All-potent nuclease variant with improved salt tolerance and application of all-potent nuclease variant

Abstract

The invention relates to the technical field of enzyme engineering, and particularly provides an all-round nuclease variant, the variant can tolerate a wider salt concentration range compared with wild type all-round nuclease, good enzyme activity is still kept under the increased salt concentration, and the application of all-round nuclease under different salt concentrations is widened.

Description

Universal nuclease variants with improved salt tolerance and their applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202311249502.3 filed with the Patent Office of China on September 26, 2023, entitled “Universal nuclease variants with improved salt tolerance and their applications,” the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present application relates to enzyme engineering technology, and in particular to a universal nuclease variant with improved salt tolerance, a method of use and applications thereof. Background Art

[0004] Universal nucleases, also known as non-restrictive endonucleases, primarily function to eliminate nucleic acids from biological products. They can reduce the viscosity of cell supernatants and cell lysates in scientific research, improving protein purification efficiency and functional studies. They can also be used in virus purification, vaccine production, and the protein and polysaccharide pharmaceutical industries as reagents for removing residual host nucleic acids, reducing them to pg levels and thus improving the efficacy and safety of biological products. They can also effectively prevent the clumping of human peripheral blood mononuclear cells (PBMCs) in cell therapy and vaccine research, making them a unique tool enzyme in the CGT field.

[0005] The most popular universal nuclease is a bacterial nuclease from Serratia marcescens, which was published by Benedik and Strych in 1998 (FEMS Microbiol Lett. 165: 1-13) and is registered by Merck as Its optimal activity temperature is 37°C, but its primary drawback is its limited tolerance to elevated salt concentrations. Data indicate that sodium or potassium ions strongly inhibit the activity of this nuclease, and when the salt concentration in the reaction system exceeds 300 mM, the enzyme activity is almost completely lost. This characteristic restricts the omnipotent nuclease to salt-free or low-salt environments, limiting its widespread application. Whether in the industrial preparation of biological products or in the scientific research process of obtaining recombinant proteins, varying levels of salt are added to the buffer system to ensure process stability. Therefore, the market urgently needs to develop and produce an omnipotent nuclease with significantly improved salt tolerance.

[0006] Enzymes from halophilic microorganisms make it possible to find salt-tolerant enzymes. Some salt-tolerant nucleases from marine microorganisms have been reported. In 2022, Marcin et al. (CN114651062A) published a new type of heat-labile nuclease that can tolerate a salt concentration of 500mM, but the enzyme can only maintain high activity at low temperatures, especially 4°C-8°C, and the yield is very low, the production cost is high, and the application environment is harsh. In addition, Salt Active nuclease (Yisheng Bio, China) is also available on the market. It shows optimal activity under 500mM NaCl conditions, but the relative enzyme activity can only reach about 25% of the optimal specific enzyme activity of Benzonase. Moreover, its activity decreases significantly with decreasing salt concentration at a salt concentration below 500mM. The tolerance range of salt concentration is also narrow. This characteristic limits its application to only 500mM salt concentration. Therefore, equivalent enzymes from halophilic organisms do not always exist.

[0007] In view of this, this application is filed.

[0008] SUMMARY OF THE INVENTION

[0009] In response to the above technical problems, this application obtains a universal nuclease variant that can tolerate a wider range of salt concentrations through rational design, solving the pain point of the narrow salt tolerance range of universal nucleases on the current market, making its application scenarios wider and more universal, and achieving the effect of removing nucleic acids under different salt concentration systems.

[0010] Specifically, this application, based on the Benzonase universal nuclease, increases its salt tolerance range, specifically to 200mM to 500mM, without affecting its original activity. This ultimately provides a nuclease with a wider salt tolerance. This application not only continues to leverage the inherent advantages of Benzonase – high activity, stability, and high yield in the absence of salt or in very low salt conditions – but also improves its tolerance to even higher salt concentrations.

[0011] Therefore, this application includes at least the following four objectives:

[0012] The first object of the present application is to provide a universal nuclease variant with improved salt tolerance;

[0013] The second object of the present application is to provide a use of a universal nuclease variant with improved salt tolerance in nucleic acid processing;

[0014] The third object of the present application is to provide a method for preparing a universal nuclease variant with improved salt tolerance;

[0015] A fourth object of the present application is to provide a method for using a universal nuclease variant with improved salt tolerance.

