or (substituted or unsubstituted) aryl group. Specifically, one of R3 and R4 is a hydrogen atom and the other is a (substituted or unsubstituted) alkyl group. Good results are obtained, especially when both R3 and R4 are hydrogen atoms.

[0139] In P 2In cases where the OR portion is used, R can represent a C-terminal protecting group or a cation, such as a monovalent cation, like a trisubstituted or tetrasubstituted ammonium ion or an alkali metal ion or H. In cases where R is a C-terminal protecting group, this can specifically be an optionally substituted alkyl group. Preferably, it is a tertiary alkyl group, although in principle it can also be any other protected ester, as known to those skilled in the art. The tertiary alkyl group can, in principle, be any protected tertiary alkyl group. Preferably, the tertiary alkyl group is selected from the group consisting of tert-butyl (2-methyl-2-propyl), tert-pentyl (2-methyl-2-butyl), and tert-hexyl (2,3-dimethyl-2-butyl).

[0140] In one embodiment, the (oligo)peptide nucleophilic group is C-terminally protected. In another embodiment, it is not C-terminally protected.

[0141] The nucleophilic group of an oligopeptide can be synthesized using methods known in the art, such as solid-phase synthesis, solution-phase synthesis, or by microbial fermentation. The N-terminal amino acid of the oligopeptide nucleophilic group and other amino acids in the group can, in principle, be any protein or non-protein amino acid. If the amino acid sequence of the N-terminal portion of the oligopeptide nucleophilic group is poorly recognized or inaccessible by the coupling enzyme due to amino acid priority or due to the secondary or tertiary structure of the oligopeptide nucleophilic group, the primary structure (amino acid sequence) can be extended at the N-terminus. Essentially, the N-terminus of the oligopeptide nucleophilic group is extended by a certain number of amino acids to ensure good recognition or accessibility by the enzyme for enzymatic coupling reactions. Those skilled in the art will recognize how to extend the oligopeptide nucleophilic group based on the information disclosed herein and common knowledge. Typically, the number of extended amino acids ranges from 1 to 10, although in principle it can be higher. Good results have been obtained by extending the oligopeptide nucleophilic group with 3 amino acid residues, for example, H-Ser-Tyr-Arg.

[0142] This invention provides an enzyme with catalytic activity (condensation activity) for forming peptide bonds, thereby possessing catalytic activity for synthesizing (oligo)peptides with a high S / H ratio. Specifically, the enzyme has ligase activity or cyclase activity, that is, catalytic activity in (oligo)peptide cyclization by catalyzing the formation of peptide bonds at the C-terminus and N-terminus of the coupled (oligo)peptide.

[0143] Specifically, the present invention provides isolated enzymes (isolated from organisms in which they have been expressed, typically recombinant organisms), if they have already been produced in the organism or derived from reaction media in which they have been synthesized.

[0144] Specifically, for the purposes of this invention, the enzymes of this invention are considered isolated if they have been clearly purified by any suitable technique, such as the single-step purification method disclosed in Smith and Johnson, Gene 67:31-40 (1988).

[0145] The enzymes of the present invention may be provided in at least substantially pure form (e.g., more than 75% by weight, more than 80% by weight) or in a mixture having one or more other components, for example, in mother liquor form, especially in an aqueous buffer solution.

[0146] This enzyme is generally a variant of subtilisin BPN' or a homolog thereof. This invention provides various examples of the enzymes of this invention, specifically considered to be variants of subtilisin BPN'. As described above, the enzymes of this invention should comprise at least: - Amino acid deletions corresponding to 75, N76, N77, S78, I79, G80, V81, L82, and G83 of BPN' (Δ75-83; therefore, the corresponding Ca is generally deleted). 2+ (binding site) - Cysteine ​​or thiocysteine ​​at position 221 corresponding to BPN' of subtilisin. - Preferably, the amino acid at position 225, which corresponds to the BPN' of subtilisin, is different from proline.

[0147] Surprisingly, a mutant possessing both a deletion of Δ75-83 at position 221 of the subtilisin BPN' and a mutation corresponding to cysteine ​​at position 221 of the subtilisin BPN' has been found to exhibit sufficient stability and an S / H ratio exceeding 1, which is an improved S / H ratio compared to, for example, subtilisin ligases. The position corresponding to S221 in the subtilisin is considered important for both enzyme stability and activity, and a single mutation corresponding to S221C in alkaline proteases has been reported to result in virtually inactive enzymes. In this respect, favorable results have been achieved by mutating to cysteine ​​at position 221.

[0148] The enzymes of the present invention may also have mutations compared to BPN' of subtilisin, provided that they have enzymatic fragment condensation or cyclization activity in the preparation of (oligo)peptides, especially one or more other mutations described elsewhere herein.

[0149] As an alternative to BPN', the enzymes of the present invention can be derived from it through mutagenesis. In particular, the template enzymes of homologs of the BPN' variants of the present invention are other BPN proteases, especially BPN proteases that have at least 50% homology with BPN'.

[0150] Suitable subtilisin sequences were obtained from the UNIPROT sequence database (http: / / www.uniprot.org / ), accessed on August 11, 2014, using subtilisin BPN' (SEQ ID 2) as a reference by BLASTing the database. However, sequence acquisition is not limited to UNIPROT or the database. Those skilled in the art know how to search alternative sequence deposits or collect other homologous sequences by sequencing (see, for example, Fecal Microbial Amplification: Molecular Microdiversity of Subtilisin in Soil). Zooming in on metagenomics: molecular microdiversity of Subtilisin Carlsberg in soil. (Gabor E, Niehaus F, Aehle W, Eck JJ Mol Biol. 20 April 2012; 418(1-2):16-20). Specifically, the present invention also relates to Figure 14 Variants of any of the subtilisins shown have at least the deletion of amino acids corresponding to L75 up to and including G83 of subtilisin BPN', a cysteine ​​at position 221 of subtilisin BPN', and an alanine at position 225 of subtilisin BPN', or another mutation (such as the mutations corresponding to P225N, 225D, P225S, P225C, P225G, P225A, P225T, P225V, P225I, P225L, P225H, P225Q of SEQUENCE ID NO: 2), the full-length sequences of which are available from the UNIPROT sequence database and are shown in alignment around positions 75-83.

[0151] Preferably, the BPN' variant or homolog of the subtilisin of the present invention contains a mutation corresponding to the P225 position. For improvement of the S / H ratio, the mutation is typically a mutation corresponding to P225 selected from the group consisting of: P225N, P225D, P225S, P225C, P225G, P225A, P225T, P225V, P225I, P225L, P225H, P225Q, P225F, and P225E. For improvement of the S / H ratio relative to, for example, Btilisin ligase, the mutation is preferably a mutation corresponding to P225 selected from the group consisting of: P225N, P225D, P225S, P225C, P225G, P225A, P225T, P225V, P225I, P225L, P225H, and P225Q. Of these, particularly good results have been achieved by mutating to one of the amino acids in the group commonly referred to as "Asx," namely, asparagine (Asn / N) and aspartic acid (Asp / D), i.e., mutations corresponding to P225N or P225D. Additionally, particularly good results have been achieved by mutations corresponding to P225S. Furthermore, particularly good results have been achieved by mutations corresponding to P225C.

[0152] Furthermore, favorable results have been achieved using mutations corresponding to P225G. Additionally, favorable results have been achieved using mutations corresponding to P225A. Furthermore, favorable results have been achieved using mutations corresponding to P225T. Additionally, favorable results have been achieved by mutating at the position corresponding to P225 to branched amino acids, namely valine (V), isoleucine (I), or leucine (L).

[0153] Preferably, the BPN' variants or homologs of the present invention comprise one or more mutations at the amino acid positions corresponding to Q2, S3, P5, S9, I31, K43, M50, A73, E156, G166, G169, S188, Q206, N212, N218S, T254, and Q271 of SEQUENCE ID NO2. The inventors have found one or more of the following mutations to be preferred in the BPN' variants of the present invention: Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, N218S, T254A, and Q271E. Specifically, for the purpose of improved activity, improved stability, or improved S / H ratio, a plurality of said mutations are preferably present in the enzyme of the present invention, such as at least 2, at least 3, more preferably 4 or more, more preferably 5 or more, more preferably 6 or more, more preferably at least 8, more preferably at least 12 mutations selected from the group consisting of: Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, N218S, T254A, and Q271E. The inventors believe that the presence of one or more of the mutations N218S, S3C-Q206C, G169A, T254A, A73L, M50F, and Q2K is particularly advantageous for improving enzyme stability. In addition, the inventors believe that the presence of one or more of the mutations I31L, E156S, G166S, and G169A is particularly advantageous for improving activity and / or the S / H ratio.

[0154] Furthermore, the BPN' variants or homologs of the present invention containing multiple mutations at the amino acid positions corresponding to Q2, S3, P5, S9, I31, K43, M50, A73, E156, G166, G169, S188, Q206, N212, N218S, T254, and Q271 of SEQUENCE ID NO 2 are easier to produce and purify than BPN ligases.

[0155] In a preferred embodiment, the enzyme includes a mutation at the amino acid position corresponding to N218, particularly N218S.

[0156] In a preferred embodiment, the enzyme includes a mutation at the amino acid position corresponding to M50, particularly M50F.

[0157] In a preferred embodiment, the enzyme includes a mutation at the amino acid position corresponding to Q2, particularly Q2K.

[0158] In a preferred embodiment, the enzyme includes a mutation at the amino acid position corresponding to A73, particularly A73L.

[0159] In a preferred embodiment, the enzyme includes a mutation at the amino acid position corresponding to P5, particularly P5S.

[0160] In a preferred embodiment, the enzyme includes a mutation at the amino acid position corresponding to G166, particularly G166S.

[0161] In a preferred embodiment, the enzyme includes a mutation at the amino acid positions corresponding to S3 and Q206, particularly S3C-Q206C.

[0162] To improve the S / H ratio, it is particularly preferred that the enzyme contains mutations at each position corresponding to Q2, P5, M50, A73 and N218, and more specifically at each position corresponding to Q2, P5, M50, A73, G166 and N218.

[0163] Specifically, good results have been achieved using BPN' variants of subtilisin containing various mutations corresponding to Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, N218S, T254A, and Q271E.

[0164] Furthermore, it has been surprisingly found that altering the S1' or S4 bag by point-specific mutations at one or more amino acid positions in the bag improves the S / H ratio for general or certain substrates. Particularly surprising is the effect of point-specific mutations in the bag, particularly in the P1' bag, on the other bag, especially the P2' bag. The inventors recognize the advantage of broadening the range of substrates that can be preferentially used for the synthetic peptides of the present invention. Therefore, this broadens the range of substrates from which the enzymes of the present invention provide high S / H ratios.

[0165] The S1' bag is primarily formed by amino acids M222 and Y217 (Strausberg L. et al., Biochemistry, 2005, 44, 3272; Estell DA et al., J. Biol. Chem., 1985, 260, 6518). The three-dimensional structure of the S1' binding bag can also be altered by more distant amino acids, such as N62, G100, S125, L126, G127, P129, N155, and N218. Substitution of one or more of these amino acids can significantly alter and improve the S / H ratio of BPN' variants or homologs of subtilisin, at least for multiple peptide sequences. In a preferred embodiment, substitution at the amino acid position corresponding to M222 or Y217 increases the substrate range for which the activity, S / H ratio, or enzyme-shown (highly) improved S / H ratio is achieved.

[0166] Preferably, the mutation present at the position corresponding to M222 is M222G, M222P, M222N, M222E, M222Q, or M222A. In a particularly preferred embodiment, the mutation corresponds to M222P or M222G.

[0167] Preferably, the mutation at the position corresponding to Y217 is Y217L, Y217N, Y217E, Y217G, Y217S, Y217F, or Y217H.

[0168] Particularly good results have been obtained with variants having mutations selected from M222G, M222P and Y217L, with significantly increased S / H ratios and / or hindracetic rates in the resulting subtilisin BPN' variants, at least for some peptide sequences.

[0169] The S4 binding bag is mainly formed by amino acids Y104, I107, L126, S101, G102, G127, and G128, but the three-dimensional structure of the S4 binding bag is also determined by more distant amino acids such as L135 and P168 (Ruan et al., Biochemistry, 2008, 47, 6628; Rheinnecker et al., Biochemistry, 1994, 33, 221).

[0170] Preferably, the enzyme contains mutations corresponding to one, two, or each of the positions Y104, I107, and L135. Particularly good results have been obtained with BPN' variants of subtilisin containing mutations selected from the group consisting of Y104F, Y104S, I107V, I107A, L135N, L135S, L135D, and L135A. Substitution of these amino acids can significantly alter and improve the S / H ratio and / or activity of the enzyme, at least for certain substrates.

[0171] Specifically, good results have been achieved for the P4 substrate range, with enzyme activity and S / H ratio obtained from BPN' variants of the subtilisin corresponding to the amino acid substitutions in I107 (I107V) and L135 substitutions (L135S or L135N).

[0172] In a preferred embodiment, the enzyme of the present invention has one or more substitutions in the S1' binding bag and one or more substitutions in the S4 binding bag, particularly having two or more substitutions in the S1' binding bag and two or more substitutions in the S4 binding bag.

[0173] It has been found that substitutions of two amino acids corresponding to both M222 and I107 preferably provide enzymes with improved activity and S / H ratios compared to variants of the invention having only one of the aforementioned mutations. Any single mutation has also been found to be beneficial to enzyme activity and S / H ratio. Specifically, good results have been achieved in this embodiment by mutating I107V and M222G. Examples of other combinations of mutations of particular interest are variants having the mutation L135N + M222G and variants having the mutation I107V + M222P. Additionally, combinations of mutations at the positions corresponding to I107 and M222 provide improvements in substrate range relative to the P4 and P1' bags.

[0174] In a preferred embodiment, the subtilisin BPN' variant or homolog of the present invention has substitutions in the S1' binding pocket at the positions corresponding to M222 and Y217. In this embodiment, the M222 mutation is preferably M222G or M222P. The Y217 mutation is preferably selected from one of Y217F, Y217H, and Y217G. This enzyme of the present invention has been found to have a wide substrate range and a good S / H ratio. Particularly good results have been achieved with subtilisin BPN' variants or homologs comprising the following: mutants M222P and Y217H; mutants M222P and Y217G; mutants M222G and Y217F; or mutants M222G and Y217G. Among these, the subtilisin BPN' variant or homolog comprising M222G and Y217F has achieved particularly good results in terms of substrate width and S / H ratio.

[0175] Good results have been achieved with the BPN' variant of the present invention or its homologs, which has a substitution in the S1' binding pocket at the position corresponding to M222 and no mutation in the S4 binding pocket at the position corresponding to Y217. However, in an alternative embodiment that also achieves good results, there are one or more mutations in the S4 binding pocket. In one specific embodiment, this BPN' variant of the BPN or its homologs has substitutions at two or more positions in the S4 binding pocket corresponding to Y104, I107, L126, L135, S101, G102, G127, and G128. The mutations in the S4 binding pocket may specifically include I107V and / or L135N or L135S.

[0176] The preferred enzyme of the present invention is specifically a variant or homolog of the subtilisin BPN' comprising any one of the sequences shown in SEQUENCE ID NO 3, 4, or 5. SEQUENCE ID NO 3 shows a preferred mutation corresponding to S221C, although in another embodiment this may be selenocysteine. The X at the position corresponding to P225 can be P, or a different amino acid, preferably one of the preferred mutations identified elsewhere herein (N / D / S / C / G / A / T / V / I / LH / Q). Compared to SEQUENCE ID NO 3, SEQUENCE ID NO 4 shows a preferred mutation site. In SEQUENCE ID NO 4, each X independently represents any protein amino acid. Specifically, any X can be an amino acid present in wild-type subtilisin BPN' at the position of that X, or a mutation described elsewhere herein. Preferably, one or more Xs represent mutations, as described elsewhere herein.

[0177] In the method of the present invention, the enzymatic coupling reaction and cyclization are carried out in a fluid containing water. Preferably, the reaction is carried out in a buffered fluid. Based on the total liquid content, the water content is typically 10-100% by volume, preferably 20% by volume or more, more preferably 40% by volume or more, especially 50% by volume or more, or even more especially 60% by volume or more.

