CN117230052A

CN117230052A - All-purpose nuclease stability protective agent, liquid enzyme preparation and preparation method thereof

Info

Publication number: CN117230052A
Application number: CN202311037182.5A
Authority: CN
Inventors: 罗漫杰; 施婧妮; 雷宵; 张雅洁; 王梁; 徐灿
Original assignee: Wuhan New Biocalysis Solution Co ltd
Current assignee: Wuhan New Biocalysis Solution Co ltd
Priority date: 2023-08-15
Filing date: 2023-08-15
Publication date: 2023-12-15

Abstract

The invention provides a omnipotent nuclease stability protective agent, a liquid enzyme preparation and a preparation method thereof. The omnipotent nuclease stability protective agent can enable the omnipotent nuclease to be stable at high temperature or extreme pH value, and keep good biological activity; the invention relates to a liquid enzyme preparation of a full-functional nuclease, which comprises the following components: the full-function nuclease and the full-function nuclease stability protective agent can stabilize the full-function nuclease at 50-80 ℃ and pH 4.0-10.0, can keep better activity stability when being treated for 1h at 60-80 ℃, keep better activity stability when being treated for 12h at 50 ℃, keep better activity stability when being treated for 12h at pH 4.0 or 10.0, and can improve the storage stability of the enzyme, keep better activity stability when being stored for 30 days at normal temperature and keep better activity stability when being stored for 1 year at 4 ℃.

Description

All-purpose nuclease stability protective agent, liquid enzyme preparation and preparation method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a comprehensive nuclease stability protective agent, a liquid enzyme preparation and a preparation method thereof.

Background

The biological product field is an unprecedented development opportunity, more and more biological products enter the treatment field, the quality control is increasingly strict, and because the biological products such as recombinant protein vaccines, vero cell vaccines, cell therapy/gene therapy drugs and the like are mostly from continuous strains or cell strains, such as bacteria, yeast, animal cells or human cells. Although these complex expression or production systems are subjected to stringent purification processes, there is a possibility that nucleic acid fragments of host cells remain in the product, and these biological products can present uncontrollable risk factors when applied to the prevention and treatment of human diseases. Therefore, from the aspects of biological product safety and effectiveness requirements, nucleic acid residues are important in various quality control standards.

The omnipotent nuclease is a genetically engineered endonuclease from Serratia marcescens (Serratia marcescens) and can degrade nucleic acid into 5' -monophosphate oligonucleotides of 3-5 bases, and can degrade DNA and RNA in any form (double-chain, single-chain, linear, circular and supercoiled) with high efficiency without protein cleavage activity. The ability of the omnipotent nuclease to hydrolyze nucleic acid rapidly can be widely applied to removing exogenous nucleic acid of vaccine products, reducing the risk of toxicity of nucleic acid residue, reducing the viscosity generated by nucleic acid release during cell/bacteria lysis, shortening the processing time and improving the yield of virus/protein.

The omnipotent nuclease is used as a nonspecific broad-spectrum nuclease, can be used for various scenes needing to remove nucleic acid, and can be challenged for the stability of the omnipotent nuclease by considering the complex conditions of solution temperature and extreme pH value in different application scenes.

The prior art discloses an expression purification method of a totipotent endonuclease, which obtains the high-purity totipotent endonuclease through the steps of prokaryotic expression inclusion body, renaturation, purification and the like of escherichia coli, can effectively reduce the production cost of the totipotent endonuclease, shortens the purification time, and is beneficial to the application of the totipotent endonuclease in the fields of scientific research and industry. The prior art discloses a protein preparation method of omnipotent nuclease, which mainly solves the problems of complex process and difficult control of renaturation process in the existing omnipotent nuclease preparation process, and provides a novel preparation method which has the advantages of simple purification process, no need of denaturation renaturation process and high activity of protein products. The prior art discloses a method for high-density fermentation expression, separation and purification of omnipotent nuclease, which mainly solves the problems of how to improve the secretion expression and high-purity separation of the omnipotent nuclease. However, the above prior art is mostly directed to expression purification methods of omnipotent nucleases.