[0016] To achieve the above objectives, this application proposes the following technical solutions:

[0017] The present application first provides a full-strength nuclease or full-strength nuclease variant with improved salt tolerance, wherein the variant comprises changes in at least one or more amino acid sites in the Benzonase nuclease sequence.

[0018] Further, the sites include any one or more of position 56, position 98, position 101 and / or position 242;

[0019] Further, preferably, the site includes position 98, and further includes any one or more of position 56, position 101 and / or position 242.

[0020] Further, the changes include any one or more of T56D, T98K, N101H and / or G242D;

[0021] Further preferably, the change includes T98K, and further includes any one or more of T56D, N101H and / or G242D.

[0022] Furthermore, the Benzonase nuclease is a wild-type Benzonase nuclease;

[0023] Further preferably, the amino acid sequence of the wild-type Benzonase nuclease is shown as SEQ ID NO.1.

[0024] In some specific embodiments, the specific sequence of the variant includes any one or more of SEQ ID NOs. 2-16.

[0025] The present application also provides a method for improving the salt tolerance of a omnipotent nuclease, comprising: introducing changes including at least one or more amino acid sites into the Benzonase nuclease sequence.

[0026] Further, the sites include any one or more of position 56, position 98, position 101 and / or position 242;

[0027] Further, preferably, the site includes position 98, and further includes any one or more of position 56, position 101 and / or position 242.

[0028] Further, the changes include any one or more of T56D, T98K, N101H and / or G242D;

[0029] Further preferably, the change includes T98K, and further includes any one or more of T56D, N101H and / or G242D.

[0030] Furthermore, the Benzonase nuclease is a wild-type Benzonase nuclease;

[0031] Further preferably, the amino acid sequence of the wild-type Benzonase nuclease is shown as SEQ ID NO.1.

[0032] In some specific embodiments, the specific sequence of any one or more variants is such as SEQ ID NO. 2-16.

[0033] The present application also provides a nuclease endonuclease gene, which encodes any of the above-mentioned full-potent nuclease variants.

[0034] The present application also provides an expression cassette, a plasmid or a vector comprising the above-mentioned endonuclease gene.

[0035] The present application also provides a host cell comprising the aforementioned endonuclease gene, or the aforementioned expression cassette, plasmid or vector.

[0036] The present application also provides a method for preparing the above-mentioned all-potent nuclease variant, which is obtained by expression using the above-mentioned expression cassette, plasmid, vector, host cell or engineered microorganism.

[0037] In some specific methods, recombinant expression plasmids containing the aforementioned variants are obtained, each recombinant expression plasmid is transformed into, for example, E. coli, and positive clones are selected to obtain recombinant expression strains expressing the wild-type and mutated full-strength nucleases. Furthermore, after activation, the strains are inoculated into fermenters, fermented using autoinduction, and the fermentation supernatant is collected.

[0038] The present application also provides the use of the above-mentioned universal nuclease variant in nuclease cleavage or removal;

[0039] Further preferably, the application is carried out at a salt concentration of 0-500 mM.

[0040] The present application also provides a method for enzymatic cleavage or removal of nucleic acids, which comprises treating nucleic acids with any of the above-mentioned universal nuclease variants; preferably, the treatment is performed at a salt concentration of 0-500 mM.

[0041] Beneficial technical effects of this application:

[0042] 1) The mutations or mutation combinations provided in this application have significantly improved salt tolerance compared to the wild type. The wild-type all-potent nuclease almost completely loses its activity under conditions above 300mM NaCl, while the variants can maintain the original enzyme activity without reduction under salt-free or very low-salt conditions, and can maintain more than 80% of the activity under 200mM NaCl conditions. Among them, the following eight variants T98K, T56D / T98K, T98K / N101H, T98K / G242D, T56D / T98K / G242D, N101H / T98K / G242D, T56D / T98K / N101H, T56D / N101H / T98K / G242D have the following activity at 300mM, 400mM, and 500mM NaCl. The activity is significantly improved under NaCl conditions, and can maintain more than 70%, 50%, and 20% activity respectively. This broadens the application of universal nucleases at different salt concentrations, can reduce their usage in high-salt buffer systems, and can achieve better nucleic acid removal effects.