[0178] In principle, any buffer is suitable. Good buffers are known to those skilled in the art. See, for example, David Sheehan, in Physical Biochemistry, 2nd ed., Wiley-VCH Verlag GmbH, Weinheim 2009; http: / / www.sigmaaldrich.com / life-science / core-bioreagents / biological-buffers / learning-center / buffer-calculator.html.

[0179] The pH of the buffer for (oligo)peptide fragment condensation can be at least 5, especially at least 6, and preferably at least 7. The maximum required pH is generally less than 11, especially less than 10, and even more preferably less than 9. Generally, the optimal pH for enzymatic reactions is 7-9. For cyclization reactions, the optimal pH may be different. The pH for cyclization reactions can be at least 3, especially at least 4, and preferably at least 5. The maximum required pH is generally less than 11, especially less than 10, and preferably less than 9. Generally, the optimal pH for enzymatic cyclization reactions is 5-9.

[0180] Due to the high S / H ratio, a large excess of the (oligo)peptide C-terminal ester or thioester or (oligo)peptide nucleophilic group is generally not required to achieve high yields in the condensation reaction. Typically, the ratio of (a) (oligo)peptide C-terminal ester or thioester to (b) (oligo)peptide nucleophilic group is 1:5 to 5:1, preferably 1:3 to 3:1, more preferably 1.0:2.5 to 2.5:1.0, especially 1:2 to 2:1, and even more especially 1:1.5 to 1.5:1. Approximate stoichiometric ratios have been found to be particularly effective.

[0181] In the method of the present invention, additives may preferably be added to the fluid, wherein the reaction is carried out to improve the solubility of the (oligo)peptide fragments or to improve the reaction yield. Such additives may be salts or organic molecules, such as guanidine hydrochloride, urea, sodium dodecyl sulfate, or Tween.

[0182] The reaction can be carried out in a completely aqueous fluid or in a water- and water-miscible cosolvent, such as N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), acetonitrile, esters such as tetrahydrofuran (THF), 2-methyltetrahydrofuran (Me-THF), or 1,2-dimethoxyethane, or (halogenated) alcohols such as methanol, ethanol, isopropanol, tert-butanol, 2,2,2-trifluoroethanol (TFE), 1,1,1,3,3,3-hexafluoroisopropanol, or mixtures of these organic solvents. Depending on the stability of the BPN' variant of the subtilisin and the solubility of the (oligo)peptide substrate, the amount of cosolvent is preferably less than 70% by volume, more preferably less than 60% by volume, even more preferably less than 50% by volume, and most preferably less than 40%.

[0183] In principle, the temperature during enzymatic fragment condensation or cyclization is not critical, as long as the selected temperature at which the subtilisin BPN' variant used exhibits sufficient activity and stability. This temperature is typically known or routinely determined by the subtilisin BPN' variant to be used, using a known substrate of the subtilisin BPN' variant under known reaction conditions. Generally, the temperature can be at least -10°C, especially at least 0°C or at least 10°C. Generally, the temperature can be 70°C or lower, especially 60°C or lower or 50°C or lower. Those skilled in the art can readily identify the optimal temperature conditions for a particular subtilisin BPN' variant for the condensation or cyclization of a particular enzymatic fragment through routine experiments based on common knowledge and the information disclosed herein. Generally, the preferred temperature range is 20-50°C.

[0184] The subtilisin BPN' variant of the present invention is generally generated by a recombination method, especially by the expression of subtilisin BPN' DNA, which has been mutated so that, after expression, it produces the subtilisin BPN' variant of the present invention with enzymatic activity.

[0185] The expression of the DNA of the BPN' variant of the present invention and its homologs is provided using available vectors and regulatory sequences. The actual choice largely depends on the specific host cell used for expression. For example, if expressing the BPN' mutant DNA in Bacillus subtilis, a Bacillus promoter and a Bacillus-derived vector are generally used.

[0186] To produce and secrete the enzymes of the present invention from host cells into a medium, a gene encoding a precursor polypeptide (enzyme) containing a signal sequence and a pre-pro sequence prior to the mature enzyme can be used. In subtilisin BPN', the additional N-terminal sequence comprises 107 amino acids. After secretion, the signal sequence can be removed first, and the pre-pro sequence can be removed after secretion to produce a fully active enzyme (James A. Wells, Nucleic Acids Research, Vol. 11, No. 22, 1983). In the case of native subtilisin BPN', the mature enzyme comprises 275 amino acids. For convenience in describing the position of individual amino acids in the polypeptide chains of subtilisin BPN' and its homologs, the so-called subtilisin BPN' numbering is used, from the N-terminus (amino acid 1) to the C-terminus (amino acid 275). The corresponding position in the homologous enzyme can be determined by comparing the homologous sequence with the sequence of subtilisin BPN'.

[0187] As those skilled in the art will know, the N- and / or C-termini of the mature polypeptide encoded as 1-275 in SEQ ID NO: 5 or the amino acids of SEQ ID NO: 2, 3 or 4 (as shown by amino acids 1-275) of the mature enzyme may be heterogeneous due to variations in treatment during maturation. Specifically, such treatment variations may occur after overexpression of the enzyme. Additionally, exoprotease activity may produce heterogeneity. The degree of heterogeneity also depends on the host and fermentation protocol used. This C-terminal treatment artifact may produce polypeptides shorter or longer than those shown by the mature wild-type subtilisin BPN' (SEQ ID NO: 2) or the mature enzymes of the present invention shown by SEQ ID NO: 3 or 4. As a result of such treatment variations, the N-terminus may also be heterogeneous. Treatment variants at the N-terminus may be due to alternative cleavage of the signal sequence by the signal peptidase.

[0188] Based on common knowledge and the information disclosed herein, the enzymes of this invention can be generated using recombinant technology. To secrete the translated enzyme into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment, a suitable secretion signal sequence can be fused with a polynucleotide encoding the enzyme of this invention. These signals can be endogenous to the enzyme or they can be exogenous signals.

[0189] The enzymes of the present invention can be produced in modified forms, such as fusion proteins, and may include not only secretory signals but also other heterologous functional regions. Thus, for example, other amino acid regions (so-called tags), especially charged amino acids, can be added to the enzyme, particularly the C-terminus, to improve stability and persistence in the host cell, during purification or subsequent operations, and during storage, or to facilitate purification. Examples of suitable tags are described by ME Kimple et al. in their review in *Current Protocols in Protein Science*, 9.9.1–9.9.23, August 2013. A well-known example of a usable tag is the so-called His tag, an amino acid sequence having multiple histidine units. The inventors have found that such tags can be used continuously in the production and purification of the enzymes of the present invention. No significant differences in functional enzyme properties have been observed between enzymes with and without His tags.

[0190] Furthermore, the enzyme of this invention can be generated through the formation of inclusion bodies and refolded in a suitable buffer solution. The enzymes of the present invention comprise naturally purified products, products of chemical synthesis processes, and products produced from prokaryotic or eukaryotic hosts, including, for example, bacteria, yeast, higher plants, insects, and mammals, through recombinant technology. Depending on the host used in the recombinant production process, the enzymes of the present invention may be glycosylated or non-glycosylated. Additionally, in some cases, due to a host-mediated process, the enzymes of the present invention may also include initially modified methionine residues.

[0191] The polynucleotides of the present invention can be incorporated into vectors, including cloning and expression vectors. The vectors can be recombinant reproducible vectors. The vectors can be used to replicate the polynucleotides of the present invention in compatible host cells. The vectors can be readily processed using recombinant DNA.

[0192] The present invention also relates to methods for growing, transforming, or transfecting such vectors in suitable host cells, for example, in the case of expression of the enzymes of the present invention. The present invention provides a method for preparing the enzymes of the present invention by introducing the polynucleotides of the present invention into a vector (in one embodiment, an expression vector), introducing the vector into a compatible host cell, and growing the host cell under conditions that produce replication of the vector.

[0193] The vector can be recovered from the host cell.

[0194] The vector of the present invention can be a self-replicating vector, that is, a vector that exists as an extrachromosomal entity and whose replication is independent of chromosome replication, for example, a plasmid.

[0195] Alternatively, the vector may be a vector that integrates into the host cell genome upon introduction into the host cell and replicates along with the integrated chromosome.

[0196] One type of vector is the "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted. Another type of vector is the viral vector, in which additional DNA segments can be inserted into the viral genome.

[0197] Some vectors are capable of autonomous replication in the host cells they are transduced into (e.g., bacterial origin of replication and bacterial vectors and episome mammalian vectors). Other vectors (e.g., bacterial integration vectors without a suitable origin of replication or non-epistone mammalian vectors) are integrated into the host cell genome after introduction into the host cell, thereby replicating together with the host genome.

[0198] The recombinant expression vector of the present invention comprises the polynucleotide of the present invention having a form in which the nucleic acid sequence is expressed in a host cell. This means that the recombinant expression vector includes one or more regulatory sequences selected based on the host cell to be used for expression, which are operatively linked to the polynucleotide sequence to be expressed. The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). The regulatory sequences are described in, for example, Goeddel; *Gene Expression Technology: Enzymatic Approaches* (…). Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990).

[0199] The vector or expression construct for a given host cell may therefore include the following elements operatively linked to each other in a continuous sequence from the 5'-end to the 3'-end of the coding strand relative to the sequence encoding the enzyme of the present invention: (1) a promoter sequence capable of guiding transcription of the nucleotide sequence encoding the enzyme in a given host cell; (2) a ribosome binding site to facilitate the translation of transcribed RNA; (3) optionally, a signal sequence capable of guiding the secretion of the enzyme from the host cell into a culture medium; (4) the polynucleotide sequence of the present invention; and preferably, also (5) a transcription termination region (terminator) capable of terminating downstream transcription of the nucleotide sequence encoding the enzyme.

[0200] Downstream of the nucleotide sequence according to the invention may be a 3' untranslated region (e.g., a terminator, also referred to herein as a stop codon) containing one or more transcription termination sites. The start point of the terminator is less critical. For example, the terminator may be native to the DNA sequence encoding the enzyme. However, bacterial terminators are preferred for bacterial host cells, and filamentous fungal terminators are preferred for filamentous fungal host cells. More preferably, the terminator is endogenous to the host cell (where the nucleotide sequence encoding the enzyme will be expressed). Within the transcribed region, ribosome-binding sites for translation may be present. The coding portion of the mature transcript expressed by the construct will include a start codon, typically AUG (or ATG), but alternative start codons such as GUG (or GTG) and UUG (or TTG) are also used in prokaryotes. A stop or translation stop codon may also be appropriately placed at the end of the polypeptide to be translated.

[0201] Enhanced expression of the polynucleotides of the present invention can also be achieved by selecting homologous and heterologous regulatory regions, such as promoters, secretion leader sequences and / or terminator regions, which can be used to increase the expression and (if desired) secretion levels of proteins of interest from the expression host and / or provide inducible control over the expression of the enzymes of the present invention.

[0202] The enzymes according to the invention can be produced in bacterial cells such as *Escherichia coli* and *Bacillus* (using baculovirus expression vectors), fungal cells, yeast cells, or mammalian cells. Suitable host cells are described herein and further described in Goeddel, *Gene Expression Technology: Methods in Enzymology*, 185, Academic Press, San Diego, CA (1990) and "Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems", 2004, Wiley-Blackwell (ed.), (http: / / eu.wiley.com / WileyCDA / Section / id-302479.html?query=Gerd+Gellissen). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using a T7 promoter regulatory sequence and a T7 polymerase.

[0203] For most bacteria, filamentous fungi, and yeasts, the vector or expression construct is preferably integrated into the host cell's genome to obtain stable transformants. When the expression construct is integrated into the host cell's genome, the construct is integrated at a random locus in the genome or at a predetermined target locus using homologous recombination, in which case the target locus preferably contains a highly expressed gene.

[0204] In this invention, bacteria, particularly Bacillus, are preferably used as host cells for expressing the enzymes of this invention. Suitable inducible promoters that can be used in such host cells include promoters primarily regulated by cofactors such as repressors or activators. A repressor is a sequence-specific DNA-binding protein that inhibits promoter activity. Transcription can be initiated from this promoter in the presence of an inducing agent, which prevents the repressor from binding to the promoter's operon. The generation of secondary sigma factors primarily leads to transcription from a specific promoter. Attenuation and antitumor agents also regulate transcription.

[0205] Strong constitutive promoters are well known, and a suitable promoter can be selected based on the specific sequence to be controlled in the host cell. A variety of promoters capable of directing transcription in the recombinant host cells of the present invention can be used. Preferably, the promoter sequence is derived from a highly expressed gene.

[0206] Vector DNA can be introduced into prokaryotic or eukaryotic cells using natural capabilities and conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing exogenous polynucleotides (e.g., DNA) into host cells, including calcium phosphate or calcium chloride coprecipitation, DEAE-glucan-mediated transfection, transduction, infection, lipid transfection, cationic lipid-mediated transfection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (ibid.) and other laboratory manuals.

[0207] To identify and select cells containing the vector, the polynucleotide of the present invention is typically introduced into host cells along with a gene encoding a selectability marker (e.g., antibiotic resistance). Preferred selectability markers include, but are not limited to, those that confer drug resistance or compensate for host cell defects. They also include, for example, multifunctional marker genes that can be used to transform most filamentous fungi and yeasts, such as acetamase genes or genes that provide resistance to antibiotics such as G418, hygromycin, bleomycin, kanamycin, methotrexate, and sphaerocephalosporin resistance (benA). Alternatively, specific selectability markers can be used, such as auxotrophic markers requiring a corresponding mutant host strain: for example, D-alanine racemicase (from Bacillus), URA3 (a similar gene from Saccharomyces cerevisiae or other yeasts), pyrG or pyrA (from Aspergillus nidus or Aspergillus niger), argB (from Aspergillus nidus or Aspergillus niger), or trpC. In one embodiment, after the introduction of the expression construct, the selectability marker is removed from the transformed host cells to obtain transformed host cells capable of producing the enzyme of the present invention without the selectability marker gene.

[0208] Protein expression in prokaryotes is typically carried out using vectors containing constitutive or inducible promoters that guide the expression of fusion or non-fusion proteins. Fusion vectors add numerous amino acids to the protein they encode, for example, to the N-terminus of the recombinant protein. Such fusion vectors generally serve three purposes: 1) to increase the expression of the recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Typically, proteolytic cleavage sites are introduced in fusion expression vectors at the junction of the fusion moiety and the recombinant protein so that the fusion protein can be separated from the fusion moiety after purification.

[0209] Preferred vectors for use with bacteria are disclosed, for example, in WO-A1-2004 / 074468, which is incorporated herein by reference. Other suitable vectors will be apparent to those skilled in the art.

[0210] The vectors of the present invention can be transformed into suitable host cells as described herein to provide expression of the polypeptides of the present invention. Therefore, in another aspect, the present invention provides a method for preparing an enzyme according to the present invention, comprising culturing host cells transformed or transfected with an expression vector encoding the enzyme, and recovering the expressed polypeptide.

[0211] When the polynucleotides of the present invention are transformed into suitable host cells, they encode the enzymes of the present invention. The present invention is characterized by cells, such as transformed host cells or recombinant host cells containing the polynucleotides of the present invention or containing the vectors of the present invention. "Transformed host cells" or "recombinant host cells" are cells in which the polynucleotides of the present invention are introduced via recombinant DNA technology.

[0212] This includes prokaryotic and eukaryotic cells, such as bacteria, fungi, yeast, insects, and mammals.

