Salt-tolerant totipotent nuclease mutants and their applications

Abstract

This application relates to the field of enzyme engineering technology and specifically provides a variant of a totipotent nuclease. Compared to the wild-type totipotent nuclease, the variant has a wider tolerance range for salt concentrations and can maintain good enzyme activity even at increasing salt concentrations, thus expanding the range of applications of the totipotent nuclease at different salt concentrations.

Description

Technical Field

[0001] The present application relates to enzymatic engineering technology, in particular to a broadly active nuclease mutant with improved salt tolerance, a method of use and use thereof.

[0002] Cross-Reference to Related Applications The present application claims priority based on the Chinese application filed with the China National Intellectual Property Administration on September 26, 2023, with the application number 202311249502.3 and the title "A broadly active nuclease mutant with improved salt tolerance and use thereof", the entire content of which is incorporated into the present disclosure by reference.

Background Art

[0003] Broadly active nuclease, also called non-restriction endonuclease, mainly functions to remove nucleic acids from biological products. In scientific research, it not only reduces the viscosity of cell supernatant and cell lysate, and improves protein purification efficiency and functional research, but also is applied as a reagent for removing host-derived residual nucleic acids in the industries of virus purification, vaccine production, production of protein and polysaccharide pharmaceutical products. By reducing host-derived residual nucleic acids to pg level, it can improve the efficacy and safety of biological products, can effectively prevent aggregation of human peripheral blood mononuclear cells (PBMC) in cell therapy and vaccine research, and is an essential tool enzyme in the field of CGT.

[0004] The most popular totipotent nuclease currently is the bacterial nuclease derived from Serratia marcescens, published by Benedik and Strych in 1998 (FEMS Microbiol Lett. 165:1-13). Registered as a trademark (Benzonase®) by Merck, its optimal activity temperature is 37°C. However, its main drawback is its low tolerance to increasing salt concentrations; data shows strong inhibition of this nuclease activity by sodium and potassium ions. Enzyme activity is almost completely lost when the salt concentration in the reaction system exceeds 300 mM. This characteristic limits the application range of totipotent nucleases, as they can only function in salt-free or low-salt environments. In both industrial bioproduct manufacturing processes and scientific research on recombinant protein acquisition, various salt concentrations are added to buffer systems to ensure process stability. Therefore, there is an urgent need for the development and production of totipotent nucleases with significantly improved salt tolerance.

[0005] Enzymes derived from halophilic microorganisms enable the search for salt-tolerant enzymes, and several salt-tolerant nucleases from marine microorganisms have been reported. In 2022 (CN114651062A), Marcin et al. disclosed a novel thermolabile nuclease that can withstand a salt concentration of 500 mM. However, this enzyme maintains high activity only at low temperatures, especially between 4°C and 8°C, and has problems such as low yield, high production costs, and severely limited application environments. Salt Active nuclease (Yi Sheng Zhuo, China) is a commercially available product, but although it shows the highest activity under 500 mM NaCl conditions, its relative specific activity is only about 25% of the optimal specific activity of benzonase. Furthermore, at salt concentrations below 500 mM, its activity decreases significantly with decreasing salt concentration, and its acceptable salt concentration range is similarly narrow. Due to these characteristics, its application is limited to salt concentrations below 500 mM. Therefore, equivalent enzymes from halophilic organisms do not always exist.

[0006] In light of the above, this application is submitted. [Overview of the project]

[0007] In response to the technical problems described above, this application provides a totipotent nuclease variant with a wide salt concentration tolerance range through rational design. This solves the practical problem of the narrow salt concentration tolerance range of current totipotent nucleases, resulting in a broader range of application, greater versatility, and the ability to achieve nucleic acid removal effects in various salt concentration systems.

[0008] Specifically, this application provides a nuclease based on benzonase, a totipotent nuclease, that improves its tolerance to salt concentrations, particularly in the 200 mM to 500 mM range, without compromising its original activity, thereby providing a nuclease with a wider salt concentration tolerance. According to this application, the inherent advantages of benzonase—high activity, stability, and high yield at salt-free or low-salt concentrations—are maintained while also improving its tolerance to high salt concentrations.

[0009] Therefore, this application has at least the following four objectives:

[0010] The first object of this application is to provide a totipotent nuclease mutant with improved salt tolerance.

[0011] A second objective of this application is to provide an application for nucleic acid processing of a totipotent nuclease mutant with improved salt tolerance.

[0012] A third object of this application is to provide a method for preparing totipotent nuclease mutants with improved salt tolerance.

[0013] A fourth object of this application is to provide a method for using a totipotent nuclease mutant with improved salt tolerance.

[0014] To achieve the above objective, this application proposes the following technical solution.

[0015] This application first provides a totipotent nuclease (or totipotent nuclease mutant) with improved salt tolerance, wherein the mutant comprises a mutation in at least one amino acid site in the sequence of the benzonase nuclease.