[0043] 2) The mutated omnipotent nuclease prepared in this application can solve the pain point of the current market omnipotent nuclease with a narrow salt tolerance range, has a wider application scenario, and is suitable for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1. 3D structure of the wild-type totipotent nuclease and mutation locations.

[0045] Figure 2. SDS-PAGE diagram of the recombinant omnipotent nuclease production process; where M represents protein marker, S represents the collected fermentation supernatant, SPFF represents the peak eluted after the sample passes through the ion exchange column, and SEC represents the peak eluted after the sample passes through the molecular sieve.

[0046] Figure 3. HPLC purity of recombinantly produced universal nuclease.

[0047] Figure 4. Comparison of the relative enzymatic activities of wild-type and variant omnipotent nucleases under different salt concentration conditions, where WT represents the wild-type omnipotent nuclease and Competitor Y represents the Yisheng high-salt-tolerant nuclease.

[0048] Detailed Description of the Invention

[0049] The present application discloses some omnipotent nuclease variants, which can tolerate a wider range of salt concentrations relative to the wild-type omnipotent nuclease and still maintain a certain activity under elevated salt concentrations. Those skilled in the art can refer to the contents of this article to realize its application. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and they are all considered to be included in this application. The preparation method and application of this application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the preparation method and application of this article without departing from the content, spirit and scope of this application to realize and apply the technology of this application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this application belongs.

[0050] The following terms or definitions are provided merely to aid understanding of the present application. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

[0051] Unless otherwise defined below, the meaning of all technical terms and scientific terms used in the specific embodiments of the present application is intended to be the same as that generally understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present application.

[0052] As used in this application, the terms "comprises," "comprising," "having," "containing," or "involving" are inclusive or open-ended and do not exclude other unrecited elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising." If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists only of these embodiments.

[0053] When referring to a singular noun an indefinite or definite article e.g. "a" or "an", "the" include a plural of that noun.

[0054] The terms "approximately" and "substantially" in this application represent an accuracy range that can be understood by those skilled in the art and can still ensure the technical effect of the characteristics discussed. This term usually means ±10%, preferably ±5%, of the deviation from the indicated value.

[0055] 47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more of the above values. Any larger number or fraction therebetween is also included.

[0056] Conversely, the term "no more than" includes every value that is less than the stated value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 56, 57, 58, 59 ... 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Also included are any smaller numbers or fractions therebetween.

[0057] The terms "plurality", "at least two", "two or more", "at least a second", etc., should be understood to include, but are not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 , 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. Any larger number or fraction therebetween is also included.

[0058] The salt-tolerance-enhanced omnipotent nuclease or omnipotent nuclease variant of the present application comprises changes in at least one or more amino acid sites in the Benzonase nuclease sequence.

[0059] It should be understood that the term "variant" herein is merely a formalism. Any sequence comprising one or more amino acid changes within the Benzonase nuclease sequence falls within the scope of the "variant sequence" herein. Variants may include both mutant and non-mutant forms. Furthermore, the specific source or preparation method of the variant is not limited. For example, in some embodiments, the variant is obtained directly through chemical synthesis; in other embodiments, the variant is obtained directly through recombinant expression; and in still other embodiments, the variant may be obtained by introducing mutant amino acids into the wild-type sequence through genetic engineering or genetic means.

[0060] In addition, the "change" in the text includes mutation forms, but is not limited to mutations. It can be understood that variant sequences obtained in non-mutation forms will also have the same effects and functions in this application, which also falls within the scope of reasonable protection of this application.

[0061] Typically, the Benzonase nucleases used herein have at least about 65% sequence identity with the nuclease of SEQ ID NO. 1, more particularly at least about 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity. These homologous differences may be due to different species origins, but it is understood that, as long as the core functional domain sequence is consistent, mutations occurring herein within these basic series are within the scope of protection of this application.