[0213] Suitable host cells include bacteria, including *Escherichia coli*, *Anabaena*, bacteria, *Staphylococcus*, *Rhodops*, *Pseudomonas*, *Paracococcus*, *Bacillus*, *Brevibacterium*, *Corynebacterium*, *Rhizobium sinense*, *Flavobacterium*, *Klebsiella*, *Enterobacter*, *Lactobacillus*, *Lactococcus*, *Bacteroides*, *Staphylococcus*, *Streptomyces*, and *Pseudomonas*. In one aspect of the vector of the present invention, the host cell is a bacterial cell selected from the group consisting of *Bacillus subtilis* (…). B. subtilis ), Bacillus pantyhose ( B. puntis ), Bacillus megaterium ( B. megaterium ), salt-resistant Bacillus ( B. halodurans ), Bacillus pumilus ( B. pumilus ), oxidized gluconic acid bacillus (G. oxydans) ), Crested bacteria CB15 ( Caulobactert crescentus CB 15 ), Methylobacterium truncatum ( Methylobacterium extorquens ), Rhodopsycetes ( Rhodobacter sphaeroides ), xanthophyllosis ( Pseudomonas zeaxanthinifaciens ), denitrifying paracocci ( Paracoccus denitrificans ), Corynebacterium glutamicum ( C. glutamicum Staphylococcus aureus ( Staphylococcus carnosus Streptomyces purpureus ( ) Streptomyces lividans ), Alfalfa rhizobia ( Sinorhizobium melioti ) and radiation rhizobium ( Rhizobium radiobacter ).

[0214] In another embodiment of the carrier of the present invention, a suitable host cell is a species of Aspergillus, Beauveria bassiana, Kluyveromyces, Penicillium, Yeast, or Orthopaedicella.

[0215] Preferably, the host cell is Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Escherichia coli, Aspergillus niger, or Aspergillus oryzae.

[0216] The recombinant host cell according to the present invention may contain the polynucleotide of the present invention or the vector of the present invention.

[0217] In one embodiment of the recombinant host cell of the present invention, the recombinant host cell is capable of expressing or overexpressing the polynucleotide of the present invention or the vector of the present invention.

[0218] The method for preparing the polynucleotide or the vector of the present invention includes the following steps: culturing host cells transformed with the polynucleotide or the vector, and isolating the polynucleotide or the vector from the host cells.

[0219] Preferred Bacillus strains of cells, such as Bacillus alkalophilus ( Bacillus alkalophilus ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), Bacillus brevis ( Bacillus brevis ), Bacillus circularis ( Bacillus circulans ), Bacillus krusei ( Bacillus clausii Bacillus coagulans ( Bacillus coagulans ), Bacillus thuringiensis ( Bacillus firmus ), Bacillus splendens ( Bacillus lautus ), Bacillus tarda ( Bacillus lentus ), Bacillus licheniformis ( Bacillus licheniformis ), Bacillus megaterium ( Bacillus megaterium ), Bacillus pumilus ( Bacillus pumilus ), thermophilic steatobacterium ( Bacillus stearothermophilus Bacillus subtilis ( Bacillus subtilis ), or Bacillus thuringiensis ( Bacillus thuringiensis ); or cells of Streptomyces strains, such as Streptomyces purpureus ( ); Streptomyces lividans ) or Streptomyces boulardii ( Streptomyces murinus ); or from Gram-negative bacteria, such as Escherichia coli or Pseudomonas (Long Liu et al., Appl Microbiol Biotechnol (2013) 97:6113–6127 and KayTerpe, Appl Microbiol Biotechnol (2006) 72:211–222).

[0220] According to another aspect, the host cell is a eukaryotic host cell. In one embodiment, the eukaryotic cell is a fungal cell, i.e., a yeast cell, such as *Candida*, *Hansenula*, *Kluyveromyces*, *Pichia pastoris*, or *Saccharomyces* strains. Preferably, the yeast cell is *Kluyveromyces lactis*, *Saccharomyces cerevisiae*, *Hansenula polymorpha*, *Yazir* lipophila, *Pichia pastoris*, or a filamentous fungal cell.

[0221] Filamentous fungi encompass all filamentous forms subdivided into the classes Fungi and Oomycetes (defined by Hawksworth et al., *Ainsworth and Bisby's Dictionary of the Fungi*, 8th ed., 1995, CAB International, University Press, Cambridge, UK). Filamentous fungi are characterized by a mycelial wall composed of chitosan, cellulose, glucan, mannan, and other complex polysaccharides. Vegetative growth is strictly aerobic, driven by hyphal elongation and carbon catabolism. Filamentous fungal strains include, but are not limited to, *Acer*, *Aspergillus*, *Bacillus*, *Bacillus*, *Salmonella*, *Coprinus*, *Cryptococcus*, *Ustilago*, *Fusarium*, *Pyrophyllus*, *Salmonella*, *Mucor*, *Hypertricis*, *Neurospora*, *Penicillium*, *Pseudomonas*, *Pseudomonas*, *Pleurotus*, *Schizophyllum*, *Hypertricis*, *Thermophyton*, *Fusarium*, *Trichoderma*, and *Aspergillus*. In one embodiment, filamentous fungal cells belonging to *Aspergillus*, *Bacillus*, *Penicillium*, *Taraxacum*, *Fusarium*, or *Trichoderma* are used, preferably *Aspergillus niger*, *Aspergillus awamori*, *Aspergillus fumigatus*, *Aspergillus sacchari*, *Aspergillus davidii*, *Aspergillus fumigatus*, *Aspergillus davidii*, *Aspergillus fumigatus*, *Aspergillus versicolor*, and *Aspergillus oryzae*. Chrysosporium lucknowense ), thermophilic filamentous fungi ( Thermocophorora thermophila Fusarium oxysporum ( Fusarium oxysporum Trichoderma reesei ( Trichoderma reesei ) or produce Penicillium chrysogenum ( Penicillium chrysogenum ).

[0222] A host cell can be selected that modifies and processes the encoded enzyme in a specific, desired manner post-translation. Such post-translational modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products promote optimal protein function. Various host cells exhibit characteristic and specific mechanisms for the post-translational processing and modification of protein and gene products. Suitable cell lines or host systems familiar to those skilled in the art of molecular biology and / or microbiology can be selected to ensure the desired and correct modification and processing of the resulting exogenous protein. For example, in one embodiment, a subtilisin BPN' variant or its homolog is initially secreted as a pre-enzyme, and the presence of a 77-amino acid pre-sequence is important for the in vivo production of mature subtilisin but must be cleaved to obtain full catalytic activity.

[0223] The method for producing the enzyme according to the present invention typically includes culturing recombinant host cells, for example, transforming or transfecting them with an expression vector under conditions that express the coding sequence encoding the enzyme, and recovering and purifying the resulting enzyme from the cells or culture medium. The polynucleotides of the present invention can be integrated into recombinant reproducible vectors, such as expression vectors or replication vectors. Transcription vectors are used to amplify their insert fragments.

[0224] The purpose of vectors that transfer genetic information to another cell is typically to isolate, proliferate, or express an insert fragment in the target cell. Vectors called expression vectors (expression constructs) are specifically designed for expressing transgenes in target cells and typically have a promoter sequence that drives transgene expression. Simpler vectors called transcription vectors can only be transcribed but not translated: unlike expression vectors, they can replicate in target cells but not express the transgene. Transcription vectors are used to amplify their insert fragments. Therefore, in another embodiment, the present invention provides a method for preparing the polynucleotides of the present invention by introducing the polynucleotides of the present invention into a reproducible vector, introducing the vector into a compatible host cell, and growing it under conditions that cause the host cell to replicate the vector. The vector can be recovered from the host cell.

[0225] Preferably, the enzyme of the present invention is produced as a secretory protein, in which case the nucleotide sequence encoding the mature form of the enzyme in the expression construct is operatively linked to the nucleotide sequence encoding the signal sequence. Preferably, the signal sequence is native (homologous), also referred herein as the “wild-type” of the nucleotide sequence encoding the enzyme. Alternatively, the signal sequence is foreign (heterologous) relative to the nucleotide sequence encoding the enzyme, in which case the signal sequence is preferably endogenous to the host cell in which the nucleotide sequence of the present invention is expressed. Examples of suitable signal sequences for Bacillus subtilis can be found in “van Dijl, JM et al., 2001. In: Sonenshein, AL, Hoch, JA and Losick, R. eds., Bacillus subtilis and its close relatives: from gene to cell (…) Bacillus subtilis and its closest relatives: from genes to cells. Washington, D.C.: ASM Press, pp. 337-355. and Degering C et al., Appl Environ Microbiol. 2010 Oct;76(19):6370-6. The expression of heterologous proteins in yeast is well-known. Sherman, F. et al., *Methods in Yeast Genetics*, Cold Spring Harbor Laboratory (1982), is a recognized work describing various methods that can be used to express proteins in yeast. Vectors, strains, and protocols for expression, such as those for *Saccharomyces* and *Pichia pastoris*, are well-known in the art and are available from commercial suppliers (e.g., Invitrogen). Suitable vectors typically have expression control sequences, such as promoters including 3-phosphoglycerate kinase or alcohol oxidase, as well as desired origin of replication, termination sequences, etc. More specifically, suitable yeast signaling sequences are sequences derived from yeast α-factor genes. Similarly, suitable signaling sequences for filamentous fungal host cells are, for example, signaling sequences from filamentous fungal amylase (AG) genes, such as the *Aspergillus niger* g / aA gene. This can be used with the amylase (also known as (gluco) amylase) promoter itself, as well as in combination with other promoters. Mixed signaling sequences can also be used in conjunction with the content of this invention. Preferred heterologous secretion leader sequences are derived from fungal amyloglucosidase (AG) genes (g / aA - 18 and 24 amino acid forms from Aspergillus), α-factor genes (yeasts such as Saccharomyces and Kluyveromyces), or [α-amylase (amyE, amyQ and amyL) and alkaline protease aprE and neutral protease genes (Bacillus).

[0226] Alternatively, heterologous host cells can be selected, wherein the enzymes of the present invention are produced in a form with substantially no enzymatic activity that could interfere with application, for example, peptides that do not degrade or modify the enzyme. Specifically, in the case of producing variants, the host cells should not produce any wild-type enzymes. This can be achieved by selecting host cells that do not normally produce such enzymes or by deliberately removing the corresponding genes using techniques known in the art.

[0227] This invention includes a method for producing the enzyme of the invention by recombinant expression of a DNA sequence encoding the enzyme of the invention. For this purpose, the DNA sequence of the invention can be used for gene amplification and / or exchange of expression signals, such as promoters, secretory signal sequences, to allow the enzyme to be economically produced in suitable homologous or heterologous host cells. Homologous host cells are host cells of the same species or variants within the same species from which the DNA sequence is obtained. The host cell may overexpress the enzyme, and techniques for engineered overexpression are well known. Therefore, the host may have two or more copies encoding the polynucleotide (and thus the vector may correspondingly have two or more copies). Therefore, in one embodiment of the invention, the recombinant host cell of the invention is capable of expressing or overexpressing the polynucleotide or vector of the invention.

[0228] Another aspect of the invention is a method for preparing the enzyme of the invention, comprising (a) culturing the recombinant host cells of the invention under the conditions for preparing the enzyme of the invention; and (b) optionally recovering the enzyme of the invention from the cell culture medium. For each combination of promoter and host cells, culture conditions suitable for expressing the DNA sequence encoding the enzyme are obtained. Once the desired cell density or enzyme titer is reached, the culture is stopped and the enzyme is recovered. The term "culture" includes maintaining and / or growing live recombinant host cells of the invention, particularly the recombinant host cells of the invention.

[0229] On one hand, the recombinant host cells of the present invention are cultured in a liquid culture medium. On the other hand, the recombinant host cells are cultured in a solid or semi-solid culture medium. Preferably, the recombinant host cells of the present invention are cultured in a liquid culture medium containing nutrients essential or beneficial to the maintenance and / or growth of the recombinant host cells. The recombinant host cells can be cultured continuously or intermittently in liquid culture medium by conventional culture methods such as vertical culture, test tube culture, shaking culture, aerated rotary culture, or fermentation. Preferably, the recombinant host cells are cultured in a fermenter. The fermentation method of the present invention includes batch, fed-batch, and continuous fermentation methods. Various such methods have been developed and are well known in the art.

[0230] The recombinant host cells are preferably cultured at a controlled pH. In one embodiment, the recombinant host cells may be cultured at a pH of 4.5 to 8.5, preferably 6.0 to 8.5, and more preferably about 7. The desired pH may be maintained by any method known to those skilled in the art.

[0231] Preferably, the recombinant host cells are further cultured under controlled ventilation and controlled temperature. In one embodiment, the controlled temperature includes a temperature between 15 and 70°C, preferably between 20 and 55°C, and more preferably between 30 and 50°C. Appropriate conditions are typically selected based on the choice of expression host and the protein to be produced.

[0232] In a specific embodiment, the enzyme is expressed in Bacillus strain GX4935 (see Examples). The strain is cultured under aerobic conditions in a suitable fermentation medium. In addition to inorganic salts, the suitable medium may contain absorbable carbon and nitrogen sources, and optionally growth-promoting nutrients such as yeast extract. Fermentation is typically carried out at 35-40°C and a pH of 6.5-7.5, preferably maintained substantially constant automatically. The enzyme is secreted into the medium. At the end of fermentation, if necessary, the production host can be killed by methods known to those skilled in the art. The subsequent fermentation broth can be used to remove bacterial cells, debris, and other solids, for example, by filtration or centrifugation. The filtrate or supernatant containing the enzyme can be further clarified, for example by filtration or centrifugation, and then concentrated as needed, for example by ultrafiltration or under reduced pressure in an evaporator, to obtain a concentrate, which can be concentrated to dryness, for example by lyophilization or spray drying, if desired.

[0233] After fermentation, cells can be removed from the fermentation broth by centrifugation or filtration if necessary. After fermentation is stopped or cells are removed, the enzymes of the present invention can then be recovered and purified and isolated by conventional methods if desired, including but not limited to treatment with conventional resins, treatment with conventional adsorbents, pH alteration, solvent extraction, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization, etc. For example, the enzymes according to the present invention can be recovered and purified from recombinant cell cultures by methods known in the art (Protein Purification Protocols, Paul Cutler's Methods in Molecular Biology series, Humana Publishing, 2004). Typically, the compound is "isolated" when the resulting formulation is substantially free of other components.

[0234] In one embodiment, an isolated enzyme preparation is provided having a purity of about 80% (dry weight) or more of the enzyme of the present invention (i.e., components or fermentation byproducts less than about 20% of the total culture medium). In a specific embodiment, the present invention provides an enzyme of the present invention with a purity of about 90% or higher, preferably 95% or higher, and particularly 98% or higher. In practice, small amounts of other components may be present in the isolated enzyme preparation of the present invention. Therefore, the purified enzyme preparation may contain 99% or less, particularly 98% or less of the enzyme.

[0235] However, alternatively, the enzyme of the present invention is not purified from recombinant host cells or cultures. The whole culture or culture supernatant can be used as the source of the enzyme. In specific embodiments, cultures or culture supernatants containing the enzyme are used without substantial modification.

[0236] It is further noted that the enzymes of the present invention, such as the subtilisin BPN' variant, can also be prepared using known chemical protein synthesis techniques, such as solid-phase peptide synthesis. However, expression of the subtilisin mutant in microbial host cells is generally preferred, as this will allow the microbial host cells to produce subtilisin in a conformational form appropriate for enzyme activity. However, it should be possible to convert improperly folded subtilisin BPN' variants or their homologs into the active conformation.

[0237] The enzymes of this invention (subtilisin BPN' variants or homologues thereof) can be chemically or biochemically modified, for example, post-translational modified. For example, they can be glycosylated or contain modified amino acid residues. As mentioned above, they can also be modified by adding tags. Such modified polypeptides and proteins fall within the scope of the term "enzyme" in this invention.

[0238] To further illustrate the present invention and its advantages, the following specific embodiments are provided, which should be understood to be for illustrative and limiting purposes only.

[0239] Example The production of enzymes according to the present invention (use) Mutagenesis, cloning and expression The gene encoding the Bacillus subtilis protease BS149 (Ruan et al., 2008) was obtained from Philip N. Bryan (University of Maryland Institute for Biotechnology, 9600 Gudelsky Drive, Rockville, Maryland 20850). Using pUB110-based Escherichia coli-Bacillus subtilis strains containing the BS149 gene... E. coli-B. subtilis A shuttle vector was used, and mutagenesis was induced using a natural promoter or alternatively, an aprE promoter and an optional C-terminal his tag (pBE-S DNA, http: / / www.clontech.com / takara). The gene encoding the enzyme of this invention was constructed by introducing the mutants S221C and P225A into the BS149 gene using site-directed mutagenesis (Sambrook et al., 1989). All primers were designed using the Agilent Primer design tool (http: / / www.genomics.agilent.com). The constructed sequence was verified by DNA sequencing and then transformed into Bacillus subtilis GX4935.