[0016] Furthermore, the said region includes one or more of the 56th, 98th, 101st, and / or 242nd regions.

[0017] More preferably, the region includes the 98th region, and further includes one or more of the 56th, 101st, and / or 242nd regions.

[0018] Furthermore, the mutations include one or more of T56D, T98K, N101H, and / or G242D.

[0019] More preferably, the mutation comprises T98K and further comprises one or more of T56D, N101H, and / or G242D.

[0020] Furthermore, the aforementioned benzonase nuclease is a wild-type benzonase nuclease.

[0021] More preferably, the amino acid sequence of the wild-type benzonase nuclease is the sequence shown in SEQ ID NO.1.

[0022] In some specific embodiments, the specific sequence of the variant includes one or more sequences shown in SEQ ID NO. 2 to 16.

[0023] This application further provides a method for improving the salt tolerance of a totipotent nuclease, the method comprising the step of introducing a mutation at least one amino acid site into the sequence of a benzonase nuclease.

[0024] Further, said site(s) comprise(s) any one or more of the 56th position, the 98th position, the 101st position and / or the 242nd position.

[0025] More preferably, said site comprises the 98th position, and further comprises any one or more of the 56th position, the 101st position and / or the 242nd position.

[0026] Further, said mutation(s) comprise(s) any one or more of T56D, T98K, N101H and / or G242D.

[0027] More preferably, said mutation comprises T98K, and further comprises any one or more of T56D, N101H and / or G242D.

[0028] Further, said Benzonase nuclease is a wild-type Benzonase nuclease.

[0029] More preferably, the amino acid sequence of said wild-type Benzonase nuclease is the sequence set forth in SEQ ID NO: 1.

[0030] In some specific embodiments, the specific sequence of any one or more of said variants is a sequence set forth in SEQ ID NOs: 2 to 16.

[0031] The present application further provides an endonuclease gene encoding any one of the above-described omnipotent nuclease variants.

[0032] The present application further provides an expression cassette, a plasmid and a vector comprising the above-described endonuclease gene.

[0033] The present application further provides a host cell comprising the above-described endonuclease gene, or the above-described expression cassette, plasmid or vector.

[0034] This application further provides a method for preparing the above-mentioned totipotent nuclease variants, which are obtained by expression using the above-mentioned expression cassette, plasmid, vector, host cell, or recombinant microorganism.

[0035] In some specific embodiments, the process includes obtaining recombinant expression plasmids containing the above-mentioned mutants, transforming each recombinant expression plasmid into, for example, E. coli, selecting positive clones, and obtaining recombinant expression strains that express wild-type and post-mutation totipotent nucleases. Furthermore, the process includes activating these strains, inoculating them into a fermenter, fermenting them by a self-induction method, and collecting the fermentation supernatant.

[0036] This application further provides the use of the above-mentioned totipotent nuclease variant in the cleavage or removal of nucleic acids.

[0037] More preferably, the use is carried out at a salt concentration of 0 to 500 mM.

[0038] This application further provides a method for cleaving or removing nucleic acids, wherein the method involves treating the nucleic acid with any one of the above-described totipotent nuclease variants, preferably the treatment being carried out at a salt concentration of 0 to 500 mM.

[0039] Beneficial technical effects of this application: 1) The mutation or combination of mutations described in this application exhibits significantly improved salt tolerance compared to the wild type. While the wild-type totipotent nuclease almost completely lost activity under conditions exceeding 300 mM NaCl, the mutant maintained its original enzyme activity under salt-free or low-salt conditions, while also maintaining over 80% activity even under 200 mM NaCl conditions. In particular, eight mutants—T98K, T56D / T98K, T98K / N101H, T98K / G242D, T56D / T98K / G242D, N101H / T98K / G242D, T56D / T98K / N101H, and T56D / N101H / T98K / G242D—show significantly improved activity under 300mM, 400mM, and 500mM NaCl conditions, maintaining activity levels of 70%, 50%, and 20% or higher, respectively. This expands the applicability range of totipotent nucleases at different salt concentrations, allowing for reduced usage in high-salt buffer systems while achieving superior nucleic acid removal efficacy.

[0040] 2) The mutated totipotent nuclease described in this application solves the problem of the narrow salt concentration tolerance range of current totipotent nucleases, resulting in a wider range of applications and suitability for industrial applications. [Brief explanation of the drawing]

[0041] [Figure 1] This shows a three-dimensional diagram of the wild-type totipotent nuclease and the location of mutations. [Figure 2] This is an SDS-PAGE diagram of the recombinant totipotent nuclease production process. M indicates the protein marker, S indicates the recovered fermentation supernatant, SPFF indicates the eluted peak after passing the sample through the ion exchange column, and SEC indicates the eluted peak after passing the sample through the molecular sieve. [Figure 3] This shows the purity of recombinant totipotent nuclease as determined by HPLC. [Figure 4]This shows a comparison of the relative enzyme specific activity of wild-type totipotent nuclease and mutant totipotent nuclease under different salt concentration conditions. WT represents the wild-type totipotent nuclease, and Competitor Y represents the high-salt-tolerant nuclease produced by the same company. [Modes for carrying out the invention]