[0062] In some embodiments, the Benzonase nuclease described herein refers to a wild-type Benzonase nuclease. In some specific embodiments, the amino acid sequence of the wild-type Benzonase nuclease is specifically shown in SEQ ID NO. 1. It is understood that the wild-type sequences of bacterial Benzonase nucleases from Serratia marcescens obtained from different sources or different habitats may have individual amino acid differences, but they can also be understood as wild-type sequences. Therefore, the amino acid sequence of the Benzonase nuclease of the present application should not be limited to the sequence SEQ ID NO. 1 in theory. Wild-type sequences having at least about 85% sequence identity, particularly at least about 90%, 95% or 99% sequence identity to the nuclease of SEQ ID NO. 1, as long as the core functional domain sequence is consistent, it can be expected that mutations described herein based on these basic wild sequences are also within the scope of protection of the present application.

[0063] As can be understood from the examples of this application, the number of mutations or altered sites in this application is not limited. When including a mutation site verified in this application: such as T56D, or T98K; or any two mutation sites: such as T56D and G242D, or T98K and G242D; or even including three sites: such as T56D, T98K and G242D, the experimental data of this application have shown that these mutations have very good salt tolerance and basic enzyme activity characteristics. Therefore, any variant containing 1, 2, 3 or 4 of the present application falls within the scope of protection of this application.

[0064] Therefore, compared with the corresponding wild-type Benzonase nuclease, the Benzonase nuclease variants of the present application include at least one change. In some embodiments, the at least one mutation occurs at one or more of positions 56, 98, 101 and 242 of the wild-type Benzonase nuclease sequence (particularly corresponding to SEQ ID NO.1).

[0065] In some embodiments, the wild-type Benzonase nuclease comprises at least one mutation, wherein the at least one mutation is at position 56 corresponding to SEQ ID NO. 1, preferably the mutation is T56D.

[0066] In some more specific embodiments, the mutant sequence is shown as SEQ ID NO.2.

[0067] SEQ ID NO.2: (T56D)

[0068] In some embodiments, the wild-type Benzonase nuclease comprises at least one mutation, wherein the at least one mutation is at position 98 corresponding to SEQ ID NO. 1, preferably the mutation is T98K.

[0069] In some more specific embodiments, the mutant sequence is shown as SEQ ID NO.3.

[0070] SEQ ID NO.3: (T98K)

[0071] In some embodiments, the wild-type Benzonase nuclease comprises at least one mutation, wherein the at least one mutation is at position 101 corresponding to SEQ ID NO. 1, preferably the mutation is N101H.

[0072] In some more specific embodiments, the mutant sequence is shown as SEQ ID NO.4.

[0073] SEQ ID NO.4: (N101H)

[0074] In some embodiments, the wild-type Benzonase nuclease comprises at least one mutation, wherein the at least one mutation is at position 242 corresponding to SEQ ID NO. 1; preferably, the mutation is G242D.

[0075] In some more specific embodiments, the mutant sequence is shown as SEQ ID NO.5.

[0076] SEQ ID NO.5: (G242D)

[0077] In some embodiments, the wild-type Benzonase nuclease comprises at least two mutations, wherein at least one mutation is at positions 56 and 98 corresponding to SEQ ID NO. 1; preferred mutations are T56D and T98K.

[0078] In some more specific embodiments, the mutant sequence is shown as SEQ ID NO.6.

[0079] SEQ ID NO.6: (T56D / T98K)

[0080] In some embodiments, the wild-type Benzonase nuclease comprises at least two mutations, wherein at least one mutation is at positions 56 and 242 corresponding to SEQ ID NO. 1; preferred mutations are T56D and G242D.

[0081] In some more specific embodiments, the mutant sequence is shown as SEQ ID NO.7.

[0082] SEQ ID NO.7: (T56D / G242D)

[0083] In some embodiments, the wild-type Benzonase nuclease comprises at least two mutations, wherein at least one mutation is T56D and N101H at positions 56 and 101 corresponding to SEQ ID NO.1.

[0084] In some more specific embodiments, the mutant sequence is shown in SEQ ID NO.8; the preferred mutation is.