[0240] To produce His-tagged BS149-DM, the gene encoding BS149-DM and its natural promoter sequence was cloned into the pBS42 shuttle vector (DSMZ, Germany) at the EcoRI / BamHI site. The ligation mixture was transformed into competent *E. coli*, and the transformants were plated on LB agar plates containing chloramphenicol (34 μg / mL). Plasmid pBS42-S5 was propagated in *E. coli*, isolated, and sequenced for verification. The sequence-verified plasmid was then used to transform *Bacillus subtilis* hosts.

[0241] The gene encoding the His-tagged BS149-DM was cloned into the pUB-110 Escherichia coli-Bacillus subtilis shuttle vector (pBES) using the MluI and BamHI sites. Figure 12 The polynucleotide sequence encoding the enzyme (peptide) of the present invention and the gene encoding the enzyme (BS149-DM) is shown in SEQUENCE ID NO 5. The corresponding amino acid sequences are numbered according to the subtilisin BPN' numbering scheme. Amino acids -107 to -1 contain the signal sequence, and the pre-sequence and initial sequence are cleaved after full maturation. Amino acids 1-275 contain the mature enzyme with full catalytic activity. To enable rapid and efficient purification, a C-terminal His tag is attached after amino acid 275, as shown in SEQ ID NO NO 5. As a result of the removal of calcium binding sites, BS149-DM contains a deletion of 9 amino acids compared to subtilisin BPN' which contains amino acids corresponding to L75, N76, N77, S78, I79, G80, V81, L82, and G83. To maintain the subtilisin BPN' numbering of BS149-DM, the numbering jumps from 74 to 83. In the shuttle vector, gene expression is controlled by the aprE promoter. The vector contains the pUB origin of replication for Bacillus and kanamycin resistance markers. It also contains the ColE1 origin of replication and an ampicillin resistance marker for maintenance in *E. coli*. The resulting plasmid pBES-BS149DMHIS was propagated in *E. coli* TOP10 and transformed into *Bacillus subtilis* GX4935 (ΔnprEΔaprE). Using pBES-BS149DMHIS as a template, mutagenesis was performed using the Quikchange method (Agilent Technologies). Alternatively, other site-directed mutagenesis methods known in the art can be used (Sambrook et al., 1989).

[0242] The gene for the Bacillus subtilis ligase (Abrahmsén et al., 1991) in the pJ201 cloning vector of DNA2.0 was ordered from DNA2.0 (https: / / www.dna20.com / ) and re-cloned into the Escherichia coli-Bacillus subtilis shuttle vector (pBS42 DSM 8748; pBS42-S5 from DSMZ). The pJ201 vector (DNA2.0) containing the Bacillus subtilis ligase and the pBS42 shuttle vector (DSMZ) were digested with EcoRI and BamHI (NEB). The linear shuttle vector and the Bacillus subtilis ligase insert (LigaFast, Promega) were isolated from the gel. The construct was transformed into Escherichia coli strain MM294 (DSMZ). The plasmid pBS42-S5 was propagated in E. coli, isolated, and validated by sequencing. Figure 13 The validated DNA was used for transformation of Bacillus subtilis DB104 or Bacillus subtilis GX4935. The Bacillus subtilis GX4935 strain exhibited reduced extracellular proteolytic activity (Kawamura and Doi 1984; Fahnestock and Fisher 1987). As reported by Abrahmsén et al. 1991, it was not necessary to add wild-type Bacillus subtilis protease to promote the production of the mature form.

[0243] Except for the Bacillus subtilis ligase in Example 23 and the His-tagged BS149-DM, enzymes were prepared using a C-terminal His tag in all experiments.

[0244] Production and purification of BPN' variant of subtilisin: Transformants from the pBS42 shuttle vector were selected and cultured at 37°C for 16 hours on LB agar plates containing 10 μg / mL chloramphenicol. Transformants were then picked and inoculated into 5 mL LB broth containing 10 μg / mL chloramphenicol. After incubation at 37°C for 16 hours, 1% (v / v) of the culture was inoculated into 1 L terrific broth (12 g / L tryptone, 24 g / L yeast extract, 0.4% (v / v) glycerol, 17 mM KH2PO4 and 72 mM K2HPO4, 50 mg / L Trp, 50 mg / L Lys, 50 mg / L Met). The culture was incubated at 37°C with vigorous shaking for another 48 hours. After 48 hours of expression, cells were isolated from the medium by centrifugation at 6,000 g for 20 minutes at 4°C. Subsequently, 5 g CaCl2 was added to the medium to adjust the pH to 7.5. The precipitate was precipitated by centrifugation at 6,000g for 20 minutes at 4°C. Ammonium sulfate was added to the supernatant to a final concentration of 45% (w / v) to precipitate the enzyme. The precipitated enzyme was collected by centrifugation at 8,000g for 40 minutes at 4°C. The precipitate was washed with 80% acetone and resuspended in 15 mL of water. The protein sample was desalted using a HiPrep 26 / 10 desalting column (GE Healthcare) in buffer (20 mM N-tris(hydroxymethyl)methylglycine, 1 mM CaCl2, pH 7.5). The desalted protein was loaded onto a HiTrap Q HP column (GE Healthcare). The flow-through containing the enzyme was collected and concentrated. Protein purity was analyzed by SDS-PAGE, and enzyme concentration was determined by measuring absorbance at 280 nm (Stoscheck, CM. Quantitative of Protein, Methods in Enzymology 182:50-69, 1990), for example, by a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc.). The specific extinction coefficients can be calculated at http: / / web.expasy.org / protparam / , according to Gasteiger E., Hoogland C., Gattiker A., ​​Duvaaud S., Wilkins MR, Appel RD, Bairoch A. Protein identification and analysis tools on the ExPASy server; (in) John M. Walker (ed.): The Proteomics Protocols Handbook, Humana Press (2005), pp. 571-607. Purity of approximately 90% or higher is feasible.The obtained enzyme was provided at a concentration of approximately 2 mg / mL in an aqueous solution of 20 mM N-tris(hydroxymethyl)methylglycine, 1 mM CaCl2, and pH 7.5. This enzyme solution was used as is for the condensation and cyclization of (oligo)peptide fragments.

[0245] Production and purification of the His-tagged synthetic subtilisin BPN' variant: Single colonies of *Bacillus subtilis* containing plasmids with the gene of the *Bacillus subtilis* protease variant of interest were inoculated in 5 mL LB broth containing kanamycin (10 μg / mL) at 37°C in a shaking incubator. 0.6 mL of overnight culture was added to 30 mL Terrific broth supplemented with antibiotics (10 μg / mL kanamycin) and amino acids (100 mg / L Trp, 100 mg / L Met, and 100 mg / L Lys). Cells were grown at 37°C in a shaking incubator (200 rpm) for 48 h. Cells were harvested by centrifugation (15 min, 4000 rpm, 4°C). The culture medium (30 mL) was decanted in two centrifugation steps (15 min, 4000 rpm, 4°C) and concentrated on an Amicon centrifuge (15 mL, 10 kDa MW cutoff). The concentrated culture medium (0.5 ml) was then replaced with buffer A (25 mM N-tris(hydroxymethyl)methylglycine, pH 7.5, 0.5 M NaCl, 20 mM imidazole) in three wash / concentration steps (14 ml buffer A, 10 min, 4000 rpm, 4 °C). For His-tag purification, Talon resin (2.5 ml, Clonetech) was added to a plastic column. The resin was washed with 5 mL of MilliQ water and equilibrated with 5 mL of buffer A. The crude enzyme was loaded onto the column and washed with 5 mL of buffer A. The enzyme was eluted with 5 mL of buffer B (25 mM N-tris(hydroxymethyl)methylglycine, pH 7.5, 0.5 M NaCl, 200 mM imidazole). The eluent was concentrated on an Amicon centrifuge unit (5 ml, 10 kDa MW cutoff) by centrifugation (15 min, 4000 rpm, 4 °C), and the buffer was replaced with 25 mM N-tris(hydroxymethyl)methylglycine, pH 7.5 (5 ml buffer, 10 min, 4000 rpm, 4 °C) during the three wash / concentration steps.

[0246] Purity and enzyme concentration were determined as described above. Purity was greater than 90%. The resulting aqueous solution containing approximately 2 mg / ml of the enzyme (25 mM N-tris(hydroxymethyl)methylglycine, pH 7.5) was used for (oligo)peptide fragment condensation and cyclization.

[0247] References Abrahmsén, L, J Tom, J Burnier, KA Butcher, A Kossiakoff, and JA Wells. 1991. Engineering Subtilisin and Its Substrates for Efficient Ligation of Peptide Bonds in Aqueous Solution. Biochemistry 30 (17) (April 30): 4151–9. http: / / www.ncbi.nlm.nih.gov / pubmed / 2021606. Fahnestock SR, Fisher KE: Expression of thestaphylococcal protein A gene in Bacillus subtilis by gene fusions utilizing the promoter from a Bacillus amyloliquefaciens alpha-amylase gene. J Bacteriol. Mar 1986;165(3):796-804 Kawamura, Fujio, and Roy H. Doi. Construction of a Bacillus subtilis double mutant deficient inextracellular alkaline and neutral proteases. J Bacteriol. Oct 1984;160(1):442-4 Ruan, Biao, Viktoriya London, Kathryn E Fisher, D Travis Gallagher, and Philip N Bryan. Engineering substrate preference in subtilisin: structural and kinetic analysis of a specificity mutant. Biochemistry. 2008, 24 Jun;47(25):6628-36. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Wells, James A, Eugenio Ferrari, Dennis J Henner, David A Estell, and Ellson Y Chen. Cloning, sequencing, and secretion of Bacillus amyloliquefaciens subtilisinin Bacillus subtilis. Nucleic Acids Res. Nov 25, 1983;11(22):7911-25. Enzymatic fragment condensation and cyclization examples Materials and methods Unless otherwise specified, chemicals obtained from commercial sources can be used without further purification. Analytical HPLC was performed at 40 °C on an HP1090 liquid chromatograph using a reversed-phase column (Phenomenex, C18, 5 μm particle size, 150 × 4.6 mm). UV detection was performed at 220 nm using a UV-VIS 204 linear spectrometer. The gradient program was: a linear gradient of 5% to 98% eluent B for 0–25 min and 5% eluent B (eluent A: 0.5 mL / L methanesulfonic acid (MSA) in H₂O, eluent B: 0.5 mL / L MSA in acetonitrile) for 25.1–30 min. The flow rate was 1 mL / min from 0–25.1 min, 2 mL / min from 25.2–29.8 min, then back to 1 mL / min until stopping at 30 min. The injection volume was 20 μL. Preparative HPLC was performed using a Varian PrepStar system with a solid-phase column (Pursuit XRs, C18, 10 μm particle size, 500 × 41.4 mm). LC-MS was performed on an Agilent 1200 series HPLC system using a reversed-phase column (Phenomenex, C18, 5 μm particle size, 150 × 4.6 mm) at 40 °C. UV detection and gradient programs were as described for analytical HPLC. Molecular weights were determined using an Agilent 6130 quadrupole LC / MS system.

[0248] Option 1: Synthesize N-Fmoc-protected (oligo)peptide-OCam ester as follows: One gram of Rink resin (4-((2,4-dimethoxyphenyl)(Fmocamino)methyl)phenoxyalkyl linker, loading 0.64 mmol / g) was washed with dichloromethane (DCM, 2 × 2 min, 10 mL) and 1-methyl-2-pyrrolidone (NMP, 2 × 2 min, 10 mL) and Fmoc deprotection was performed using piperidine / NMP (1 / 4, v / v, 2 × 8 min, 10 mL). After washing with NMP (2 × 2 min, 10 mL), DCM (2 × 2 min, 10 mL), and NMP (2 × 2 min, 10 mL), iodoacetic acid (4 equivalents) was coupled to the resin using HOAt (4 equivalents) and DCC (4 equivalents) in DCM (45 min, 10 mL). After washing with NMP (2 × 2 min, 10 mL), DCM (2 × 2 min, 10 mL), and THF (2 × 2 min, 10 mL), the resin was loaded with 10 equivalents of DiPEA and 4 equivalents of Fmoc-Xxx-OH protected with Fmoc in DMF / THF (1 / 1, v / v, 10 mL) for 20 hours at 50 °C. In this and other parts of this disclosure, “Xxx” represents an amino acid (the variant shown in the figures below).

[0249] After washing with DMF (2×2 min, 10 mL), DCM (2×2 min, 10 mL), and NMP (2×2 min, 10 mL), the peptide was extended following the standard SPPS protocol (Weng C. Chan and Peter White, OUP Oxford, 2000). Resin cleavage and side-chain deprotection were performed for 120 min using a mixture of trifluoroacetic acid (TFA), triisopropylsilane (TIS), and water (95 / 2.5 / 2.5, v / v / v, 15 mL). The crude peptide was precipitated using methyl tert-butyl ether (MTBE) / n-heptane (1 / 1, v / v, 50 mL). The precipitated peptide was collected by centrifugation, washed twice with MTBE / n-heptane (1 / 1, v / v, 50 mL), and then freeze-dried in acetonitrile / water (1 / 1, v / v, 50 mL).

[0250] Option 2: Synthesize the N-Fmoc-protected (oligo)peptide -OCam-Xxx-NH as described below. 2 ester: 1 g of Rink resin was washed with DCM (2 x 2 min, 10 mL) and NMP (2 x 2 min, 10 mL) and then Fmoc deprotected with piperidine / NMP (1 / 4, v / v, 2 x 8 min, 10 mL). After washing with NMP (2 x 2 min, 10 mL), DCM (2 x 2 min, 10 mL), and NMP (2 x 2 min, 10 mL), Fmoc-Xxx-OH (4 equivalents) was coupled to the resin with HBTU (4 equivalents), HOBt (4 equivalents), and DiPEA (8 equivalents) in NMP (45 min, 10 mL). After washing with NMP (2 x 2 min, 10 mL), DCM (2 x 2 min, 10 mL), and NMP (2 x 2 min, 10 mL), the resin was then Fmoc-deprotected with piperidine / NMP (1 / 4, v / v, 2 x 8 min, 10 mL). After washing with NMP (2×2 min, 10 mL), DCM (2×2 min, 10 mL), and NMP (2×2 min, 10 mL), iodoacetic acid (4 equivalents) was coupled with DCC (4 equivalents) and HOAt (4 equivalents) in DCM (45 min, 10 mL). After washing with NMP (2×2 min, 10 mL), DCM (2×2 min, 10 mL), and THF (2×2 min, 10 mL), Fmoc-protected amino acids were coupled with 10 equivalents of DiPEA and 4 equivalents of Fmoc-Xxx-OH in DMF / THF (1 / 1, v / v, 10 mL) at 50 °C for 20 h. After washing with DMF (2×2 min, 10 mL), DCM (2×2 min, 10 mL), and NMP (2×2 min, 10 mL), the peptide was extended following the standard SPPS protocol (Weng C. Chan and Peter White, OUP Oxford, 2000). Resin cleavage and side chain deprotection were performed for 120 minutes using a mixture of TFA / TIS / water (95 / 2.5 / 2.5, v / v / v, 15 mL). Crude peptides were precipitated using MTBE / n-heptane (1 / 1, v / v, 50 mL). The precipitated peptides were collected by centrifugation, washed twice with MTBE / n-heptane (1 / 1, v / v, 50 mL), and then freeze-dried in acetonitrile / water (1 / 1, v / v, 50 mL).