[0042] This application discloses several totipotent nuclease variants, which, compared to wild-type totipotent nucleases, have a wider range of tolerable salt concentrations and can maintain a certain level of activity even as salt concentrations increase. Those skilled in the art can realize the applications of this specification by reference to its contents, and in particular, all similar substitutions and modifications are obvious to those skilled in the art and are included in this application. The preparation methods and uses relating to this application are illustrated by preferred embodiments. Those skilled in the art can realize and apply the technology of this application by modifying, changing, or combining the preparation methods and uses relating to this application without departing from the contents, spirit and scope of this application. Unless otherwise noted, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.

[0043] The following terms and definitions are provided for the ease of understanding this application. These definitions should not be understood to be less than what a person skilled in the art would understand.

[0044] Unless otherwise defined below, all technical and scientific terms used in the specific embodiments of this application are intended to have the same meaning as those commonly understood by those skilled in the art. While the following terms are expected to be readily understood by those skilled in the art, their definitions are provided below for the purpose of better illustrating this application.

[0045] The terms “inclusive,” “includes,” “have,” “contains,” and “concern” as used in this application are inclusive or open, and do not exclude any other unlisted elements or steps of the method. The term “consisting of” is considered a preferred embodiment of the term “includes.” Where a group is defined below as containing at least a certain number of embodiments, this should be understood to mean a preferred group consisting only of these embodiments.

[0046] Indefinite or definite articles used with singular nouns, such as "one," "one kind," and "the aforementioned," also include the plural form of the noun.

[0047] In this application, the terms "approximately" and "about" indicate a range of precision that can be understood by a person skilled in the art, which guarantees the technical effect of the described features. This term usually means allowing a deviation of ±10%, preferably ±5%, from the stated value.

[0048] For example, terms like "more than," "at least," and "exceeding" such as "at least one" are expressed as "at least 1, 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 This means that the range includes, but is not limited to, values ​​of 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, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or values ​​exceeding the listed values. It also includes any larger numbers or fractions in between.

[0049] Conversely, the term "less than or equal to" includes all values ​​smaller than the listed value. For example, "less than or equal to 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, 54 It contains 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. It also includes any smaller numbers or fractions in between.

[0050] Terms such as "multiple," "at least two," "more than two," and "at least the second" are used for 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, 6 It should be understood that this includes, but is not limited to, 0, 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, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. It also includes any larger numbers or fractions in between.

[0051] The salt-tolerant totipotent nuclease described in this application is also called a totipotent nuclease variant, and the variant includes a mutation in at least one amino acid site in the sequence of the benzonase nuclease.

[0052] Furthermore, the term "mutant" as used herein is merely a representational term, and any sequence containing mutations in one or more amino acid sites within a benzonase nuclease sequence is included in the category of "mutant sequences" as described herein. Therefore, mutants may include both mutants and non-mutants. The specific methods for obtaining or preparing mutants are also not limited. For example, in some specific embodiments, the mutants are obtained directly by chemical synthesis. In another specific embodiment, the mutants are obtained directly by recombination. In yet another specific embodiment, the mutants are obtained by introducing mutant amino acids from a wild-type sequence using genetic engineering or genetic methods.

[0053] Furthermore, the term "mutation" as used herein includes but is not limited to spontaneous mutations. Mutant sequences obtained through non-mutation also have similar effects and functions in this application and fall within the scope of protection of this application.

[0054] Typically, the benzonase nucleases applied herein have at least about 65% sequence identity with the nuclease of SEQ ID NO. 1, and in particular, at least about 70%, 75%, 80%, 85%, 90%, 95%, and 99% sequence identity. While these differences in homology may be due to species differences, all mutations referred to herein that occur in these basic sequences are within the scope of protection of this application, provided that the sequences of the core functional domains match.

[0055] In some embodiments, the benzonase nuclease described herein is the wild-type benzonase nuclease, and in some specific embodiments, the amino acid sequence of the wild-type benzonase nuclease is specifically the sequence shown in SEQ ID NO. 1. Furthermore, bacterial benzonase nucleases derived from Serratia marcescens, acquired from different origins or habitats, may have some amino acid differences in their wild-type sequences, but these can also be understood as wild-type sequences. Therefore, the amino acid sequences of the benzonase nucleases relating to this application should theoretically not be limited to sequence SEQ ID NO. 1, but rather wild-type sequences having at least about 85% sequence identity with the nuclease of SEQ ID NO. 1, and in particular, at least about 90%, 95%, and 99% sequence identity. As long as the sequences of the core functional domains match, foreseeable mutations described herein arising from these basic wild-type sequences are also within the scope of protection of this application.