[0085] SEQ ID NO.8: (T56D / N101H)

[0086] In some specific embodiments, the wild-type Benzonase nuclease comprises at least two mutations, wherein at least one mutation is at positions 98 and 242 corresponding to SEQ ID NO. 1; preferred mutations are T98K and G242D.

[0087] In some more specific embodiments, the mutant sequence is shown as SEQ ID NO.9.

[0088] SEQ ID NO.9: (T98K / G242D)

[0089] In some specific embodiments, the wild-type Benzonase nuclease comprises at least two mutations, wherein at least one mutation is at positions 98 and 101 corresponding to SEQ ID NO. 1; preferred mutations are T98K and N101H.

[0090] In some more specific embodiments, the mutant sequence is shown as SEQ ID NO.10.

[0091] SEQ ID NO.10: (T98K / N101H)

[0092] In some specific embodiments, the wild-type Benzonase nuclease comprises at least two mutations, wherein at least one mutation is at positions 101 and 242 corresponding to SEQ ID NO. 1; preferred mutations are G242D and N101H.

[0093] In some more specific embodiments, the mutant sequence is shown as SEQ ID NO.11.

[0094] SEQ ID NO.11: (G242D / N101H)

[0095] In some embodiments, the wild-type Benzonase nuclease comprises at least three mutations, wherein at least one mutation is at positions 56, 98, and 101 corresponding to SEQ ID NO. 1; preferred mutations are T56D, T98K, and N101H.

[0096] In some more specific embodiments, the mutant sequence is shown as SEQ ID NO.12.

[0097] SEQ ID NO.12: (T56D / T98K / N101H)

[0098] In some embodiments, the wild-type Benzonase nuclease comprises at least three mutations, wherein at least one mutation is at positions 56, 98, and 242 corresponding to SEQ ID NO. 1; preferred mutations are T56D, T98K, and G242D.

[0099] In some more specific embodiments, the mutant sequence is shown as SEQ ID NO.13.

[0100] SEQ ID NO.13: (T56D / T98K / G242D)

[0101] In some embodiments, the wild-type Benzonase nuclease comprises at least three mutations, wherein at least one mutation is at positions 98, 101, and 242 corresponding to SEQ ID NO. 1; preferred mutations are T98K, G242D, and N101H.

[0102] In some more specific embodiments, the mutant sequence is shown as SEQ ID NO.14.

[0103] SEQ ID NO.14: (T98K / G242D / N101H)

[0104] In some embodiments, the wild-type Benzonase nuclease comprises at least three mutations, wherein at least one mutation is at positions 56, 101, and 242 corresponding to SEQ ID NO. 1; preferred mutations are T98K, G242D, and N101H.

[0105] In some more specific embodiments, the mutant sequence is shown as SEQ ID NO.15.

[0106] SEQ ID NO.15: (T56D / N101H / G242D)

[0107] In some embodiments, the wild-type Benzonase nuclease comprises at least four mutations, wherein at least one mutation is at positions 56, 98, 101 and 242 corresponding to SEQ ID NO. 1; preferred mutations are T56D, T98K, G242D, and N101H.

[0108] In some more specific embodiments, the mutant sequence is shown as SEQ ID NO.16.

[0109] SEQ ID NO.16: (T56D / T98K / G242D / N101H)

[0110] As shown in the data of the examples of the present application, the following eight mutants T98K, T56D / T98K, T98K / N101H, T98K / G242D, T56D / T98K / G242D, N101H / T98K / G242D, T56D / T98K / N101H, T56D / N101H / T98K / G242D have significantly improved activity under 300mM, 400mM, and 500mM NaCl conditions, and can maintain more than 70%, 50%, and 20% activity, respectively. Therefore, in some preferred embodiments of the present application, the wild-type Benzonase nuclease comprises at least a 98-position mutation, such as T98K, and further comprises mutations at one or more other sites (T56D / G242D / N101H).

[0111] In addition, it is also understandable that, on the basis of satisfying the usual basic enzymatic activity of Benzonase, those skilled in the art may further perform other known effective site mutations based on the mutations of the present application to meet the needs of practical optimization and improvement. Such mutations, because they occur on the basis of the present application, also fall within the scope of protection of the present application, and their corresponding effects are reasonably anticipated by those skilled in the art based on their basic understanding of the enzyme structure.