[0251] Option 3: Synthesize the N-Fmoc-protected (oligo)peptide-OCam-Xxx-OH ester as follows: 1 g of triphenylmethyl resin (2-chlorotriphenylmethyl linker, loading 1.0 mmol / g) was washed with DCM (2 x 2 min, 10 mL) and Fmoc-Xxx-OH (2 equivalents) was coupled to the resin using DiPEA (5 equivalents) in DCM (30 min, 10 mL). After washing with DMF (2 x 2 min, 10 mL), unreacted chlorotriphenylmethyl groups were capped with DCM / MeOH / DiPEA (80 / 15 / 5, v / v / v, 2 x 10 min, 10 mL). The resin was washed with NMP (2 x 2 min, 10 mL), DCM (2 x 2 min, 10 mL), and NMP (2 x 2 min, 10 mL), and then Fmoc-deprotected with piperidine / NMP (1 / 4, v / v, 2 x 8 min, 10 mL). After washing with NMP (2×2 min, 10 mL), DCM (2×2 min, 10 mL), and NMP (2×2 min, 10 mL), iodoacetic acid (4 equivalents) was coupled with DCC (4 equivalents) and HOAt (4 equivalents) in DCM (45 min, 10 mL). After washing with NMP (2×2 min, 10 mL), DCM (2×2 min, 10 mL), and THF (2×2 min, 10 mL), Fmoc-protected amino acids were coupled with 10 equivalents of DiPEA and 4 equivalents of Fmoc-Xxx-OH in DMF / THF (1 / 1, v / v, 10 mL) at 50 °C for 20 h. After washing with DMF (2×2 min, 10 mL), DCM (2×2 min, 10 mL), and NMP (2×2 min, 10 mL), the peptide was extended following the standard SPPS protocol (Weng C. Chan and Peter White, OUP Oxford, 2000). Resin cleavage and side chain deprotection were performed for 120 minutes using a mixture of TFA / TIS / water (95 / 2.5 / 2.5, v / v / v, 15 mL). Crude peptides were precipitated using MTBE / n-heptane (1 / 1, v / v, 50 mL). The precipitated peptides were collected by centrifugation, washed twice with MTBE / n-heptane (1 / 1, v / v, 50 mL), and then freeze-dried in acetonitrile / water (1 / 1, v / v, 50 mL).

[0252] Option 4: Synthesize the C-terminal amide nucleophilic group of the (oligo)peptide as follows: 1 g of Rink resin (4-((2,4-dimethoxyphenyl)(Fmocamino)methyl)phenoxyalkyl linker, loading 0.64 mmol / g) was washed with DCM (2 × 2 min, 10 mL) and NMP (2 × 2 min, 10 mL) and Fmoc deprotection was performed using piperidine / NMP (1 / 4, v / v, 2 × 8 min, 10 mL). The peptide was extended according to the standard SPPS protocol (Weng C. Chan and Peter White, OUP Oxford, 2000). Resin cleavage and side-chain deprotection were performed for 120 min using a mixture of TFA / TIS / water (95 / 2.5 / 2.5, v / v / v, 15 mL). The crude peptide was precipitated using MTBE / n-heptane (1 / 1, v / v, 50 mL). The precipitated peptides were collected by centrifugation, washed twice with MTBE / n-heptane (1 / 1, v / v, 50 mL), and then freeze-dried in acetonitrile / water (1 / 1, v / v, 50 mL).

[0253] Option 5: Synthesize N-acetyl-protected (oligo)peptide activated esters as follows: After SPPS of the desired sequence according to any of protocols 1-3, the resin-bound peptide was Fmoc-deprotected using piperidine / NMP (1 / 4, v / v, 2 x 8 min, 10 mL). The resin was washed with NMP (2 x 2 min, 10 mL), DCM (2 x 2 min, 10 mL), and NMP (2 x 2 min, 10 mL), and the N-terminal amine of the peptide was acetylated using a mixture of Ac2O (10 v / v), DiPEA (5 v / v), and HOBt (0.2 w / v) in NMP (2 x 10 min, 10 mL). The resin was washed with NMP (3 x 2 min, 10 mL) and DCM (3 x 2 min, 10 mL). The resin was cleaved and the side chains were deprotected for 120 min using a mixture of TFA / TIS / water (95 / 2.5 / 2.5, v / v / v, 15 mL). The crude peptide was precipitated using MTBE / n-heptane (1 / 1, v / v, 50 mL). The precipitated peptide was collected by centrifugation, washed twice with MTBE / n-heptane (1 / 1, v / v, 50 mL), and then freeze-dried in acetonitrile / water (1 / 1, v / v, 50 mL).

[0254] Option 6: Synthesize the C-terminal acid of the (oligo)peptide as follows: 1 g of triphenylmethyl resin (2-chlorotriphenylmethyl linker, loading 1.0 mmol / g) was washed with DCM (2 x 2 min, 10 mL) and Fmoc-Xxx-OH (2 equivalents) was coupled to the resin with DiPEA (5 equivalents) in DCM (30 min, 10 mL). After washing with DMF (2 x 2 min, 10 mL), unreacted chlorotriphenylmethyl groups were capped with DCM / MeOH / DiPEA (80 / 15 / 5, v / v / v, 2 x 10 min, 10 mL). The resin was washed with DMF (2 x 2 min, 10 mL), DCM (2 x 2 min, 10 mL), and NMP (2 x 2 min, 10 mL) and the peptide was extended following the standard SPPS protocol (Weng C. Chan and Peter White, OUP Oxford, 2000). Resin cleavage and side chain deprotection were performed for 120 minutes using a mixture of TFA / TIS / water (95 / 2.5 / 2.5, v / v / v, 15 mL). Crude peptides were precipitated using MTBE / n-heptane (1 / 1, v / v, 50 mL). The precipitated peptides were collected by centrifugation, washed twice with MTBE / n-heptane (1 / 1, v / v, 50 mL), and then freeze-dried in acetonitrile / water (1 / 1, v / v, 50 mL).

[0255] Option 7: Synthesize partially protected (oligo)peptide fragments During SPPS of the peptide sequence in one of schemes 1-6, different (TFA-stabilized) protected amino acids are coupled at the desired positions, such as Fmoc-Asp(OcHex)-OH, Fmoc-Glu(OBn)-OH, or Fmoc-Lys(Alloc)-OH. Resin cleavage and side-chain deprotection are performed for 120 minutes using a mixture of TFA / TIS / water (95 / 2.5 / 2.5, v / v / v, 15 mL), except for TFA-stabilized cHex, Bn, or Alloc, which remain unaffected. The crude peptide is precipitated using MTBE / n-heptane (1 / 1, v / v, 50 mL). The precipitated peptide is collected by centrifugation, washed twice with MTBE / n-heptane (1 / 1, v / v, 50 mL), and then freeze-dried in acetonitrile / water (1 / 1, v / v, 50 mL).

[0256] Coupling Examples Note: Compared with SEQUENCE ID NO.2, the enzyme represented as BS149-DM (SEQ ID NO: 5) contains deletions and mutations of amino acids 75-83: Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, Y217L, N218S, S221C, P225A, T254A, and Q271E. Based on this disclosure, common knowledge, and optional limited-quantity routine (route) testing, those skilled in the art can restore one or more of the following mutations: Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, Y217L, N218S, T254A, and Q271E, or make different substitutions at one or more of the following positions: Q2, S3, P5, S9, I31, K43, M50, A73, E156, G166, G169, S188, Q206, N212, N218S, T254, and Q271, while still exhibiting significantly improved properties compared to Bacillus subtilis ligase (see, for example, Example 24).

[0257] Compared to SEQUENCE ID NO: 2, the enzyme represented as Bacillus subtilis ligase contains the mutants S221C and P225A.

[0258] The enzymes of the present invention used in Examples 1-23 have all the mutations of BS149-DM, as well as other optional mutations mentioned in the examples.

[0259] As shown below, enzymes with other mutations are prepared using the above techniques.

[0260] Example 1: Enzymatic oligopeptide fragment conjugation using different BS149-DM mutants: To test the activity and S / H ratio of different mutants, the following standard reaction was performed. 800 μL of phosphate buffer (100 mM, pH 8.0) was added to 100 μL of a tripeptide C-terminal amide stock solution (0.01 mmol H-Ala-Leu-Arg-NH2.2TFA in 300 μL water) and 100 μL of a pentapeptide C-terminal cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 μL water). 5.5 μg of enzyme was added to the mixture, and the reaction mixture was shaken at room temperature (150 rpm). After 30 minutes, 500 μL aliquots of the reaction mixture were withdrawn, quenched with 500 μL MSA / water (1 / 99, v / v), and analyzed by LC-MS. The product, the hydrolyzed pentapeptide C-terminal cam-ester, and the remaining pentapeptide C-terminal cam-ester peaks were integrated.

[0261] The activity of different BS149-DM mutants was defined as the sum of the amount of product and the amount of hydrolyzed pentapeptide C-terminal cam-ester within a specified reaction time, divided by the sum of the amount of product, the hydrolyzed pentapeptide C-terminal cam-ester, and the remaining cam-ester. The most active mutant was set at 100% (see [link to relevant documentation]). Figure 1A The S / H ratio for different BS149-DM mutants is defined as the amount of product over a specified time divided by the amount of hydrolyzed pentapeptide C-terminal cam-ester (see [reference]). Figure 1B ).

[0262] Unless otherwise specified, the activity and S / H ratio in other examples were determined in the same manner.

[0263] Conclusion: Clearly, BS149-DM exhibits twice the activity and an improved S / H ratio (1.8 vs. 0.9) compared to Bacillus subtilis ligase. The M222 position is crucial for the enzyme's S / H ratio. Excellent results were obtained using the M222G and M222P mutants of BS149-DM. All BS149-DM variants containing P4 bag mutations (positions Y104, I107, and L135) had S / H ratios comparable to BS149-DM. However, for certain mutations, the activity of BS149-DM variants was significantly improved. Mutations Y104S, I107V, L135D, L135N, and L135S yielded particularly good results. When combined with P4 bag mutations, BS149-DM variants with even higher activity were obtained, namely I107V + L135S and I107V + L135N. When the P4 bag mutation is combined with the P1' bag mutation, a very active BS149-DM variant with an increased S / H ratio compared to BS149-DM is obtained, for example, I107V + M222G.

[0264] Example 2: Enzymatic oligopeptide fragment conjugation using different BS149-DM + M222P + L217 mutants: To test the activity and S / H ratio of different mutants, the same reaction as described in Example 1 was performed. The activity of different BS149-DM + M222P + L217 mutants was defined as the total amount of product and hydrolyzed oligopeptide C-terminal cam-ester divided by the total amount of product, hydrolyzed oligopeptide C-terminus, cam-ester, and remaining cam-ester. The most active mutant was set at 100% (see [link to relevant documentation]). Figure 2A The S / H ratio of different BS149-DM+M222P+L217 mutants is defined as the amount of product divided by the amount of hydrolyzed C-terminal Cam-ester (see [link to relevant documentation]). Figure 2B ).

[0265] Conclusion: Clearly, all BS149-DM+M222P+L217 mutants exhibited improved S / H ratios and similar or improved activities compared to Bacillus subtilis ligases, with some showing increased activity compared to BS149-DM+M222P. Mutants L217N, L217T, L217E, L217I, L217V, and L217A yielded particularly good results. The L217 position proved important not only for activity and S / H ratio but also for substrate range, as described in Example 5.

[0266] Example 3: P4 bag substrate specificity plotting for different BS149-DM mutants containing P4 bag mutations (positions Y104, I107, and L135): To determine the P4 bag substrate specificity of different mutants, the following standard reaction was performed. 800 μL of phosphate-buffered saline (100 mM, pH 8.0) was added to 100 μL of a tripeptide C-terminal amide stock solution (0.01 mmol H-Ala-Leu-Arg-NH2.2TFA in 300 μL water) and 200 μL of a pentapeptide C-terminal cam-ester stock solution (0.01 mL Ac-Asp-Xxx-Ser-Lys-Leu-OCam.TFA in 1 mL Acn). All these peptides and esters were conjugated, distinguished by the amino acid at that position, such as... Figures 3A-3C As shown.

[0267] Add 5.5 μg of enzyme to the mixture and shake the reaction mixture at room temperature (150 rpm). After 30 minutes, withdraw 500 μL aliquots of the reaction mixture and quench with 550 μL MSA / water (1 / 99, v / v) and analyze by LC-MS. Integrate the peaks of the product, the hydrolyzed pentapeptide C-terminal cam-ester, and the remaining pentapeptide C-terminal cam-ester. Activity is defined as the amount of product divided by the total amount of product, hydrolyzed pentapeptide C-terminal cam-ester, and remaining pentapeptide C-terminal cam-ester within a specified reaction time. The most active substrate is set at 100%, see [link to relevant documentation]. Figure 3A -C.

[0268] Conclusion: From Figure 3A As can be seen from the -C pattern, the P4 substrate range of BS149-DM mutants with P4 mutations (positions Y104, I107, and / or L135) differs significantly from that of BS149-DM, potentially offering advantages for a wide range of peptide sequences. Several mutants exhibit a broader P4 substrate range than BS149-DM, particularly the mutations I107V, L135D, L135N, and L135S.

[0269] Example 4: P1' and P2' bag substrate specificity of different BS149-DM+M222 mutants: To determine the specificity of the P1' and P2' bag substrates for different mutants, the following two standard reactions were performed. 800 μL of phosphate buffer (100 mM, pH 8.0) was added to 100 μL of a tripeptide C-terminal amide stock solution (H-Xxx-Leu-Arg-NH2.2TFA in 300 μL of water for P1' and H-Ala-Xxx-Arg-NH2.2TFA for P2') and 100 μL of a pentapeptide C-terminal cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 μL of water). 5.5 μg of enzyme was added to the mixture, and the reaction mixture was shaken at room temperature (150 rpm). After 30 minutes, 500 μL aliquots of the reaction mixture were withdrawn, quenched with 550 μL MSA / water (1 / 99, v / v), and analyzed by LC-MS. The product, the hydrolyzed pentapeptide C-terminal cam-ester, and the remaining pentapeptide C-terminal cam-ester peaks were integrated. Activity was defined as the amount of product divided by the total amount of product, hydrolyzed pentapeptide C-terminal cam-ester, and remaining pentapeptide C-terminal cam-ester within a specified reaction time. The most active substrate was set to 100%. The P1' and P2' bag substrate specificities for different BS149-DM+M222 mutants are shown in [Figure / Table / Insert Table ... Figure 4A-D. Coupling to tryptophan at the P1' position was not determined due to overlap in the LC-MS peaks.

[0270] Conclusion: From Figure 4A -D it is clear that the P1' and P2' substrate scope of BS149-DM mutants with a P1' mutation at position M222 is significantly different from that of BS149-DM, which is favorable for various specific peptide sequences. Several mutants showed a broader P1' and P2' substrate scope than BS149-DM. This is particularly the case for mutations M222G and M222P.

[0271] Example 5: Mapping P1' and P2' pocket substrate specificity of different BS149-DM + M222P + L217 mutants: To determine the P1' and P2' pocket substrate specificity of different mutants, the same reactions and analyses were performed as described in Example 4. In Figure 5A -L shows the P1' and P2' pocket substrate specificity of different BS149-DM + M222P + L217 mutants.

[0272] Conclusion: From Figure 5A -L it can be seen that the P1' and P2' substrate scope of BS149-DM + M222P mutants with a P1' mutation at position L217 is significantly different from that of BS149-DM + M222P, which is favorable for various specific peptide sequences. Several mutants showed a broader P1' and P2' substrate scope than BS149-DM. This is particularly the case for mutations L217G and L217H. Several mutants showed significantly improved activity for certain specific substrates. For example, improved activity of Phe in the P1' pocket for mutants BS149-DM + M222P + L217N, E, G, Y, F or H. Mutant BS149-DM + M222P + L217H also showed greatly increased activity of Asn in the P1' pocket. Mutants BS149-DM + M222P + L217E and A have improved activity for Leu, Ile and Val in the P1' pocket. Mutants BS149-DM + M222P + L217T and S have improved activity for Asp in the P1' pocket.

[0273] Example 6: Mapping P1' pocket substrate specificity of different BS149-DM + M222G + L217 mutants: To determine the P1' pocket substrate specificity of different mutants, the same reactions and analyses were performed as described in Example 4. The P1' pocket substrate specificity of different BS149-DM + M222G + L217 mutants is shown in Figure 6A -F.