[0056] As can be seen from the examples of this application, the number of mutations or mutant sites in this application is not limited. Experimental data in this application have shown that any mutations containing one mutant site, such as T56D or T98K, or any two mutant sites, such as T56D and G242D, or T98K and G242D, or three sites, such as T56D, T98K, and G242D, all exhibit excellent salt tolerance and basal enzyme activity. Therefore, any mutant containing one, two, three, or four variants as defined in this application falls within the scope of protection.

[0057] Therefore, the benzonase nuclease variant according to this application contains at least one mutation compared to the corresponding wild-type benzonase nuclease. In some embodiments, the at least one mutation occurs at one or more of the following sites in the wild-type benzonase nuclease sequence (particularly corresponding to SEQ ID NO. 1): 56, 98, 101, and 242.

[0058] In some specific embodiments, the wild-type benzonase nuclease contains at least one mutation, the at least one mutation occurring at the 56th position of the sequence corresponding to SEQ ID NO.1, and the preferred mutation is T56D.

[0059] In some more specific embodiments, the mutant sequence is the sequence shown in SEQ ID NO.2.

[0060] SEQ ID NO.2:(T56D) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSDTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYTGANAALKVDRGHQAPLASLAGVSDWESLNYLSNIT PQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN.

[0061] In some specific embodiments, the wild-type benzonase nuclease comprises at least one mutation, the at least one mutation occurring at the 98th position of the sequence corresponding to SEQ ID NO.1, and a preferred mutation is T98K.

[0062] In some more specific embodiments, the mutant sequence is the sequence shown in SEQ ID NO.3.

[0063] SEQ ID NO.3:(T98K) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYKGANAALKVDRGHQAPLASLAGVSDWESLNYLSNIT PQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN.

[0064] In some specific embodiments, the wild-type benzonase nuclease comprises at least one mutation, the at least one mutation occurring at the 101st position of the sequence corresponding to SEQ ID NO.1, and a preferred mutation is N101H.

[0065] In some more specific embodiments, the mutant sequence is the sequence shown in SEQ ID NO.4.

[0066] SEQ ID NO.4:(N101H) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYTGAHAALKVDRGHQAPLASLAGVSDWESLNYLSNIT PQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN.

[0067] In some specific embodiments, the wild-type benzonase nuclease contains at least one mutation, the at least one mutation occurring at the 242nd position of the sequence corresponding to SEQ ID NO.1, and a preferred mutation is G242D.

[0068] In some more specific embodiments, the mutant sequence is the sequence shown in SEQ ID NO. 5.

[0069] SEQ ID NO.5:(G242D) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYTGANAALKVDRGHQAPLASLAGVSDWESLNYLSNIT PQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWADLPDDVQASLKSKPGVLPELMGCKN.

[0070] In some specific embodiments, the wild-type benzonase nuclease comprises at least two mutations, the at least two mutations occurring at positions 56 and 98 of the sequence corresponding to SEQ ID NO.1, with preferred mutations being T56D and T98K.

[0071] In some more specific embodiments, the mutant sequence is the sequence shown in SEQ ID NO. 6.

[0072] SEQ ID NO.6:(T56D / T98K) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSDTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYKGANAALKVDRGHQAPLASLAGVSDWESLNYLSNIT PQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN.

[0073] In some specific embodiments, the wild-type benzonase nuclease comprises at least two mutations, the at least two mutations occurring at positions 56 and 242 of the sequence corresponding to SEQ ID NO.1, with preferred mutations being T56D and G242D.

[0074] In some more specific embodiments, the mutant sequence is the sequence shown in SEQ ID NO.7.

[0075] SEQ ID NO.7:(T56D / G242D) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSDTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYTGANAALKVDRGHQAPLASLAGVSDWESLNYLSNIT PQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWADLPDDVQASLKSKPGVLPELMGCKN.

[0076] In some specific embodiments, the wild-type benzonase nuclease contains at least two mutations, the at least two mutations occurring at positions 56 and 101 of the sequence corresponding to SEQ ID NO.1, with preferred mutations being T56D,N101H.

[0077] In some more specific embodiments, the mutant sequence is the sequence shown in SEQ ID NO. 8.

[0078] SEQ ID NO.8:(T56D / N101H) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSDTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYTGAHAALKVDRGHQAPLASLAGVSDWESLNYLSNIT PQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN.

[0079] In some specific embodiments, the wild-type benzonase nuclease contains at least two mutations, the at least two mutations occurring at positions 98 and 242 of the sequence corresponding to SEQ ID NO.1, with preferred mutations being T98K and G242D.

[0080] In some more specific embodiments, the mutant sequence is the sequence shown in SEQ ID NO.9.

[0081] SEQ ID NO.9:(T98K / G242D) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYKGANAALKVDRGHQAPLASLAGVSDWESLNYLSNIT PQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWADLPDDVQASLKSKPGVLPELMGCKN.