[0112] The method of improving the salt tolerance of the omnipotent nuclease of the present invention comprises the step of introducing at least one or more amino acid site mutations or changes into the Benzonase nuclease sequence. The logic of these mutations or changes is the same as above.

[0113] For example, in some embodiments, the sites include position 56, position 98, position 101 and / or position 242; in some preferred embodiments, the sites include position 98 and further include position 56, position 101 and / or position 242.

[0114] In some specific embodiments, the mutation includes one or more of T56D, T98K, N101H and / or G242D; in some preferred embodiments, the mutation includes T98K and further includes one or more of T56D, N101H and / or G242D.

[0115] The endonuclease gene of the present application comprises any gene sequence that can encode any of the above-mentioned full-potent nuclease variants.

[0116] The expression cassette of the present application may be an expression cassette comprising any of the above-mentioned endonuclease genes.

[0117] The plasmid of the present application may be a plasmid comprising any of the above-mentioned endonuclease genes, preferably an expression plasmid.

[0118] The vector of the present application may be a vector comprising any of the above-mentioned endonuclease genes, preferably an expression vector.

[0119] The cell of the present application is a cell capable of expressing any of the above-mentioned vectors of the endonuclease gene, preferably a host cell.

[0120] The following will clearly and completely describe the technical solutions of this application in conjunction with the embodiments. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application. Example

[0121] The following will clearly and completely describe the technical solution of this application in conjunction with the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of this application.

[0122] Example 1 Design of mutants

[0123] The preliminary study of this application found that in the wild-type non-specific nuclease Based on this, mutations at certain sites can produce mutants with significantly improved salt tolerance. Specifically, when designing mutants, by exploring the amino acid preferences of salt-tolerant enzymes on the external surface of their structure and combining them with their three-dimensional structure, we selected the following multiple point mutations after appropriate design and screening. Mutations predicted to have salt tolerance were introduced into the wild-type all-potent nuclease through site-directed mutagenesis.

[0124] After batch mutation design and screening, this application finally obtained four optimal mutation sites with a wider range of potential salt tolerance, namely positions 56, 98, 101 and 242 (see Figure 1).

[0125] Therefore, based on the wild-type sequence (SEQ ID NO. 1), the present application designed various combination mutations for testing at positions 56, 98, 101 and 242 as key sites.

[0126] SEQ ID NO.1:

[0127] The specifically designed mutation combinations include: T56D / T98K / N101H / G242D, that is, based on SEQ ID NO. 1, an amino acid sequence is designed that simultaneously has one, more, or all of the T56D, T98K, N101H, and G242D mutations.

[0128] The specific information is as follows:

[0129] The specific sequence information is as follows:

[0130] SEQ ID NO.2: (T56D)

[0131] SEQ ID NO.3: (T98K)

[0132] SEQ ID NO.4: (N101H)

[0133] SEQ ID NO.5: (G242D)

[0134] SEQ ID NO.6: (T56D / T98K)

[0135] SEQ ID NO.7: (T56D / G242D)

[0136] SEQ ID NO.8: (T56D / N101H)

[0137] SEQ ID NO.9: (T98K / G242D)

[0138] SEQ ID NO.10: (T98K / N101H)

[0139] SEQ ID NO.11: (G242D / N101H)

[0140] SEQ ID NO.12: (T56D / T98K / N101H)

[0141] SEQ ID NO.13: (T56D / T98K / G242D)

[0142] SEQ ID NO.14: (T98K / G242D / N101H)

[0143] SEQ ID NO.15: (T56D / N101H / G242D)

[0144] SEQ ID NO.16: (T56D / T98K / G242D / N101H)