[0274] Conclusion: From Figure 6A As can be seen from the -F pattern, the P1' substrate range of the BS149-DM + M222G mutant with the P1' mutation at position L217 is significantly different from that of BS149-DM + M222G, potentially favoring a variety of specific peptide sequences. Several mutants exhibit a broader P1' substrate range than BS149-DM. This is particularly true for the L217G and L217F mutants. Several mutants show significantly improved activity for certain specific substrates. For example, the improved activity of Phe in the P1' pocket of the BS149-DM + M222G + L217N, E, G, Y, F, I, or H mutants. The BS149-DM + M222G + L217F mutant also shows a significantly increased activity for Asn in the P1' pocket. The BS149-DM + M222G + L217F, G, A, and Y mutants have improved activity for Leu, Ile, and Val in the P1' pocket. The mutant BS149-DM + M222G + L217R, with T and S exhibiting improved activity against Asp in the P1' bag.

[0275] Example 7: P1', P2', and P4 bag substrate specificities of the BS149-DM + M222G + I107V mutant: To determine the P1' and P2' bag-bottom specificities of BS149-DM+M222G+I107V, the same reactions and analyses were performed as described in Example 4. The P1' and P2' bag-bottom specificities of the BS149-DM+M222G+I107V mutant were shown in […]. Figure 7A and 7B To determine the P4 bag substrate specificity of BS149-DM + M222G + I107V, the same reactions and analyses were performed as described in Example 3. The P4 bag substrate specificity of the BS 7C-M + M222G + I107V mutant is shown in... Figure 7C .

[0276] Conclusion: From Figure 7A As can be seen from -C, the P1' and P2' substrate ranges, as well as the P4 substrate range, of the BS149-DM + M222G + I107V mutant are wider than those of the BS149-DM. Clearly, favorable mutations in the P1' and P2' bags (i.e., M222G) and the P4 bag (i.e., I107V) can bind successfully because the substrate width is comparable to that of the BS149-DM + M222G mutant, but the S / H ratio is significantly higher (see Example 1).

[0277] Example 8: Enzymatic coupling reaction of oligopeptides with different N-acetyl-protected C-terminal cam-esteracyl donors: The peptide ligation reaction was carried out at 25 °C in 100 mM Tricine buffer (pH 8.0) containing 15 μM BS149-DM, 10 mM C-terminal cam-ester of the peptide (Ac-Asp-Leu-Ser-Lys-Gln-OCam.TFA, Ac-Thr-Ser-Asp-Leu-Ser-Lys-Gln-OCam.TFA, Ac-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-OCam.TFA or Ac-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-OCam.TFA), and 15 mM C-terminal amide of the dipeptide (H-Ala-Phe-NH2). After 180 minutes, the reaction mixture was analyzed by LC-MS. The peaks of the product, the hydrolyzed C-terminal Cam-ester, and the remaining Cam-ester are integrated. The S / H ratio for different reactions is defined as the amount of product divided by the amount of hydrolyzed C-terminal Cam-ester within a specified reaction time.

[0278] Table 1: Coupling of different acyl donors with H-Ala-Phe-NH2

[0279] Conclusion: Oligopeptidyl donors of varying lengths can be used. The S / H ratio increases with the length of the oligopeptidyl donor.

[0280] Example 9: Enzymatic coupling reaction using different C-terminal amide nucleophilic groups of oligopeptides: The peptide linkage reaction was carried out at 25 °C in a solution containing 15 μM BS149-DM, 1 mM C-terminal pentapeptide cam-ester (Ac-Phe-Ile-Glu-Trp-Leu-OCam), and 3 mM peptide amine nucleophiles (H-Ala-Phe-NH2, H-Ala-Phe-Ala-NH2, or H-Ala-Phe-Ala-Tyr-NH2). After 60 minutes, the reaction mixture was analyzed by LC-MS. The peaks of the product, the hydrolyzed C-terminal pentapeptide cam-ester, and the remaining C-terminal pentapeptide cam-ester were integrated. The S / H ratio for different reactions was defined as the amount of product divided by the amount of hydrolyzed C-terminal pentapeptide cam-ester within a specified reaction time.

[0281] Table 2: Coupling of different oligopeptide nucleophilic groups with Ac-Phe-Ile-Glu-Trp-Leu-OCam

[0282] Conclusion: Nucleophilic groups of oligopeptides of varying lengths can be used. The S / H ratio increases with the length of the oligopeptide nucleophilic group.

[0283] Example 10: Effect of pH on the S / H ratio of the BS149-DM + M222G mutant: To examine the effect of pH on the S / H ratio of the BS149-DM+ M222G mutant, the following standard reaction was performed. 800 μL of phosphate buffer (1 M, pH 7.0–8.8), N-tris(hydroxymethyl)methylglycine buffer (1 M, pH 7.9–8.9), or carbonate buffer (1 M, pH 9.2–10.6) was added to 100 μL of a tripeptide C-terminal amide stock solution (0.01 mmol H-Ala-Leu-Arg-NH2.2TFA in 300 μL water) and 100 μL of a pentapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1.2 mL water). 5.5 μg of enzyme was added to this mixture, and the reaction mixture was shaken at room temperature (150 rpm). After 30 minutes, a 550 μL aliquot of the reaction mixture was extracted, quenched with 50 μL MSA, and analyzed by LC-MS. The peaks of the product, the hydrolyzed pentapeptide C-terminal cam-ester, and the remaining pentapeptide C-terminal cam-ester were integrated, and the S / H ratio was defined as the amount of product divided by the amount of hydrolyzed pentapeptide C-terminal cam-ester within the specified reaction time. (See [reference]). Figure 8 .

[0284] Conclusion: The S / H ratio of BS149-DM + M222G depends on pH, with a clear optimal value between pH 8 and pH 9. However, lower or higher pH values ​​can also be used, depending on the solubility and stability of the oligopeptide.

[0285] Example 11: Effect of acyl donor and nucleophilic group concentrations on the S / H ratio of BS149-DM + M222G: To examine the effect of substrate concentration on the S / H ratio of the mutant BS149-DM + M222G, the following reaction was performed. C-terminal tripeptide amide (12.9 mg H-Glu-Leu-Arg-NH2.2TFA or 11.7 mg H-Ala-Leu-Arg-NH2.2TFA) and C-terminal pentapeptide cam-ester (4.2 mg Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA) were prepared in 150 μL of water. The mixture was adjusted to neutral pH with 5.1 μL NaOH (32 wt% in water). To prepare reaction mixtures with different substrate concentrations, 10 μL of one of the above stock solutions was diluted with 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, and 10000 μL phosphate buffer (1 M, pH 8.5). Add 11 μg BS149-DM + M222G to these reaction mixtures and shake the mixture at room temperature (150 rpm). After 30 minutes, withdraw 550 μL aliquots of the reaction mixture, quench with 50 μL MSA, and analyze by LC-MS. Integrate the peaks of the product, the hydrolyzed pentapeptide C-terminal cam-ester, and the remaining pentapeptide C-terminal cam-ester. Define the S / H ratio as the amount of product divided by the amount of hydrolyzed pentapeptide C-terminal cam-ester within the specified reaction time. See [reference needed]. Figure 9A And B.

[0286] Conclusion: The S / H ratio depends on the substrate concentration. Based on the nucleophilic group's affinity for the enzyme and the solubility and stability of the oligopeptide, each individual substrate has an optimal substrate concentration.

[0287] Example 12: Effect of acyl donor on the S / H ratio of BS149-DM + M222G: To examine the effect of acyl donor administration on the S / H ratio of the mutant BS149-DM + M222G, the following two reactions were performed. 100 μL of a tripeptide C-terminal amide stock solution (0.01 mmol H-Ala-Leu-Arg-NH2.2TFA in 300 μL water) and 5.5 μg of BS149-DM + M222G were added to two 800 µL phosphate buffer (100 mM, pH 8.0). 100 μL of a pentapeptide C-terminal Cam-ester stock solution (0.01 mL Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1.2 mL water) was added to one of the mixtures, and the reaction mixture was shaken at room temperature (150 rpm). To another mixture, 100 μL of pentapeptide C-terminal cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1.2 mL water) was added at a fraction of 3.5 μL per minute while the reaction mixture was shaken at room temperature (150 rpm). After 30 minutes, 550 μL aliquots of both reaction mixtures were extracted and quenched with 50 μL MSA and analyzed by LC-MS. For both reactions, the conversion of the pentapeptide C-terminal cam-ester starting material was 100%. The product and hydrolyzed pentapeptide C-terminal cam-ester peaks were integrated, and the S / H ratio was defined as the amount of product divided by the amount of hydrolyzed pentapeptide C-terminal cam-ester within a specified reaction time. The S / H ratio for the reaction with all acyl donors added at t = 0 was 2.45, and the S / H ratio for the reaction with acyl donors added per minute was 2.73.

[0288] Conclusion: Timely administration of the C-terminal cam-ester of oligopeptide can improve the S / H ratio.

[0289] Example 13: Cyclation of C-terminal Cam-esters of oligopeptides using different enzymes: The following experiments were conducted to determine the S / H ratio of cyclization of the C-terminal Cam-ester of oligopeptides by Bacillus subtilis ligase, BS149-DM, and BS149-DM + M222G.

[0290] 800 μL of phosphate buffer (100 mM, pH 8.0) was added to 100 μL of a stock solution (1 mL water, 0.01 mmol H-Ala-Cys-Lys-Asn-Gly-Gln-Thr-Asn-Cys-Tyr-Gln-Ser-Tyr-OCam.2TFA) containing 5 mg / mL dithiothreitol and an N-terminal free amine (0.01 mmol H-Ala-Cys-Lys-Asn-Gly). 5.5 μg of enzyme was added to the mixture, and the reaction mixture was shaken at room temperature (150 rpm). After 30 minutes, 500 μL aliquots of the reaction mixture were withdrawn, quenched with 550 μL MSA / water (1 / 99, v / v), and analyzed by LC-MS. The peaks of the product, the hydrolyzed C-terminal cam-ester, and the remaining cam-ester starting material were integrated. The S / H ratio of different enzymes was defined as the amount of product divided by the amount of hydrolyzed C-terminal cam-ester within a specified reaction time. See [reference needed]. Figure 10 .

[0291] Conclusion: Clearly, for peptide cyclization, BS149-DM exhibits an improved S / H ratio compared to Bacillus subtilis ligase. The BS-149-DM + M222G mutant even has an even higher S / H ratio.

[0292] Example 14: Effect of pH on S / H ratio during C-terminal cyclization of oligopeptides using BS149-DM + M222G: To determine the effect of pH on the S / H ratio of BS149-DM + M222G during the C-terminal cyclization of the oligopeptide, the following standard reaction was performed. 800 μL of phosphate buffer (1 M, pH 5, 6, 7, 8, and 9) was added to 100 μL of a stock solution containing 5 mg / mL dithiothreitol for the C-terminal cyclization of a decatheptide with an N-terminal free amine (0.01 mmol H-Ala-Cys-Lys-Asn-Gly-Gln-Thr-Asn-Cys-Tyr-Gln-Ser-Tyr-OCam.2TFA in 1 mL of water). 5.5 μg of BS149-DM + M222G was added to this mixture, and the reaction mixture was shaken at room temperature (150 rpm). After 30 minutes, 550 μL aliquots of the reaction mixture were withdrawn, quenched with 50 μL of MSA, and analyzed by LC-MS. The peaks of the product, the hydrolyzed C-terminal cam-ester, and the remaining C-terminal cam-ester starting material were integrated, and the S / H ratio was defined as the amount of product divided by the amount of hydrolyzed C-terminal cam-ester within a specified reaction time. (See [reference]). Figure 11 .

[0293] Conclusion: The S / H ratio of BS149-DM + M222G used for enzymatic oligopeptide cyclization is pH-dependent, although it is less than the degree of condensation of enzymatic oligopeptide fragments.

[0294] Example 15: Condensation with fragments of oligopeptides longer than 10 amino acids: To examine the feasibility of enzymatic cleavage condensation with longer oligopeptides in aqueous solution, the following standard reaction was performed. 800 μL of phosphate buffer (1M, pH 8.0) was added to 100 μL of decapeptide C-terminal amide stock solution (0.01 mmol H-Ala-Leu-Met-Lys-Tyr-Asn-Ser-Thr-Glu-Val-NH2.2TFA in 300 μL water) and 200 μL of tridecapeptide C-terminal cam-ester stock solution (0.01 mmol Fmoc-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-OCam.2TFA in 1 mL water + 1 mL DMF). 5.5 μg of BS149-DM + M222G was added to the mixture, and the reaction mixture was shaken at room temperature (150 rpm). After 30 minutes, a 550 μL aliquot of the reaction mixture was withdrawn, quenched with 50 μL MSA, and analyzed by LC-MS. The peaks of the product, the hydrolyzed C-terminal Cam-ester, and the remaining C-terminal Cam-ester were integrated. The amount of product (Fmoc-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Ala-Leu-Met-Lys-Tyr-Asn-Ser-Thr-Glu-Val-NH2) was 68% of the area within the specified reaction time.

[0295] Conclusion: It is feasible to use fragment condensation of longer peptides.

[0296] Example 16: Fragment condensation using oligopeptides without N- or C-terminal protecting groups: To examine the feasibility of condensing enzyme fragments without significant byproduct formation and without N- or C-terminal protecting groups, the following standard reaction was performed. 800 μL of phosphate buffer (1 M, pH 8.0) was added to 100 μL of a C-terminal carboxylic acid stock solution of a tripeptide (0.01 mmol H-Ala-Leu-Arg-OH.2TFA in 300 μL water) and 100 μL of a C-terminal cam-ester stock solution of an N-terminal free amine pentapeptide (0.01 mmol H-His-Ala-Glu-Gly-Thr-OCam.TFA in 1.2 mL water). 5.5 μg of BS149-DM + M222G was added to the mixture, and the reaction mixture was shaken at room temperature (150 rpm). After 30 minutes, 550 μL aliquots of the reaction mixture were withdrawn, quenched with 50 μL of MSA, and analyzed by LC-MS. The product, the hydrolyzed pentapeptide C-terminal Cam-ester, and the remaining pentapeptide C-terminal Cam-ester peak were integrated. Within the specified reaction time, the amount of product (H-His-Ala-Glu-Gly-Thr-Ala-Leu-Arg-OH) was 74% by area. No byproducts were observed, indicating that no side reactions occurred at the C-terminal carboxylic acid functional group of H-Ala-Leu-Arg-OH.2TFA, nor at the N-terminal amine functional group of H-His-Ala-Glu-Gly-Thr-OCam.

[0297] Conclusion: Some oligopeptide sequences can be successfully enzymatically ligated without the use of N- or C-terminal protecting groups.

[0298] Example 17: Fragment condensation using C-terminal Cam-Xxx-NH2 or Cam-Xxx-OH esters of oligopeptides: To test whether the enzymatic fragment condensation of Cam-Xxx-NH2 or Cam-Xxx-OH esters is feasible, the following standard reaction was performed. Add 800 µL of phosphate buffer (1M, pH 8.0) to a mixture of 100 µL of tripeptide C-terminal amide stock solution (0.01 mmol H-Ala-Leu-Arg-NH2.2TFA in 300 µL of water) and 100 µL of pentapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-OH.TFA, Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-NH2.TFA, Ac-Asp-Phe-Ser-Lys-Leu-OCam-Lys-NH2.2TFA or Ac-Asp-Phe-Ser-Lys-Leu-OCam-Glu-NH2.TFA). Add 5.5 μg of BS149-DM + M222G to each of the four mixtures, and shake the reaction mixture at room temperature (150 rpm). After 30 minutes, extract 550 μL aliquots of the reaction mixture, quench with 50 μL MSA, and analyze by LC-MS. Integrate the peaks of the product, the hydrolyzed pentapeptide C-terminal cam-ester, and the remaining tetrapeptide C-terminal cam-ester. The amounts of the product (Ac-Asp-Phe-Ser-Lys-Leu-Ala-Leu-Arg-NH2) within the specified reaction time are 86 area % for Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-OH, 83 area % for Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-NH2, 78 area % for Ac-Asp-Phe-Ser-Lys-Leu-OCam-Lys-NH2, and 83 area % for Ac-Asp-Phe-Ser-Lys-Leu-OCam-Glu-NH2.

