KR-20260062958-A - Universal nuclease variant with improved salt tolerance and applications

Abstract

This application relates to the field of enzyme engineering technology, specifically providing a variant of a universal nuclease. Compared to a wild-type universal nuclease, said variant can have tolerance to a wider range of salt concentrations, maintains good enzyme activity under increased salt concentrations, and expands the application of the universal nuclease according to salt concentration.

Inventors

• 톈, 리
• 왕, 레이
• 친, 리리
• 장, 먀오먀오
• 궁, 빙쉐
• 지, 수셴
• 천, 이딩
• 먀오, 징윈

Assignees

• 베이징 아크로바이오시스템즈 바이오테크놀로지 컴퍼니., 리미티드

Dates

Publication Date

20260507

Application Date

20240821

Priority Date

20230926

Claims (10)

1. In a universal nuclease variant with improved salt tolerance, The above variant is a universal nuclease variant with improved salt tolerance, characterized by including a variation in at least one amino acid site in the Benzonase nuclease sequence.
2. In a method for improving the salt resistance activity of a general-purpose nuclease, A method for improving the salt resistance activity of a universal nuclease, characterized by including the step of introducing a mutation at least one amino acid site into the benzonase nuclease sequence.
3. In the universal nuclease variant according to paragraph 1 or the method according to paragraph 2, The above portion includes one or more of position 56, position 98, position 101 and/or position 242; Preferably, the universal nuclease variant or method is characterized in that the above-mentioned site includes position 98, and additionally includes position 56, position 101, and/or position 242.
4. In the universal nuclease variant according to paragraph 1 or the method according to paragraph 2, The above variant includes one or more of T56D, T98K, N101H and/or G242D; Preferably, the universal nuclease variant or method is characterized by the variant including T98K and additionally including one or more of T56D, N101H and/or G242D.
5. In the universal nuclease variant according to paragraph 1 or the method according to paragraph 2, The above Benzonase nuclease is a wild-type Benzonase nuclease; Preferably, the universal nuclease variant according to claim 1 or the method according to claim 2 is characterized in that the sequence of the wild-type Benzonase nuclease is as shown in SEQ ID NO. 1.
6. In endonuclease genes, The above gene is an endonuclease gene characterized by encoding a universal nuclease variant according to any one of claims 1, 3 to 5.
7. In expression cassettes, plasmids, vectors, host cells, or engineered microorganisms, An expression cassette, plasmid, vector, host cell, or engineered microorganism characterized by containing an endonuclease gene according to claim 6.
8. In a method for manufacturing a general-purpose nuclease variant, A method for producing a universal nuclease variant characterized by obtaining it by expression using an expression cassette, plasmid, vector, host cell, or engineered microorganism according to claim 7.
9. Application in the cleavage or removal of nucleic acids of a universal nuclease variant according to any one of claims 1, 3 to 5.
10. In a method for cutting or removing nucleic acids, Treating nucleic acids using a universal nuclease variant according to any one of paragraphs 1, 3 to 5; Preferably, a nucleic acid cleavage or removal method characterized by performing the treatment at a salt concentration of 0 to 500 mM.

Description

Universal Nuclease Variant with Improved Salt Tolerance and Applications **Universal Nuclease Variant with Improved Salt Tolerance and Applications** The present application relates to the field of enzyme engineering technology, specifically to a general-purpose nuclease variant with improved salt tolerance, a method of use, and an application thereof. Universal nucleases, also known as non-restrictive endonucleases, primarily serve to remove nucleic acids from biological products. In scientific research, they can lower the viscosity of cell supernatants and lysates and improve protein purification efficiency and functional studies. Furthermore, they are applied as reagents for removing host residual nucleic acids in virus purification, vaccine production, and the manufacturing of protein and polysaccharide pharmaceuticals. By reducing host residual nucleic acids to pg levels, they can enhance the efficacy and safety of biological products. Additionally, they can effectively prevent the agglutination of human peripheral blood mononuclei (PBMCs) in cell therapy and vaccine research, making them indispensable tool enzymes in the field of CGT. The most widely used universal nuclease currently is the bacterial nuclease derived from Serratia marcescens, disclosed by Benedik and Strych in 1998 (FEMS Microbiol Lett. 165:1–13) and trademarked by Merck as Benzonase®. Although its optimal activity temperature is 37°C, its most significant drawback is its limited tolerance to increased salt concentrations. Data indicates that sodium or potassium ions exhibit the strongest inhibitory effect on the activity of this nuclease, and almost all enzyme activity is lost when the salt concentration in the reaction system exceeds 300 mM. Due to these characteristics, universal nucleases can only function in salt-free or low-salt environments, which has limited their widespread application. This is because varying degrees of salt are added to buffer systems to ensure process stability, whether in the industrial manufacturing of biological products or in scientific research processes for obtaining recombinant proteins. Therefore, there is an urgent need in the current market for the development and production of general-purpose nucleases with significantly improved salt resistance. Enzymes derived from halophilic microorganisms have enabled the search for salt-tolerant enzymes, and some salt-tolerant nucleases derived from marine microorganisms have been reported. Marcin et al. disclosed a novel thermally unstable nuclease in 2022 (CN114651062A). Although this enzyme can withstand a salt concentration of 500 mM, it maintains high activity only at low temperatures (especially 4°C to 8°C), has very low production yield, high production costs, and high requirements for the application environment. In addition, Salt Active nuclease (Yeasen Biotechnology, China) has been released on the market. Although it exhibits optimal activity under 500 mM NaCl conditions, the relative specific activity of the enzyme is only about 25% of the optimal specific activity of benzonase. Furthermore, under salt concentrations below 500 mM, activity decreases significantly as the salt concentration decreases, and the tolerance range to salt concentration is also narrow. Due to these characteristics, the application of this product is limited to a salt concentration of 500 mM. Therefore, halophilic biological equivalent enzymes are not always present. In light of this, the present application is proposed. Figure 1 shows the three-dimensional structure and mutant sites of a wild-type universal nuclease. Figure 2 is an SDS-PAGE image of the recombinant universal nuclease production process, where M represents a protein marker, S represents the collected fermentation supernatant, SPFF represents the peak eluted after the sample passed through ion-exchange chromatography, and SEC represents the peak eluted after the sample passed through a molecular sieve. Figure 3 shows the HPLC purity of the recombinant universal nuclease. Figure 4 is a comparison of the relative enzyme inactivity of wild-type and variant universal nucleases according to salt concentration. Here, WT represents the wild-type universal nuclease, and Competitor Y represents the high-salt tolerant nuclease from Yeasen Biotechnology. Cross-reference of related applications The present application claims priority to a Chinese patent application filed with the Chinese Intellectual Property Office on September 26, 2023, with application number 202311249502.3 and the invention title “General-purpose nuclease variant with improved salt resistance and application,” the entire contents of said application are incorporated into the present application by reference. This application discloses a variant of a universal nuclease, which, compared to the wild-type universal nuclease, may have tolerance to a wider range of salt concentrations and maintains constant activity under increased salt concentrations.