[0145] Example 2 Production of recombinant mutants

[0146] The gene fragments corresponding to SEQ ID NO. 1 and SEQ ID NO. 2-16 in Example 1 were respectively inserted into the pET41a vector to obtain recombinant expression plasmids. Each recombinant expression plasmid was transformed into E. coli BL21 (DE3) competent cells by heat shock transformation. Positive clones were selected and cultured overnight at 37°C in LB medium containing kanamycin antibiotics to obtain recombinant expression strains expressing wild-type and mutant totipotent nucleases. Take 600 μl of the first-level recombinant expression bacterial solution and inoculate it into 200 ml of LB medium containing kanamycin, and culture it at 37°C until the OD600 range is 1.5-3.5; prepare the animal-free Escherichia coli self-induction medium for amplification culture in advance, treat it aseptically, and add the secondary seed liquid fermented in the previous step to the prepared culture medium at a volume ratio of 1.4%. Set the fermentation temperature to 37°C and the speed to 200 rpm. Adjust the pressure and air flow according to the situation. Monitor the pH value changes during the fermentation process. End the fermentation after 11 hours of culture. Use a centrifuge at 4000 rpm / min and collect the fermentation supernatant for 15 minutes.

[0147] The supernatant was adjusted to pH 6.0 and filtered through a 0.22 μm filter membrane. The supernatant was then concentrated and diafiltered. After 4-5 times concentration, the equilibration buffer (20 mM NaAC, 2 mM MgCl2, 20% Glycerol, pH 6.0) was added for dilution, and the sample was concentrated again to the original volume. The dilution and concentration cycle was repeated 4-5 times. The concentrated and diafiltered sample was loaded onto a cation exchange column, and the target protein bound to the column was eluted with a buffer containing 20 mM NaAC, 2 mM MgCl2, 350 mM NaCl, and 20% Glycerol (pH 6.0). To obtain a protein with a single conformation, the peak collected in the previous elution step was further passed through a molecular sieve and eluted with a buffer containing 25 mM Tris, 5 mM MgCl2, 500 mM NaCl, and 20% Glycerol (pH 7.5). The fractions were collected and sent for SDS-PAGE and HPLC analysis.

[0148] According to the test results (exemplary, as shown in Figures 2 and 3), peaks with SDS-PAGE and HPLC purity higher than 95% were pooled, supplemented with 50% glycerol, and stored at -20°C until use. Protein concentration was measured by spectrophotometry at a wavelength of 280 nm.

[0149] Example 3 Comparison of enzyme activity between wild type and mutant at different salt concentrations

[0150] To determine the enhanced activity of the mutant ubiquitinase at different salt concentrations, the wild-type and mutant strains were diluted to the appropriate multiple (within the linear range after dilution). 400 μl of salmon sperm DNA (1 mg / ml) was used as substrate, followed by an equal volume of the diluted enzyme solution. The reaction volume was then made up to 500 μl with reaction buffer (50 mM Tris, 5 mM MgCl2, 0.1 mg / ml BSA, pH 8.0). The reaction solution was mixed and incubated in a 37°C water bath for 30 min. The reaction was terminated by the addition of 500 μl of 20% trichloroacetic acid. After incubation on ice for at least 10 min, 200 μl of each reaction solution was added to a centrifuge tube containing 1800 μl of double-distilled water and mixed. The absorbance of the dilutions was measured at 260 nm using a UV spectrophotometer. One activity unit (U) is defined as the amount of enzyme required to increase the absorbance at 260 nm by 1.0 over 30 minutes at 37°C in the presence of excess substrate. The enzymatic activity of the universal nuclease at different salt concentrations was determined according to the above-described embodiment by adjusting the NaCl concentration in the reaction system to 0 mM, 100 mM, 200 mM, 300 mM, 400 mM, and 500 mM, and the specific enzyme activity was calculated based on the test results.

[0151] The results are shown in Figure 4: the wild-type omnipotent nuclease is more sensitive to salt, with only 40% and 30% relative enzyme activity left under 200mM and 300mM NaCl conditions, respectively. Under conditions above 300mM NaCl, the activity is almost lost. Compared with the wild-type, the mutant can maintain the original enzyme activity without reduction under salt-free or very low-salt conditions, and at the same time has a significantly improved tolerance to salt.