[0299] Conclusion: This example demonstrates that Cam-Xxx-NH2 and Cam-Xxx-OH esters can be successfully used for enzymatic oligopeptide fragment condensation.

[0300] Example 18: Fragment condensation using C-terminal oligopeptide thioester and BS149DM + I107V + M222G: To verify the feasibility of enzymatic oligopeptide fragment condensation using C-terminal thioesters, the following standard reaction was performed. 1 mL of N-tris(hydroxymethyl)methylglycine buffer (100 mM, pH 7.5) containing 2.5 mM pentapeptide C-terminal thioester (Suc-Ala-Ala-Pro-Phe-SBzl), 25 mM dipeptide C-terminal amide (H-Gly-Phe-NH2), and 5 µg BS149-DM + L107V + M222G was shaken (150 rpm) at 25°C. After 30 minutes, 550 μL aliquots of the reaction mixture were withdrawn, quenched with 50 μL MSA, and analyzed by LC-MS. The product, the hydrolyzed tetrapeptide C-terminal thioester, and the remaining tetrapeptide C-terminal thioester peak were integrated. The amount of product (Suc-Ala-Ala-Pro-Phe-Gly-Phe-NH2) within the specified reaction time was 85% of the area.

[0301] Conclusion: This example demonstrates that C-terminal thioesters of oligopeptides can be successfully used for enzymatic oligopeptide fragment condensation.

[0302] Example 19: Fragment condensation using C-terminal oligopeptide alkyl ester and BS149DM + I107V + M222G: To verify the feasibility of enzymatic oligopeptide fragment condensation using C-terminal alkyl esters, the following standard reaction was performed. The reaction mixture was shaken (150 rpm) at 25°C in 1 mL of N-tris(hydroxymethyl)methylglycine buffer (100 mM, pH 7.5) containing 2.5 mM pentapeptide C-terminal alkyl ester (Ac-Asp-Phe-Ser-Lys-Leu-OTFE (TFE = 2,2,2-trifluoroethyl)), 25 mM tripeptide C-terminal amide (H-Ala-Leu-Arg-NH2), and 5 µg BS149-DM + L107V + M222G. After 30 minutes, 550 μL aliquots of the reaction mixture were withdrawn, quenched with 50 μL MSA, and analyzed by LC-MS. The product, the hydrolyzed pentapeptide C-terminal alkyl ester, and the remaining pentapeptide alkyl ester peaks were integrated. Within the specified reaction time, the amount of product (Ac-Asp-Phe-Ser-Lys-Leu-Ala-Leu-Arg-NH2) was 55% of the area.

[0303] Conclusion: This example demonstrates that C-terminal alkyl esters of oligopeptides can be successfully used for enzymatic oligopeptide fragment condensation.

[0304] Example 20: Condensation of enzymatic oligopeptide fragments with partial side chain protection: To demonstrate the benefit of partial protection of the P1' side chain, the following reaction was performed. 800 μL of phosphate buffer (100 mM, pH 8.0) was added to a mixture of 100 μL of a tripeptide C-terminal amide stock solution (0.01 mmol H-Asp-Leu-Arg-NH2.2TFA or 0.01 mmol H-Asp(OcHex)-Leu-Arg-NH2.2TFA in 300 μL water) and 100 μL of a pentapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 μL water). 5.5 μg of BS149-DM + M222G was added to this mixture, and the reaction mixture was shaken at room temperature (150 rpm). After 30 minutes, 500 μL aliquots of the reaction mixture were withdrawn and quenched with 500 μL MSA / water (1 / 99, v / v) and analyzed by LC-MS. The product, the hydrolyzed pentapeptide C-terminal cam-ester, and the remaining pentapeptide cam-ester peaks were integrated. The amount of product with unprotected substrate (H-Asp-Leu-Arg-NH2) within the specified reaction time was 18% by area, and the amount of product with partially protected substrate (H-Asp(OcHex)-Leu-Arg-NH2) within the specified reaction time was 73% by area.

[0305] To demonstrate the benefit of partial P2' side chain protection, the following reaction was performed. 800 μL of phosphate buffer (100 mM, pH 8.0) was added to a mixture of 100 μL of a tripeptide C-terminal amide stock solution (0.01 mmol H-Asp-Glu(OBn)-Arg-NH2.2TFA or 0.01 mmol H-Asp-Glu-Arg-NH2.2TFA in 300 μL water) and 100 μL of a pentapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 μL water). 5.5 μg of BS149-DM + M222G was added to this mixture, and the reaction mixture was shaken at room temperature (150 rpm). After 30 minutes, 500 μL aliquots of the reaction mixture were withdrawn and quenched with 500 μL MSA / water (1 / 99, v / v) and analyzed by LC-MS. The product, the hydrolyzed pentapeptide C-terminal cam-ester, and the remaining pentapeptide cam-ester peaks were integrated. The amount of product using the unprotected substrate (H-Asp-Glu-Arg-NH2) within the specified reaction time was 15% by area, and the amount of product using the partially protected side-chain substrate (H-Asp-Glu(OBn)-Arg-NH2) within the specified reaction time was 58% by area.

[0306] To demonstrate the benefit of partial protection of the P1 side chain, the following reaction was performed. 800 μL of phosphate buffer (100 mM, pH 8.0) was added to 100 μL of a tripeptide C-terminal amide stock solution (0.01 mmol H-Asp-Leu-Arg-NH2.2TFA in 300 μL water) and 100 μL of a pentapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Leu-Lys-OCam.TFA or 0.01 mmol Ac-Asp-Phe-Ser-Leu-Lys(Alloc)-OCam in 1200 μL water). 5.5 μg of BS149-DM + M222G was added to the mixture, and the reaction mixture was shaken at room temperature (150 rpm). After 30 minutes, 500 μL aliquots of the reaction mixture were withdrawn and quenched with 500 μL MSA / water (1 / 99, v / v) and analyzed by LC-MS. The product, the hydrolyzed pentapeptide C-terminal cam-ester, and the remaining pentapeptide cam-ester peaks were integrated. The amount of product with unprotected substrate (Ac-Asp-Phe-Ser-Leu-Lys-OCam.TFA) within the specified reaction time was 5% by area, and the amount of product with partially protected substrate (Ac-Asp-Phe-Ser-Leu-Lys(Alloc)-OCam) within the specified reaction time was 84% ​​by area.

[0307] To demonstrate the benefit of partial P4 side-chain protection, the following reaction was performed. 800 μL of phosphate buffer (100 mM, pH 8.0) was added to 100 μL of a tripeptide C-terminal amide stock solution (0.01 mmol H-Ala-Leu-Arg-NH2.2TFA in 300 μL water) and 100 μL of a tetrapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Ser-Lys-Leu-OCam.TFA or 0.01 mmol Ac-Asp(OBn)-Ser-Lys-Leu-OCam.TFA in 1200 μL water). 5.5 μg of BS149-DM + M222G was added to the mixture, and the reaction mixture was shaken at room temperature (150 rpm). After 30 minutes, 500 μL aliquots of the reaction mixture were withdrawn and quenched with 500 μL MSA / water (1 / 99, v / v) and analyzed by LC-MS. The product, the hydrolyzed tetrapeptide C-terminal cam-ester, and the remaining tetrapeptide cam-ester peaks were integrated. The amount of product with unprotected substrate (mmolAc-Asp-Ser-Lys-Leu-OCam.TFA) within the specified reaction time was 32% of the area, and the amount of product with partially protected substrate (Ac-Asp(OBn)-Ser-Lys-Leu-OCam.TFA) within the specified reaction time was 78% of the area.

[0308] Conclusion: This example demonstrates that partial side-chain protection can improve the yield and / or reaction rate of enzymatic oligopeptide fragment condensation.

[0309] Example 21: Thermal stability of BS149-DM: The apparent melting temperatures of BS149-DM and Bacillus subtilis ligase were determined using a fluorescence-based thermostability assay. 20 μL of protein solution in buffer (20 mM N-tris(hydroxymethyl)methylglycine buffer, pH 7.5) and either metal ions (10 mM) or EDTA (10 mM) were mixed with 5 μL of 100-fold diluted Sypro Orange (Molecular Probes, Life Technologies, USA) dye in a thin-walled 96-well PCR plate. The plate was sealed with optical quality sealing tape and heated from 20 °C to 99 °C at a heating rate of 1.75 °C / min in a CFX 96 real-time PCR system (BioRad, Hercules, CA). Fluorescence changes were monitored using a charge-coupled device (CCD) camera. Excitation and emission wavelengths were 490 and 575 nm, respectively. The thermostability of purified BS149-DM was determined as described above. Thermal stability was also measured after adding different metal ions and chelating agents, as shown in Table 3 below. An apparent transition temperature (T0) of 66 °C was observed. m This indicates that the enzyme BS149-DM retains the thermostability of BS149 very well. In contrast, the T... m The value was determined to be 59℃.

[0310] Table 3. Effects of metal ions (10 mM) and chelating agent EDTA (10 mM) on the thermal stability of BS149-DM.

[0311] Conclusion: Compared with Bacillus subtilis ligase, BS149-DM exhibits improved thermostability. The enzyme BS149-DM is also resistant to metal ions and chelating agents, as its thermostability increases significantly in their presence. m The value is almost unaffected.

[0312] Example 22: Effects of organic solvents and different additives on the activity of BS149-DM: The peptide linkage reaction was carried out at 25 °C in 100 mM N-tris(hydroxymethyl)methylglycine buffer (pH 8.0) containing 15 μM BS149-DM, 1 mM pentapeptide C-terminal cam-ester (Ac-Phe-Ile-Glu-Trp-Leu-OCam), and 3 mM dipeptide C-terminal amide (H-Ala-Phe-NH2). Different amounts of metal ions (10 mM), EDTA (10 mM), or organic solvents were added, and the reaction mixture was analyzed by LC-MS after 60 minutes. The peaks of the product, hydrolyzed pentapeptide C-terminal cam-ester, and remaining pentapeptide cam-ester were integrated. The activity of BS149-DM was defined as the total amount of product and hydrolyzed pentapeptide C-terminal cam-ester within a specified reaction time divided by the total amount of product, hydrolyzed pentapeptide C-terminal cam-ester, and remaining cam-ester. The highest activity was defined as 100%, as shown in Table 4-8.

[0313] Table 4. Effects of metal ions (10 mM) and chelating agent EDTA (10 mM) on the activity of BS149-DM.

[0314] Table 5. Effect of THF on the activity of BS149-DM.

[0315] Table 6. Effect of DMF on the activity of BS149-DM.

[0316] Table 7. Effect of DMSO on the activity of BS149-DM.

[0317] Table 8. Effect of GndCl on the activity of BS149-DM.

[0318] Example 23: Coupling using BS149-DM enzymatic oligopeptide fragments with or without His-tag: To test the activities and S / H ratios of different enzymes, the following two standard reactions were performed. 800 μL of phosphate buffer (100 mM, pH 8.0) was added to 100 μL of a tripeptide C-terminal amide stock solution (0.01 mmol H-Ala-Leu-Arg-NH2.2TFA in 300 μL water) and 100 μL of a pentapeptide C-terminal Cam-ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 μL water). 5.5 µg of BS149-DM with or without the His-tag was added to the mixture, and the reaction mixture was shaken at room temperature (150 rpm). After 30 minutes, 500 μL aliquots of the reaction mixture were withdrawn, quenched with 500 μL MSA / water (1 / 99, v / v), and analyzed by LC-MS. The product, the hydrolyzed pentapeptide C-terminal Cam-ester, and the remaining pentapeptide C-terminal Cam-ester peak were integrated.

[0319] The S / H ratio of His-tagged and His-free BS149-DM is defined as the amount of product (synthesized oligopeptide) divided by the amount of hydrolyzed pentapeptide C-terminal cam-ester over a specified reaction time. The S / H ratio of His-tagged BS149 is 1.91 and that of His-free BS149 is 1.98.

[0320] The activity of BS149-DM with and without the His-tag is defined as the total amount of product and hydrolyzed pentapeptide C-terminal cam-ester within a specified reaction time, divided by the total amount of product, hydrolyzed pentapeptide C-terminal cam-ester, and remaining cam-ester. The activity of BS149-DM with the His-tag is 97.3%, and that of BS149-DN without the His-tag is 98.6%.

[0321] Conclusion: The presence or absence of the His-tag has no significant effect on the S / H ratio and activity.

[0322] Example 24: S / H ratio of enzymes corresponding to SEQ ID NO 3 with different mutations: To test the activity and S / H ratio of different mutants, the following standard reaction was performed. 800 μL of phosphate buffer (100 mM, pH 8.0) was added to 100 μL of a tripeptide C-terminal amide stock solution (0.01 mmol H-Ala-Leu-Arg-NH2.2TFA in 300 μL water) and 100 μL of a pentapeptide C-terminal cam ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 μL water). 5.5 μg of enzyme was added to the mixture, and the reaction mixture was shaken at room temperature (150 rpm). After 30 minutes, 500 μL aliquots of the reaction mixture were withdrawn, quenched with 500 μL MSA / water (1 / 99, v / v), and analyzed by LC-MS. The product, the hydrolyzed pentapeptide C-terminal cam ester, and the remaining pentapeptide C-terminal cam ester peak were integrated.

[0323] The S / H ratio of different mutants is defined as the amount of product over a specified time divided by the amount of hydrolyzed pentapeptide C-terminal cam-ester (see Table 9).

[0324] Table 9. S / H ratios of enzymes corresponding to SEQ ID NO 3 with different mutations

[0325] Conclusion: It is evident that several enzymes with the S221C mutation corresponding to SEQ ID NO 3 (X = A) have a two-fold increased S / H ratio compared to Bacillus subtilis ligase (S / H Bacillus subtilis ligase = 0.9, see Example 1). The S / H ratio is unaffected for X = P, G, or A.

[0326] Example 25: S / H ratio of the BS149-DM + M222P + L217H + X225 mutant: To test the activity and S / H ratio of different mutants, the following standard reaction was performed. 800 μL of phosphate buffer (100 mM, pH 8.0) was added to 100 μL of a tripeptide C-terminal amide stock solution (0.01 mmol H-Ser-Leu-Arg-NH2.2TFA in 300 μL water) and 100 μL of a pentapeptide C-terminal cam ester stock solution (0.01 mmol Ac-Asp-Phe-Ser-Lys-Leu-OCam.TFA in 1200 μL water). 5.5 μg of enzyme was added to the mixture, and the reaction mixture was shaken at room temperature (150 rpm). After 30 minutes, 500 μL aliquots of the reaction mixture were withdrawn, quenched with 500 μL MSA / water (1 / 99, v / v), and analyzed by LC-MS. The product, the hydrolyzed pentapeptide C-terminal cam ester, and the remaining pentapeptide C-terminal cam ester peak were integrated.

[0327] The S / H ratio of different mutants is defined as the amount of product over a specified time divided by the amount of hydrolyzed pentapeptide C-terminal cam-ester (see Table 10).

[0328] Table 10. S / H ratio of the BS149-DM + M222P + L217H + X225 mutant

[0329] Conclusion: Clearly, mutations at position X225 have a significant impact on the S / H ratio. Many mutations, such as X = N, D, S, C, G, and A, show excellent results. Several other enzymes, such as X = L, I, V, and T, exhibit more than a three-fold increase in S / H ratio compared to Bacillus subtilis ligase (S / H Bacillus subtilis ligase = 0.9, see Example 1). Meanwhile, mutations at X225 to H, Q, and to a lesser extent F and E show improvements over the wild-type enzyme with X225 at P.

[0330] Example 26: Selective coupling of a pentapeptide to the N-terminus of the A chain of human insulin.

[0331] 5 mg of human insulin (CAS # 11061-68-0) and 2.5 mg of Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-OH.TFA were dissolved in 200 µL of DMF. Subsequently, 200 µL of phosphate-buffered saline (1 M, pH 8.0) containing 20 µg of the BS149-DM+ M222G mutant and 200 µL of H2O were added, and the reaction mixture was shaken at room temperature (150 rpm). After 60 minutes, 100 µL aliquots of the reaction mixture were withdrawn and quenched with 500 µL MSA / water (1 / 99, v / v). LC-MS analysis showed that 92% of the insulin initiating material was converted to a single product, namely Ac-Asp-Phe-Ser-Lys-Leu- conjugated to the N-terminus of the insulin A-chain.