[0082] In some specific embodiments, the wild-type benzonase nuclease comprises at least two mutations, the at least two mutations occurring at positions 98 and 101 of the sequence corresponding to SEQ ID NO.1, with preferred mutations being T98K,N101H.

[0083] In some more specific embodiments, the mutant sequence is the sequence shown in SEQ ID NO. 10.

[0084] SEQ ID NO.10: (T98K / N101H) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYKGAHAALKVDRGHQAPLASLAGVSDWESLNYLSNIT PQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN.

[0085] In some specific embodiments, the wild-type benzonase nuclease comprises at least two mutations, the at least two mutations occurring at positions 101 and 242 of the sequence corresponding to SEQ ID NO.1, with preferred mutations being G242D and N101H.

[0086] In some more specific embodiments, the mutant sequence is the sequence shown in SEQ ID NO. 11.

[0087] SEQ ID NO.11:(G242D / N101H) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYTGAHAALKVDRGHQAPLASLAGVSDWESLNYLSNIT PQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWADLPDDVQASLKSKPGVLPELMGCKN.

[0088] In some specific embodiments, the wild-type benzonase nuclease comprises at least three mutations, the at least three mutations occurring at positions 56, 98, and 101 of the sequence corresponding to SEQ ID NO.1, with preferred mutations being T56D, T98K, and N101H.

[0089] In some more specific embodiments, the mutant sequence is the sequence shown in SEQ ID NO. 12.

[0090] SEQ ID NO.12:(T56D / T98K / N101H) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSDTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYKGAHAALKVDRGHQAPLASLAGVSDWESLNYLSNIT PQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN In some specific embodiments, the wild-type benzonase nuclease comprises at least three mutations, the at least three mutations occurring at positions 56, 98, and 242 of the sequence corresponding to SEQ ID NO.1, with preferred mutations being T56D, T98K, and G242D.

[0091] In some more specific embodiments, the mutant sequence is the sequence shown in SEQ ID NO. 13.

[0092] SEQ ID NO.13:(T56D / T98K / G242D) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSDTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYKGANAALKVDRGHQAPLASLAGVSDWESLNYLSNIT PQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWADLPDDVQASLKSKPGVLPELMGCKN

[0093] In some specific embodiments, the wild-type benzonase nuclease comprises at least three mutations, the at least three mutations occurring at positions 98, 101, and 242 of the sequence corresponding to SEQ ID NO.1, with preferred mutations being T98K, G242D, and N101H.

[0094] In some more specific embodiments, the mutant sequence is the sequence shown in SEQ ID NO. 14.

[0095] SEQ ID NO.14:(T98K / G242D / N101H) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYKGAHAALKVDRGHQAPLASLAGVSDWESLNYLSNIT PQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWADLPDDVQASLKSKPGVLPELMGCKN.

[0096] In some specific embodiments, the wild-type benzonase nuclease contains at least three mutations, the at least three mutations occurring at positions 56, 101, and 242 of the sequence corresponding to SEQ ID NO.1, with preferred mutations being T56D, G242D, and N101H.

[0097] In some more specific embodiments, the mutant sequence is the sequence shown in SEQ ID NO. 15.

[0098] SEQ ID NO.15:(T56D / N101H / G242D) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSDTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYTGAHAALKVDRGHQAPLASLAGVSDWESLNYLSNIT PQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWADLPDDVQASLKSKPGVLPELMGCKN

[0099] In some specific embodiments, the wild-type benzonase nuclease comprises at least four mutations, the at least four mutations occurring at positions 56, 98, 101, and 242 of the sequence corresponding to SEQ ID NO.1, with preferred mutations being T56D, T98K, G242D, and N101H.

[0100] In some more specific embodiments, the mutant sequence is the sequence shown in SEQ ID NO. 16.

[0101] SEQ ID NO.16:(T56D / T98K / G242D / N101H) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSDTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYKGAHAALKVDRGHQAPLASLAGVSDWESLNYLSNIT PQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWADLPDDVQASLKSKPGVLPELMGCKN.

[0102] According to the data in the examples of this application, the eight mutants T98K, T56D / T98K, T98K / N101H, T98K / G242D, T56D / T98K / G242D, N101H / T98K / G242D, T56D / T98K / N101H, and T56D / N101H / T98K / G242D show significantly improved activity under 300 mM, 400 mM, and 500 mM NaCl conditions, maintaining activity of 70%, 50%, and 20% or more, respectively. Therefore, in some preferred embodiments of this application, the wild-type benzonase nuclease includes at least the 98th mutation, for example, T98K, and further includes mutations at one or more other sites (T56D / G242D / N101H).

[0103] Furthermore, in order to satisfy the normal basic enzyme activity of Benzonase, those skilled in the art may further apply other known effective site mutations based on the mutations described in this application for practical optimization and improvement. Since such mutations are made based on this application, they fall within the scope of protection of this application, and their corresponding effects can be reasonably predicted by those skilled in the art based on a basic understanding of enzyme structure.