[0152] As can be seen from Figure 4, all mutants can maintain 80% or even higher activity under 200 mM NaCl conditions, which is significantly improved compared to the wild type; among them, the eight mutants: T98K, T56D / T98K, T98K / N101H, T98K / G242D, T56D / T98K / G242D, N101H / T98K / G242D, T56D / T98K / N101H, T56D / N101H / T98K / G242D can maintain 80% or even higher activity under 200 mM NaCl conditions, which is significantly improved compared to the wild type; The activity was significantly improved under NaCl conditions, and the activities could be maintained at 70%, 50%, 20% or more, respectively, or even higher. All of the above mutants contained the T98K mutation, indicating the importance of this mutation in improving the salt tolerance of the all-potent nuclease. The other seven mutants, T56D, N101H, G242D, T56D / N101H, T56D / G242D, N101H / G242D, and T56D / N101H / G242D, showed limited activity improvement under 300mM, 400mM, and 500mM NaCl conditions, and could maintain activities at 43%, 25%, 11% or more, respectively, but were still improved compared to the wild type.

[0153] Compared to commercial products in the prior art, such as Salt Active nuclease (Yisheng Bio, China), which exhibits optimal enzymatic activity at 500 mM, the relative enzymatic activity is only about 25% of the optimal activity of the wild-type all-potent nuclease (0 mM NaCl). At NaCl concentrations below 500 mM, the relative enzymatic activity is far below 25%. The variants screened in this application not only exhibit relative enzymatic activities far exceeding 25% at NaCl concentrations below 500 mM, but some even exhibit enzymatic activities superior to commercial Salt Active nuclease at 500 mM NaCl.

[0154] In summary, the new variants of the universal nuclease screened in this application have a wider range of uses and better activity.

[0155] The results of the above examples demonstrate that mutating amino acids at specific sites within the wild-type sequence can effectively increase the tolerance of the universal nuclease to varying salt concentrations, thereby enabling its wider application in environments with varying salt concentrations. Mutants with the highest cumulative number of mutations demonstrated superior performance compared to the wild-type, but individual single, double, and triple mutations also achieved similar results. This suggests that more mutations are not necessarily better; the key lies in identifying the amino acid sites that play a decisive role.

[0156] The foregoing descriptions of specific exemplary embodiments of the present application are for purposes of illustration and description. These descriptions are not intended to limit the present application to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments are selected and described for the purpose of explaining the specific principles of the present application and their practical application, thereby enabling those skilled in the art to realize and utilize the various exemplary embodiments of the present application and various options and modifications. The scope of the present application is intended to be defined by the claims and their equivalents.

Claims

1. A universal nuclease variant with improved salt tolerance, characterized in that The variant comprises at least one or more amino acid changes in the Benzonase nuclease sequence.

2. A method for improving the salt tolerance activity of a universal nuclease, characterized in that: The method comprises introducing changes in at least one or more amino acid sites into the Benzonase nuclease sequence.

3. The all-purpose nuclease variant according to claim 1 or the method according to claim 2, characterized in that: The sites include any one or more of the 56th, 98th, 101st and / or 242nd positions; Preferably, the site includes position 98, and further includes position 56, position 101 and / or position 242.

4. The all-purpose nuclease variant according to claim 1 or the method according to claim 2, characterized in that: The changes include any one or more of T56D, T98K, N101H and / or G242D; Preferably, the change includes T98K, and further includes any one or more of T56D, N101H and / or G242D.

5. The all-purpose nuclease variant according to claim 1 or the method according to claim 2, characterized in that: The Benzonase nuclease is a wild-type Benzonase nuclease; Preferably, the sequence of the wild-type Benzonase nuclease is shown as SEQ ID NO.

1.

6. A nuclease gene, characterized in that The gene encodes the all-potent nuclease variant according to any one of claims 1 and 3-5.

7. An expression cassette, plasmid, vector, host cell or engineered microorganism, characterized in that: Contains the nuclease gene according to claim 6.

8. A method for preparing a universal nuclease variant, characterized in that it is obtained by expression using the expression frame, plasmid, vector, host cell or engineered microorganism according to claim 7.

9. Use of the all-potent nuclease variant according to any one of claims 1, 3-5 in nuclease cleavage or removal.

10. A method for enzymatic cleavage or removal of nucleic acids, characterized in that: The nucleic acid is treated with the universal nuclease variant according to any one of claims 1 and 3 to 5; preferably, the treatment is carried out at a salt concentration of 0-500 mM.

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