[0332] Example 27: Selective coupling of pentapeptides to the N-terminus of the A- and B-chains of human insulin.

[0333] 5 mg of human insulin (CAS # 11061-68-0) and 5 mg of Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-OH.TFA were dissolved in 200 µL of DMF. Subsequently, 200 µL of phosphate buffer (1 M, pH 8.0) containing 55 µg of the BS149-DM+M222G+L217F mutant and 200 µL of H2O were added, and the reaction mixture was shaken at room temperature (150 rpm). After 60 minutes, 100 µL of the reaction mixture was withdrawn and quenched with 500 µL MSA / water (1 / 99, v / v). LC-MS analysis showed that the insulin initiating material was completely consumed and converted into three product peaks: 1) Ac-Asp-Phe-Ser-Lys-Leu- coupled to the N-terminus of the insulin A-chain (22 area %), 2) Ac-Asp-Phe-Ser-Lys-Leu- coupled to the N-terminus of the insulin B-chain (3 area %), and 3) Ac-Asp-Phe-Ser-Lys-Leu- coupled to the N-terminus of both the insulin A-chain and B-chain (75 area %).

[0334] sequence SEQ ID NO 1: Wild-type gene encoding amino acids -107 to 275 of BPN' of subtilisin ENA|K02496|K02496.1 B. Bacillus subtilis protease BPN' Bacillus amyloliquefaciens GTGAGAGGCAAAAAAGTATGGATCAGTTTGCTGTTTGCTTTAGCGTTAATCTTTACGATGG CGTTCGGCAGCACATCCTCTGCCCAGGCGGCAGGGAAATCAAACGGGGAAAAGAAATATAT TGTCGGGTTTAAACAGACAATGAGCACGATGAGCGCCGCTAAGAAGAAAGATGTCATTTCT GAAAAAGGCGGGAAAGTGCAAAAGCAATTCAAATATGTAGACGCAGCTTCAGCTACATTAA ACGAAAAAGCTGTAAAAGAATTGAAAAAAGACCCGAGCGTCGCTTACGTTGAAGAAGATCA CGTAGCACATGCGTACGCGCAGTCCGTGCCTTACGGCGTATCACAAATTAAAGCCCCTGCT CTGCACTCTCAAGGCTACACTGGATCAAATGTTAAAGTAGCGGTTATCGACAGCGGTATCG ATTCTTCTCATCCTGATTTAAAGGTAGCAGGCGGAGCCAGCATGGTTCCTTCTGAAACAAA TCCTTTCCAAGACAACAACTCTCACGGAACTCACGTTGCCGGCACAGTTGCGGCTCTTAAT AACTCAATCGGTGTATTAGGCGTTGCGCCAAGCGCATCACTTTACGCTGTAAAAGTTCTCG GTGCTGACGGTTCCGGCCAATACAGCTGGATCATTAACGGAATCGAGTGGGCGATCGCAAA CAATATGGACGTTATTAACATGAGCCTCGGCGGACCTTCTGGTTCTGCTGCTTTAAAAGCG GCAGTTGATAAAGCCGTTGCATCCGGCGTCGTAGTCGTTGCGGCAGCCGGTAACGAAGGCA CTTCCGGCAGCTCAAGCACAGTGGGCTACCCTGGTAAATACCCTTCTGTCATTGCAGTAGG CGCTGTTGACAGCAGCAACCAAAGAGCATCTTTCTCAAGCGTAGGACCTGAGCTTGATGTC ATGGCACCTGGCGTATCTATCCAAAGCACGCTTCCTGGAAACAAATACGGGGCGTACAACG GTACGTCAATGGCATCTCCGCACGTTGCCGGAGCGGCTGCTTTGATTCTTTCTAAGCACCC GAACTGGACAAACACTCAAGTCCGCAGCAGTTTAGAAAACACCACTACAAAACTTGGTGAT TCTTTCTACTATGGAAAAGGGCTGATCAACGTACAGGCGGCAGCTCAGTAA SEQ ID NO 2: Wild-type Bacillus subtilis protease BPN' (mature) >SUBT_BACAM Bacillus subtilis protease BPN' Bacillus amyloliquefaciens maturation 1 to 275 AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETNPFQDNNSHGTHVAGTVAALNNSIGVLGVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKA AVDKAVASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPNWTNTQVRSSLENTTTKLGDSFYYGKGLINVQAAAQ SEQ ID NO 3: Contains Ca 2+ The BPN' variant of the subtilisin combines loop deletion with S221C and the preferred P225 mutation (denoted as P225X). AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETNPFQDNNSHGTHVAGTVAAVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKA VASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTCMASXHVAGAAALILSKHPNWTNTQVRSSLENTTTKLGDSFYYGKGLINVQAAAQ SEQ ID NO 4: A variant of BPN' of subtilisin with a preferred mutation site compared to SEQ ID NO 3. AXXVXYGVXQIKAPALHSQGYTGSNVKVAVXDSGIDSSHPDLXVAGGASXVPSETNPFQDNNSHGTHVAGTVXAVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKA VASGVVVVAAAGNXGTSGSSSTVXYPXKYPSVIAVGAVDSSNQRAXFSSVGPELDVMAPGVSIXSTLPGXKYGAXXGTCMASXHVAGAAALILSKHPNWTNTQVRSSLENTXTKLGDSFYYGKGLINVXAAAQ SEQ ID NO 5: Contains the Bacillus subtilis protease (aprE) promoter region (bp 1-197, Takara) from Bacillus subtilis, BPN' signal sequence (bp 198-287), BPN' pre-domain (bp 288-518), mature BS149-DM, 6xHis tag, and stop codon of the E. coli / Bacillus subtilis shuttle vector pBES: Pt149DM His. From nucleotide 1590 onwards, the sequence follows that of pBES from Takara.

[0335] Compared to BPN', amino acids 72-80 are deleted (Val-Ala-Ala-Leu-Asn-Asn-Ser-Ile-Gly); GTTGCG GCT CTT AAT AAC TCA ATC GGT.

Claims

1. A method for the enzymatic synthesis of (oligo)peptides, comprising coupling (a) a C-terminal ester or thioester of the (oligo)peptide and (b) a nucleophilic group of the (oligo)peptide having an N-terminal deprotected amine. The coupling is performed in a fluid containing water, and The coupling is catalyzed by a variant of subtilisin BPN' or its homolog, which contains the following mutations compared to the subtilisin BPN' or its homologous sequence shown in SEQUENCE ID NO: 2: - Corresponds to the deletion of amino acids at positions 75-83; - A mutation corresponding to the amino acid position of S221, wherein the mutation is S221C or S221 selenocysteine; - Preferably, the mutation corresponds to the amino acid position of P225; The amino acid positions are defined according to the sequence of BPN', the subtilisin shown in SEQUENCE ID NO:

2.

2. The method as described in claim 1, characterized in that, The mutation at the amino acid position corresponding to S221 is S221C.

3. The method as described in claim 1 or 2, characterized in that, The BPN' variant of the subtilisin or its homolog contains a mutation at the amino acid position corresponding to P225, the mutation being selected from the group consisting of: P225N, P225D, P225S, P225C, P225G, P225A, P225T, P225V, P225I, P225L, P225H, P225Q, P225F, and P225E.

4. The method as described in any one of the preceding claims, characterized in that, Provides an N-terminal and / or one or more side chain functional groups of a (oligo)peptide C-terminal ester or thioester with a protective group, and / or The C-terminus of the (oligo)peptide nucleophilic group with a protective group and / or one or more side chain functional groups of the (oligo)peptide nucleophilic group with a protective group are provided.

5. A method for the enzymatic synthesis of a cyclic (oligo)peptide of at least 12 amino acids, comprising a cyclization step of a C-terminal ester or thioester of an (oligo)peptide having an N-terminal deprotected amine, wherein the cyclization is carried out in a fluid containing water, and The cyclization is catalyzed by a variant of subtilisin BPN' or its homolog, which contains the following mutations compared to the subtilisin BPN' or its homologous sequence shown in SEQUENCE ID NO: 2: - Corresponds to the deletion of amino acids at positions 75-83; - A mutation corresponding to the amino acid position of S221, wherein the mutation is S221C or S221 selenocysteine; - Preferably, the mutation corresponds to the amino acid position of P225; The amino acid positions are defined according to the sequence of BPN', the subtilisin shown in SEQUENCE ID NO:

2.

6. The method as described in claim 5, characterized in that, The mutation at the amino acid position corresponding to S221 is S221C.

7. The method as described in claim 5 or 6, characterized in that, The BPN' variant of the subtilisin or its homolog contains a mutation at the amino acid position corresponding to P225, the mutation being selected from the group consisting of: P225N, P225D, P225S, P225C, P225G, P225A, P225T, P225V, P225I, P225L, P225H, P225Q, P225F, and P225E.

8. The method according to any one of claims 5-7, characterized in that, Provides one or more side chain functional groups for the C-terminal ester or thioester of an oligopeptide with a protective group.

9. The method as described in any one of the preceding claims, characterized in that, The BPN' variant of the subtilisin or its homolog contains one or more mutations at the amino acid positions corresponding to Q2, S3, P5, S9, I31, K43, M50, A73, E156, G166, G169, S188, Q206, N212, N218, T254, or Q271 of SEQUENCE ID NO 2. Preferably, the one or more mutations are selected from the group consisting of: Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, N218S, T254A, and Q271E.

10. The method as described in any one of the preceding claims, characterized in that, The BPN' variant of the subtilisin or its homolog contains one or more mutations selected from the group of mutations corresponding to the following amino acids: N62, G100, S125, L126, G127, P129, N155, Y217, N218 or M222 of SEQUENCEID NO 2. Preferably, the mutation at the position corresponding to M222 is M222G, M222P, M222N, M222E, M222Q, or M222A. Preferably, the mutation at Y217 is Y217L, Y217N, Y217E, Y217G, Y217F, Y217S, Y217A, Y217R, Y217W, Y217C, Y217D, Y217P, or Y217H.

11. The method as described in any one of the preceding claims, characterized in that, The BPN' variant of the subtilisin or its homolog contains at least one mutation selected from the mutation group corresponding to the following amino acid positions: Y104, I107, L126, S101, G102, G127, G128, L135, or P168 of SEQUENCE IDNO 2. Preferably, the mutation is selected from the group consisting of Y104, I107 and L135, more preferably from the group consisting of Y104F, Y104S, I107V, I107A, L135N, L135S, L135D or L135A.

12. The method as described in any one of the preceding claims, characterized in that, The (oligo)peptide C-terminal ester is defined as peptide-(C=O)O-CX2-C(=O)N-R1R2, where each X independently represents a hydrogen atom or an alkyl group; and R1 represents a hydrogen atom or an alkyl group and R2 represents a peptide residue or hydrogen atom or alkyl group or amino acid having a C-terminal carboxamide or carboxylic acid functional group, optionally protected on a side chain functional group of an amino acid or on one or more of the side chain functional groups of multiple amino acids. Preferably, each X represents a hydrogen atom. Preferably, both R1 and R2 represent hydrogen atoms. Preferably, R1 represents a hydrogen atom and R2 represents a peptide residue or amino acid having a C-terminal carboxylamide or carboxylic acid functional group, optionally protected on a side chain functional group of the amino acid or on one or more of the side chain functional groups of multiple amino acids. Preferably, the C-terminal ester group of the (oligo)peptide ester is attached to the solid phase.

13. The method as described in any one of the preceding claims, characterized in that, Synthetic oligopeptides are oligopeptides containing up to 200 amino acid units, especially oligopeptides containing 5-100 amino acid units, and even more particularly oligopeptides containing 10-50 amino acid units. Preferably, the synthesized (oligo)peptide is a peptide containing more than 200 amino acid units, especially 201-35000 amino acid units, more particularly 201-5000 amino acid units, and more preferably 201-1000 amino acid units. Preferably, the (oligo)peptide is a protein. Preferably, the (oligo)peptide has no secondary or tertiary protein structure.

14. An enzyme, wherein the enzyme is a variant of subtilisin BPN' or a homolog thereof, comprising the following mutations compared to the subtilisin BPN' or a homolog thereof shown in SEQUENCE ID NO: 2: - Corresponds to the deletion of amino acids at positions 75-83; - A mutation corresponding to the amino acid position of S221, wherein the mutation is S221C or S221 selenocysteine; - Preferably, the mutation corresponds to the amino acid position of P225; The amino acid positions are defined according to the sequence of BPN', the subtilisin shown in SEQUENCE ID NO:

2.

15. The enzyme according to claim 14, characterized in that, The mutation at the amino acid position corresponding to S221 is S221C.

16. The enzyme as described in claim 14 or 15, characterized in that, The BPN' variant of the subtilisin or its homolog contains a mutation at the amino acid position corresponding to P225, the mutation being selected from the group consisting of: P225N, P225D, P225S, P225C, P225G, P225A, P225T, P225V, P225I, P225L, P225H, and P225Q.

17. The enzyme according to any one of claims 14-16, characterized in that, Contains one or more mutations at the amino acid positions corresponding to Q2, S3, P5, S9, I31, K43, M50, A73, E156, G166, G169, S188, Q206, N212, N218, T254, and Q271 of SEQUENCE ID NO 2. Preferably, the one or more mutations are selected from the group consisting of: Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, N218S, T254A, and Q271E. Optionally, the enzyme contains mutations corresponding to the N218 and M50 positions, preferably N218S and M50F. Optionally, the enzyme contains mutations at amino acid positions corresponding to S3C and Q206C, preferably wherein cysteine ​​residues at positions 3 and 206 form disulfide bridges.

18. The enzyme according to any one of claims 14-17 further comprises mutations corresponding to positions N218, M50, Q2, A73 and P5, wherein the mutations are preferably N218S, M50F, Q2K, A73L, and P5S.

19. The enzyme according to any one of claims 14-18, comprising one or more mutations at the amino acid positions corresponding to N62, G100, S125, L126, G127, P129, N155, Y217, N218, or M222 of SEQUENCE ID NO 2. Preferably, the enzyme contains a mutation at position M222 corresponding to SEQUENCE ID NO 2; more preferably, the mutation at position M222 is M222G, M222P, M222N, M222E, M222Q, or M222A, preferably M222G or M222P. Preferably, the enzyme contains a mutation at the amino acid position corresponding to Y217 of SEQUENCE ID NO 2; more preferably, the mutation at Y217 is Y217L, Y217N, Y217E, Y217G, Y217F, Y217A, Y217S, Y217R, Y217W, Y217C, Y217D, Y217P, or Y217H. Preferably, the enzyme contains a mutation at the amino acid positions corresponding to M222 and Y217, wherein the mutation is: - M222P and Y217H; - M222P and Y217G; - M222P and Y217R; - M222G and Y217F; - M222G and Y217G; or - M222G and Y217R.

20. The enzyme according to any one of claims 14-19, comprising at least one mutation selected from the mutant group corresponding to the amino acid positions Y104, I107, L126, S101, G102, G127, G128, L135, and P168 of SEQUENCE ID NO 2. Preferably, the mutation is selected from the group consisting of: Y104F, Y104S, I107V, I107A, L135N, L135S, L135D or L135A.

21. The enzyme according to any one of claims 14-20, having 50-100%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, especially at least 90%, and even more especially at least 95% sequence identity with Sequence ID 3, 4 or 5.

22. A method for preparing the recombinant enzyme of any one of claims 14-21, the method comprising: a) Provide a recombinant host cell that functionally expresses a gene encoding the enzyme; b) The host cells are cultured under conditions that provide expression of functionally active enzymes; and c) Recover the expressed enzymes from the microbial host.

23. A recombinant polynucleotide comprising a sequence encoding the enzyme of any one of claims 14-21.

24. A host cell comprising the polynucleotide of claim 23.

25. Use of the enzyme as a catalyst as described in any one of claims 14-21, Preferably, the enzyme is used as a catalyst in peptide synthesis. Preferably, the peptide is an oligopeptide. Preferably, the peptide comprises at least 201 amino acid units. Preferably, the peptide is a protein.

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