Regarding related technical information of the omnipotent nuclease preparation, as disclosed in the prior art, the formula of the nucleic acid pollution cleaning solution comprises omnipotent nuclease, buffer salt, magnesium ions, tween-80, sodium chloride, vitamin C, preservative and glycerol, the nucleic acid pollution cleaning solution provided by the omnipotent nuclease preparation can be used for removing nucleic acid pollution in a molecular biology laboratory and a PCR detection chamber, but the provided formula can only ensure that the omnipotent nuclease keeps stable activity at about pH 8.0, the enzyme activity gradually decreases after the pH value deviates from 8.0, and the enzyme activity decreases more obviously when the time is longer. The prior art also discloses a biological enzyme scavenger with good elimination performance, no strong oxidability and corrosiveness, safety and no toxicity and no nucleic acid pollution and a preparation method thereof. The biological enzyme scavenger comprises omnipotent nuclease, buffer solution, auxiliary factors and stabilizing agents, can effectively play roles at normal temperature, can be applied to a low-temperature environment, and can rapidly remove nucleic acid and aerosol pollution on the surface of an object, but the application effect of the biological enzyme scavenger at a high temperature such as 50 ℃ is unknown.

As mentioned above, the omnipotent nuclease is often applied in the pretreatment process of biological products including vaccine and protein medicines, and is inevitably affected by extreme pH and temperature, so a new protective agent of the omnipotent nuclease is needed to be found to improve the stability.

Disclosure of Invention

In view of the above, the invention provides a protective agent for the stability of omnipotent nuclease, a liquid enzyme preparation and a preparation method thereof, so as to solve the technical problems in the prior art.

In a first aspect, the present invention provides a omnipotent nuclease stability protectant comprising at least one of a saccharide, a sugar alcohol, and an amino acid.

Preferably, the omnipotent nuclease stability protective agent comprises disaccharide substances and/or polysaccharide substances, and the amino acid is basic amino acid.

Preferably, the omnipotent nuclease stability protective agent comprises at least one of trehalose and sucrose;

the polysaccharide substance comprises dextran;

the basic amino acid comprises at least one of lysine and arginine.

Preferably, the omnipotent nuclease stability protectant, the sugar alcohol comprises at least one of mannitol, sorbitol and xylitol.

Preferably, the omnipotent nuclease stability protective agent further comprises buffer solution, salt substance and Mg ²⁺ And a surfactant.

Preferably, the omnipotent nuclease stability protective agent is a buffer solution with the buffer capacity of 4.0-10.0;

And/or the salt substance comprises at least one of NaCl and KCl;

and/or the Mg ²⁺ Provided by Mg salts;

and/or the surfactant is a nonionic surfactant.

Preferably, the omnipotent nuclease stability protective agent comprises at least one of acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, disodium hydrogen phosphate-citric acid buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, glycine-sodium hydroxide buffer, tris-hydrochloric acid buffer and HEPES buffer;

and/or the Mg salt comprises at least one of magnesium chloride, magnesium acetate, magnesium sulfate, magnesium citrate and magnesium glycinate;

and/or the nonionic surfactant comprises at least one of Tween 20, tween 80, triton X-100 and octyl phenoxy polyethanol;

and/or the concentration of the buffer solution in the omnipotent nuclease stability protective agent is 20-100 mM, the concentration of the salt substance is 40-200 mM, and the concentration of the Mg ²⁺ The concentration of (2) is 4-20 mM, the volume concentration of the surfactant is 0.1% -1%, and the total concentration of the sugar substance and/or sugar alcohol and/or amino acid is 400-1000 mM.

In a second aspect, the present invention also provides a liquid enzyme preparation of a holoendose comprising: a omnipotent nuclease and a protective agent for the stability of the omnipotent nuclease.