[0104] The method for improving the salt tolerance of a totipotent nuclease according to this application includes the step of introducing a mutation or modification of at least one amino acid site into the sequence of a benzonase nuclease. The theory of these mutations and modifications is the same as described above.

[0105] For example, in some embodiments, the portion includes the 56th, 98th, 101st, and / or 242nd portion. In some preferred embodiments, the portion includes the 98th portion, and further includes the 56th, 101st, and / or 242nd portion.

[0106] In some specific embodiments, the mutation comprises one or more of T56D, T98K, N101H, and / or G242D. In some preferred embodiments, the mutation comprises T98K and further comprises one or more of T56D, N101H, and / or G242D.

[0107] The endonuclease gene relating to this application includes any gene sequence capable of encoding any one of the above-mentioned totipotent nuclease variants.

[0108] The expression cassette relating to this application may be an expression cassette containing any one of the above-mentioned endonuclease genes.

[0109] The plasmid relating to this application is a plasmid containing any one of the above-mentioned endonuclease genes, and is preferably an expression plasmid.

[0110] The vector relating to this application is a vector containing any one of the above-mentioned endonuclease genes, and is preferably an expression vector.

[0111] The cells relating to this application are cells having a vector capable of expressing any one of the above-mentioned endonuclease genes, and are preferably host cells.

[0112] The technical invention of this application will be clearly and completely described below with reference to the examples, and it goes without saying that the examples described are only a selection of examples of this application, not all of them. All other examples that a person skilled in the art could obtain without using their inventive ability based on the examples of this application are all within the scope of protection of this application.

[0113] Examples The technical invention of this application will be clearly and completely described below with reference to the drawings, and it goes without saying that the embodiments described are only a selection of embodiments of this application, not all embodiments. All other embodiments that a person skilled in the art could obtain without using their inventive ability based on the embodiments of this application are all within the scope of protection of this application.

[0114] Example 1: Mutant Design In the early stages of the research for this application, we discovered that by introducing mutations at specific sites based on the wild-type nonspecific nuclease Benzonase®, we could obtain mutants with significantly improved salt tolerance. Specifically, in designing the mutants, we studied the preference for amino acid selection on the structural surface of salt-tolerant enzymes, and after appropriate design and selection considering their three-dimensional structure, we selected mutations at the following multiple sites. We introduced mutations predicted to impart salt tolerance into the wild-type totipotent nuclease using site-directed mutagenesis.

[0115] Through extensive mutation design and selection, this application identifies four optimal mutation sites that potentially broaden the acceptable range of salt concentrations, which are sites 56, 98, 101, and 242, respectively (see Figure 1).

[0116] Therefore, this application designed and validated various combination mutations based on the wild-type sequence (SEQ ID NO.1), with positions 56, 98, 101, and 242 being key sites.

[0117] SEQ ID NO.1: MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYTGANAALKVDRGHQAPLASLAGVSDWESLNYLSNIT PQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN.

[0118] The specific combination mutations designed include T56D / T98K / N101H / G242D. That is, based on SEQ ID NO.1, amino acid sequences were designed that simultaneously contain one, more, or all of the T56D, T98K, N101H, and G242D mutations.

[0119] The specific information is as follows: [Table 1]

[0120] The specific sequence information is as follows:

[0121] SEQ ID NO.2:(T56D) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSDTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYTGANAALKVDRGHQAPLASLAGVSDWESLNYLSNIT PQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN

[0122] SEQ ID NO.3:(T98K) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYKGANAALKVDRGHQAPLASLAGVSDWESLNYLSNIT PQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN

[0123] SEQ ID NO.4:(N101H) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALPADTLAPADYTGAHAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN

[0124] SEQ ID NO.5:(G242D) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALPADTLAPADYTGANAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWADLPDDDVQASLKSKPGVLPELMGCKN

[0125] SEQ ID NO.6:(T56D / T98K) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSDTKFANWVAYHITKDTPASGKTRNWKTDPALPADTLAPADYKGANAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN

[0126] SEQ ID NO.7:(T56D / G242D) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSDTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYTGANAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWADLPDDVQASLKSKPGVLPELMGCKN

[0127] SEQ ID NO.8:(T56D / N101H) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSDTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYTGAHAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN

[0128] SEQ ID NO.9:(T98K / G242D) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALPADTLAPADYKGANAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWADLPDDDVQASLKSKPGVLPELMGCKN

[0129] SEQ ID NO.10:(T98K / N101H) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALPADTLAPADYKGAHAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN

[0130] SEQ ID NO.11:(G242D / N101H) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALPADTLAPADYTGAHAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWADLPDDDVQASLKSKPGVLPELMGCKN

