CN-121986160-A - Omnipotent nuclease variant with improved salt tolerance and application

Abstract

The application relates to the technical field of enzyme engineering, and particularly provides a variant of a omnipotent nuclease, which can tolerate a wider salt concentration range than a wild omnipotent nuclease, still keeps good enzyme activity under the condition of increased salt concentration, and widens the application of the omnipotent nuclease under different salt concentrations.

Inventors

• TIAN LI
• WANG LEI
• QIN LILI
• ZHANG MIAOMIAO
• GONG BINGXUE
• JI SHUXIAN
• CHEN YIDING
• Miao Jingbin

Assignees

• 北京百普赛斯生物科技股份有限公司

Dates

Publication Date

20260505

Application Date

20240821

Priority Date

20230926

Claims (10)

1. A omnipotent nuclease variant having improved salt tolerance, wherein the variant comprises a change in the sequence of a Benzonase nuclease of at least 1 or more amino acid positions.
2. A method for increasing salt tolerance of a totipotent nuclease comprising introducing a modification comprising at least 1 or more amino acid positions into a Benzonase nuclease sequence.
3. The omnipotent nuclease variant of claim 1 or the method of claim 2, wherein the locus comprises any one or more of position 56, position 98, position 101 and/or position 242; preferably, the locus comprises position 98 and further comprises position 56, position 101 and/or position 242.
4. The omnipotent nuclease variant of claim 1 or the method of claim 2, wherein the alteration comprises any one or more of T56D, T98K, N H and/or G242D; Preferably, the alteration comprises T98K, and further comprises any one or more of T56D, N101H and/or G242D.
5. The omnipotent nuclease variant of claim 1 or the method of claim 2, wherein the Benzonase nuclease is a wild-type Benzonase nuclease; Preferably, the sequence of the wild-type Benzonase nuclease is shown in SEQ ID NO. 1.
6. An endonuclease gene, characterized in that said gene encodes a omnipotent nuclease variant according to any one of claims 1, 3 to 5.
7. An expression cassette, plasmid, vector, host cell or engineered microorganism comprising the endonuclease gene of claim 6.
8. A method for preparing a omnipotent nuclease variant, characterized in that the omnipotent nuclease variant is obtained by expression in an expression cassette, a plasmid, a vector, a host cell or an engineered microorganism according to claim 7.
9. Use of the omnipotent nuclease variant of any one of claims 1, 3 to 5 in nuclease cleavage or removal.
10. A method for the cleavage or removal of nucleic acids, characterized in that the nucleic acids are treated with a omnipotent nuclease variant according to any one of claims 1,3 to 5, preferably at a salt concentration of 0 to 500 mM.

Description

Omnipotent nuclease variant with improved salt tolerance and application Cross Reference to Related Applications The present application claims priority from the chinese patent application filed at 26, 9, 2023, under the application number 202311249502.3, entitled "omnipotent nuclease variant with improved salt tolerance and application", the entire contents of which are incorporated herein by reference. Technical Field The application relates to an enzyme engineering technology, in particular to a omnipotent nuclease variant with improved salt tolerance, a use method and application thereof. Background Totipotent nucleases, also known as non-limiting endonucleases, primarily function to eliminate nucleic acids in biological products. The method can not only reduce the viscosity of cell supernatant and cell lysate in scientific research and improve protein purification efficiency and function research, but also be applied to virus purification, vaccine production and protein and polysaccharide pharmaceutical industry as a host residual nucleic acid removal reagent to reduce host residual nucleic acid to pg level so as to improve the efficacy and safety of biological products, and can effectively prevent the agglomeration of human Peripheral Blood Mononuclear Cells (PBMC) in cell therapy and vaccine research, thus being a tool enzyme which cannot be obtained in the CGT field. The most popular omnipotent nucleases at present are bacterial nucleases from Serratia marcescens (SERRATIA MARCESCENS) disclosed by Benedik and Strych in 1998 (FEMS Microbiol Lett.165:1-13) and registered trademark by Merck companyThe optimal activity temperature is 37 ℃, but the most important disadvantage is the limited tolerance to increased salt concentration, and data indicate that sodium ions or potassium ions have a strong inhibition of the nuclease activity, and when the salt concentration in the reaction system is higher than 300mM, the enzyme activity is almost completely lost. This property makes the omnipotent nuclease only work in salt-free or low-salt environments, limiting its wide application. Because salt with different degrees can be added into a buffer system to ensure the stability of the process no matter the preparation process of industrial biological products or the process of scientifically researching and obtaining recombinant proteins, development and production of an all-purpose nuclease with obviously improved salt tolerance are urgently needed in the market at present. Enzymes from halophilic microorganisms have made it possible to find salt tolerant enzymes, and some salt tolerant nucleases derived from marine microorganisms have been reported. Marcin et al (CN 114651062 a) disclose a novel thermostable nuclease which can tolerate 500mM salt concentration, but which maintains high activity at low temperature, especially 4-8 ℃, and which is low in yield, high in production cost and harsh in application environment, and furthermore, SALT ACTIVE nucleic (next holothurian, china) which shows optimal activity at 500mM NaCl, but relative enzyme activity can only reach about 25% of the best enzyme activity of Benzonase, and which decreases significantly with decreasing salt concentration below 500mM salt concentration, and which is also narrow in tolerance range of salt concentration, which limits its application to only 500mM salt concentration. Thus, equivalent enzymes from halophiles are not always present. In view of this, the present application has been proposed. Summary of The Invention Aiming at the technical problems, the application obtains the universal nuclease variant capable of tolerating a wider salt concentration range through rational design, solves the problem of narrow use pain point of the universal nuclease salt tolerance range in the current market, ensures wider application scene and stronger universality, and can achieve the effect of removing nucleic acid under different salt concentration systems. Specifically, the application is based on the Benzonase omnipotent nuclease, improves the tolerance range of the Benzonase omnipotent nuclease to salt concentration under the condition of not affecting the original activity, especially the salt concentration of 200mM to 500mM, and finally provides the nuclease with wider salt concentration tolerance. The application not only continuously plays the advantages of Benzonase, but also has high activity, stability and high yield under no or very low salt, and improves the tolerance of Benzonase to higher salt concentration. Thus, the present application includes at least the following four objects: A first object of the present application is to provide a omnipotent nuclease variant with improved salt tolerance; A second object of the present application is to provide the use of a omnipotent nuclease variant with improved salt tolerance in nucleic acid treatment; a third object of the present application is to provide a method for preparing a omnip