Preferably, the liquid enzyme preparation of the holoendose comprises at least one of sugar substances, sugar alcohol and amino acid, holoendose, buffer solution, salt substances and Mg ²⁺ A surfactant;

wherein the concentration of the buffer solution is 20-50 mM, the concentration of the salt substance is 20-100 mM, and the concentration of the Mg ²⁺ The concentration of the surfactant is 2-10 mM, the volume concentration of the surfactant is 0.05-0.5%, and the total concentration of the saccharide and/or sugar alcohol and/or amino acid is 200-500 mM.

In a third aspect, the invention also provides a preparation method of the full-functional nuclease liquid enzyme preparation, which comprises the following steps:

salt material, mg ²⁺ Adding surfactant, saccharide and/or sugar alcohol and/or amino acid into buffer solution to obtain mixed protecting agent solution;

and adding the omnipotent nuclease into the protective agent mixed solution to obtain the omnipotent nuclease liquid enzyme preparation.

The invention relates to a comprehensive nuclease stability protective agent in the prior art, which has the following beneficial effects:

1. The invention discloses a protective agent for the stability of omnipotent nuclease, which comprises at least one of sugar substances, sugar alcohol and amino acid, and can ensure that the omnipotent nuclease is stable at high temperature (50-80 ℃) or extreme pH (pH is 4.0-10.0) and keeps good biological activity;

2. the invention relates to a liquid enzyme preparation of a full-functional nuclease, which comprises the following components: the full-function nuclease and the full-function nuclease stability protective agent can stabilize the full-function nuclease at 50-80 ℃ and pH 4.0-10.0, can keep better activity stability when being treated for 1h at 60-80 ℃, keep better activity stability when being treated for 12h at 50 ℃, keep better activity stability when being treated for 12h at pH 4.0 or 10.0, and can improve the storage stability of the enzyme, keep better activity stability when being stored for 30 days at normal temperature and keep better activity stability when being stored for 1 year at 4 ℃.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It is evident that the drawings in the following description are only some embodiments of the present invention and that other drawings may be obtained from these drawings without inventive effort for a person of ordinary skill in the art.

FIG. 1 shows the preservation rate of the enzyme activity of the liquid enzyme preparations of examples 2, 5, 7 to 10 and comparative examples 1 to 2 under different treatment conditions.

Detailed Description

The following description of the embodiments of the present application will be made in detail and with reference to the embodiments of the present application, but it should be apparent that the described embodiments are only some embodiments of the present application, and not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present application without making any inventive effort, are intended to fall within the scope of the present application.

For a better understanding of the present application, and not to limit its scope, all numbers expressing quantities, percentages, and other values used in the present application are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

The following description of the embodiments is not intended to limit the preferred embodiments. In addition, in the description of the present application, the term "comprising" means "including but not limited to". Various embodiments of the application may exist in a range of forms; it should be understood that the description in a range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the application; it is therefore to be understood that the range description has specifically disclosed all possible sub-ranges and individual values within that range. For example, it should be considered that a description of a range from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the ranges, such as 1, 2, 3, 4, 5, and 6, wherever applicable. In addition, whenever a numerical range is referred to herein, it is meant to include any reference number (fractional or integer) within the indicated range.

The embodiment of the application provides a omnipotent nuclease stability protective agent, which comprises at least one of saccharide substances, sugar alcohol and amino acid.

The application relates to a protective agent for the stability of omnipotent nuclease, which comprises at least one of sugar substances, sugar alcohols and amino acids, and the applicant discovers that one or more substances of the sugar substances, the sugar alcohols or the amino acids can ensure that the omnipotent nuclease is stable at high temperature or extreme pH and keeps good biological activity.

Specifically, the saccharide substance includes disaccharide substance and/or polysaccharide substance, and the amino acid is basic amino acid.