[0131] SEQ ID NO.12:(T56D / T98K / N101H) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSDTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYKGAHAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN

[0132] SEQ ID NO.13:(T56D / T98K / G242D) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSDTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYKGANAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWADLPDDVQASLKSKPGVLPELMGCKN

[0133] SEQ ID NO.14:(T98K / G242D / N101H) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALPADTLAPADYKGAHAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWADLPDDDVQASLKSKPGVLPELMGCKN

[0134] SEQ ID NO.15: (T56D / N101H / G242D) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSDTKFANWVAYHITKDTPASGKTRNWKTDPALPADTLAPADYTGAHAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWADLPDDDVQASLKSKPGVLPELMGCKN

[0135] SEQ ID NO.16: (T56D / T98K / G242D / N101H) MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSDTKFANWVAYHITKDTPASGKTRNWKTDPALPADTLAPADYKGAHAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWADLPDDDVQASLKSKPGVLPELMGCKN.

[0136] Example 2: Production of Recombinant Mutants Gene fragments corresponding to SEQ ID NO.1 and SEQ ID NO.2-16 in Example 1 were inserted into the pET41a vector to obtain recombinant expression plasmids. Each recombinant expression plasmid was introduced into E. coli BL21(DE3) competent cells by heat shock transformation, and positive clones were selected. Recombinant expression strains expressing wild-type and mutant totipotent nucleases were obtained by culturing overnight at 37°C in LB medium containing kanamycin antibiotic. 600 μl of the primary recombinant expression suspension was taken and inoculated into 200 ml of LB medium containing kanamycin, and cultured at 37°C until the OD600 was in the range of 1.5-3.5. Animal component-free E. coli autoinducing medium for amplification culture was prepared in advance and sterilized. The secondary seed liquid fermented in the previous step was inoculated into the prepared medium at a volume ratio of 1.4%, the fermentation temperature was set to 37°C, the rotation speed to 200 rpm, and the pressure and air flow rate were adjusted as needed. During the fermentation process, pH changes were monitored. After 11 hours of incubation, fermentation was terminated, and the supernatant of the fermentation liquid was collected by centrifugation at 4000 rpm / min for 15 minutes using a centrifuge.

[0137] The supernatant was then adjusted to pH 6.0 and filtered through a 0.22 μm filter. Afterward, the solution was concentrated and washed, and concentrated 4-5 times. It was then diluted with equilibrium buffer (20 mM NaAC, 2 mM MgCl2, 20% Glycerol, pH 6.0), re-concentrated to the original volume, and this dilution and concentration process was repeated 4-5 times. The concentrated and washed sample was injected into 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 proteins with a single conformation, the peaks eluted and recovered in the previous step were further subjected to molecular sieving, eluted with a buffer containing 25 mM Tris, 5 mM MgCl2, 500 mM NaCl, and 20% Glycerol (pH 7.5), and then fractionated and recovered. Each fraction was then analyzed by SDS-PAGE and HPLC.

[0138] Based on the measurement results (as shown in Figures 2 and 3 for illustrative purposes), peaks with purity exceeding 95% in both SDS-PAGE and HPLC were selected, glycerol was added to 50%, and the samples were stored at -20°C. The protein concentration was measured at a wavelength of 280 nm using spectrophotometric spectroscopy.

[0139] Example 3: Comparison of enzyme activity between wild-type and mutant at different salt concentrations. To confirm the activity enhancement effect of the mutated totipotent nuclease at different salt concentrations, the wild-type and mutant strains were diluted to appropriate multiples (the measured values ​​after dilution were within a linear range). First, 400 μl of salmon testis-derived DNA (1 mg / ml) was taken as the substrate, then the same volume of diluted enzyme solution was added, and finally the reaction system was replenished to 500 μl with reaction buffer (50 mM Tris, 5 mM MgCl2, 0.1 mg / ml BSA, pH 8.0). After homogeneous mixing of the reaction solution, it was incubated in a 37°C water bath for 30 minutes. Immediately after incubation, 500 μl of 20% trichloroacetic acid was added to the reaction solution to stop the reaction, and after being placed in an ice bath for at least 10 minutes, 200 μl of the reaction solution was added to a centrifuge tube containing 1800 μl of redistilled water and homogeneously mixed, and the absorbance of the diluted solution at 260 nm was measured using a UV-Vis spectrophotometer. One active unit (U) is defined as the amount of enzyme required to increase the absorbance at 260 nm by 1.0 within 30 minutes at 37°C under substrate excess conditions. The NaCl concentrations in the reaction system were adjusted to 0 mM, 100 mM, 200 mM, 300 mM, 400 mM, and 500 mM, respectively. The enzyme activity of the totipotent nuclease at different salt concentrations was measured according to the above embodiment, and the specific enzyme activity was calculated based on the measurement results.

[0140] The results are shown in Figure 4. The wild-type totipotent nuclease was sensitive to salt, retaining only 40% and 30% of its specific enzyme activity under 200 mM and 300 mM NaCl conditions, respectively, and almost completely lost activity under NaCl conditions exceeding 300 mM. On the other hand, compared to the wild type, the mutant showed significantly improved salt tolerance while maintaining its original enzyme activity under salt-free or low-salt conditions.

[0141] Furthermore, as can be seen from Figure 4, all mutants maintained over 80% activity under 200 mM NaCl conditions, demonstrating a clear improvement in activity compared to the wild type. In particular, eight mutants—T98K, T56D / T98K, T98K / N101H, T98K / G242D, T56D / T98K / G242D, N101H / T98K / G242D, T56D / T98K / N101H, and T56D / N101H / T98K / G242D—showed significantly improved activity under 300 mM, 400 mM, and 500 mM NaCl conditions, maintaining over 70%, 50%, and 20% or higher activity, respectively. Since all of these mutants contain the T98K mutation, this mutation plays an important role in improving the salt tolerance of the totipotent nuclease. The other seven mutants, T56D, N101H, G242D, T56D / N101H, T56D / G242D, N101H / G242D, and T56D / N101H / G242D, showed slightly lower activity enhancements under 300mM, 400mM, and 500mM NaCl conditions, maintaining activity levels of 43%, 25%, and 11% or higher, respectively, but still showing improvement over the wild type.

[0142] Compared to commercially available products of the conventional technology, for example, Salt Active nuclease (Yi Sheng Zhuo, China) shows the highest enzyme activity at 500 mM, but its relative specific activity is only about 25% of the highest enzyme activity of the wild-type totipotent nuclease (0 mM NaCl), and at NaCl concentrations below 500 mM, the relative specific activity falls significantly below 25%. In contrast, the mutants selected in this application not only show a relative specific activity significantly above 25% at NaCl concentrations below 500 mM, but some mutants also exhibit enzyme activity exceeding that of conventional commercially available Salt Active nucleases even at a NaCl concentration of 500 mM.

[0143] As described above, the novel mutants of totipotent nucleases selected in this application have a broader range of applications and superior activity.

[0144] The results of the above examples demonstrate that by mutating amino acids at specific sites based on the wild-type sequence, the tolerance of totipotent nucleases to different salt concentrations can be effectively improved, allowing for broader application in various salt concentration environments. Compared to the wild type, the mutant with the most mutations showed the best effect, but single, double, and triple mutations also yielded similar effects. This indicates that a larger number of mutations is not necessarily better, and that identifying the amino acid site that plays a decisive role is crucial.

[0145] The above description of specific embodiments of this application is for illustrative purposes only. These descriptions are not limited to the precise forms disclosed in this application, and many modifications and variations are possible based on the above teachings. The purpose of selecting and describing exemplary embodiments is to illustrate the specific principles and applications of this application, enabling those skilled in the art to realize and utilize various exemplary embodiments, options, and modifications of this application. The scope of this application is limited by the claims and their equivalent forms.

Claims

1. The sequence of the benzonase nuclease contains mutations at least one amino acid site. A totipotent nuclease mutant with improved salt tolerance, characterized by the following features.

2. The process includes introducing a mutation at least one amino acid site into the sequence of the benzonase nuclease. A method for improving the salt tolerance of a totipotent nuclease, characterized by the following:

3. The aforementioned region includes one or more of the 56th, 98th, 101st, and / or 242nd regions. Preferably, the region includes the 98th region, and further includes the 56th, 101st, and / or 242nd regions. The totipotent nuclease mutant according to claim 1, or the method according to claim 2.

4. The aforementioned mutations include one or more of T56D, T98K, N101H and / or G242D, Preferably, the mutation includes T98K and further includes one or more of T56D, N101H, and / or G242D. The totipotent nuclease mutant according to claim 1, or the method according to claim 2.

5. The aforementioned Benzone nuclease is a wild-type Benzone nuclease. Preferably, the sequence of the wild-type Benzonase nuclease is the sequence shown in SEQ ID NO.

1. The totipotent nuclease mutant according to claim 1, or the method according to claim 2.

6. Encoding a totipotent nuclease mutant according to any one of claims 1, 3 to 5. An endonuclease gene characterized by the following:

7. The endonuclease gene described in claim 6 is included An expression cassette, plasmid, vector, host cell, or recombinant microorganism characterized by these features.

8. A method for preparing a totipotent nuclease variant, A method for preparing a totipotent nuclease variant obtained by expression using the expression cassette, plasmid, vector, host cell, or recombinant microorganism described in claim 7.

9. Use of the totipotent nuclease variant according to any one of claims 1, 3 to 5 in cleavage or removal of nucleic acids.

10. The nucleic acid is treated with the totipotent nuclease mutant described in any one of claims 1, 3 to 5, preferably the treatment is carried out at a salt concentration of 0 to 500 mM. A method for cleaving or removing nucleic acids, characterized by the following:

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