Wherein disaccharide means a carbohydrate that upon hydrolysis by an acid or enzyme produces two monosaccharide molecules that may be the same or different; polysaccharide substances are generally complexes composed of 8 to more than 10 monosaccharides.

Basic amino acids refer to amino acids having two amino groups and one carboxyl group, with a net positive charge at neutral pH, such as lysine, arginine, histidine, and the like.

In some embodiments, the disaccharide substance comprises at least one of trehalose, sucrose.

In some embodiments, the polysaccharide comprises dextran; dextran is a polysaccharide polymerized from several glucose molecules.

In some embodiments, the basic amino acid comprises at least one of lysine, arginine.

In some embodiments, the sugar alcohol comprises at least one of mannitol, sorbitol, xylitol.

Furthermore, in order to make the omnipotent nuclease more stable, the omnipotent nuclease stability protective agent of the invention also comprises buffer solution, salt substances and Mg ²⁺ And a surfactant.

Specifically, the omnipotent nuclease (Benzonase) is from Serratia marcescens (Serratia marcescens), and the genetically engineered endonuclease can degrade all DNA (including single strand, double strand, linear and circular) and RNA without degrading protein, and has high specificity.

Preferably, the omnipotent nuclease of the invention is derived from Serratia marcescens (Serratia marcescens) and is prepared by fermenting, expressing and purifying in Escherichia coli (E.coli) through genetic engineering.

Specifically, the high temperature described above refers to a 50-80 ℃ environment, and in some embodiments, the high temperature experiment is performed by a water bath treatment.

Specifically, the extreme pH mentioned above means a pH of 4.0 to 10.0. For the purposes of the present invention, pH stability experiments use buffer systems with buffering capacity at pH 4.0-10.0, including but not limited to acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, disodium hydrogen phosphate-citric acid buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, glycine-sodium hydroxide buffer, tris-hydrochloric acid buffer, HEPES buffer.

In some embodiments, the buffer is a buffer with a buffering capacity at a pH of 4.0 to 10.0.

In some embodiments, the salt species includes at least one of NaCl, KCl.

In some embodiments, mg ²⁺ Provided by Mg salts.

In some embodiments, the surfactant is a nonionic surfactant.

In some embodiments, the buffer system described above may also be used as a stability protectant for the omnipotent nuclease to maintain storage of the omnipotent nuclease at a relatively stable pH, but without affecting its use at each pH. Namely, the buffer solution in the all-purpose nuclease stability protective agent comprises at least one of acetic acid-sodium acetate buffer solution, citric acid-sodium citrate buffer solution, citric acid-sodium hydroxide-hydrochloric acid buffer solution, disodium hydrogen phosphate-citric acid buffer solution, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, glycine-sodium hydroxide buffer solution, tris-hydrochloric acid buffer solution and HEPES buffer solution.

In some embodiments, the Mg salt comprises at least one of magnesium chloride, magnesium acetate, magnesium sulfate, magnesium citrate, magnesium glycinate.

In some embodiments, the nonionic surfactant includes tween 20, tween 80, triton X-100 (triton X-100), octylphenoxy polyethanol (i.e., octylphenyl-polyethylene glycol, 4-nonylphenyl-polyethylene glycol,CA-630).

Based on the same inventive concept, the invention also provides a full-functional nuclease liquid enzyme preparation, which comprises the following components: omnipotent nuclease and the above-mentioned omnipotent nuclease stability protectant.

In some embodiments, the holomonuclease liquid enzymeThe preparation comprises at least one of saccharide, sugar alcohol, amino acid, total nuclease, buffer solution, salt substance, and Mg ²⁺ A surfactant;

wherein the concentration of the buffer solution is 20-50 mM, the concentration of the salt substance is 20-100 mM, and the concentration of Mg ²⁺ The concentration of (2) to (10) mM, the volume concentration of the surfactant is 0.05% to 0.5%, and the total concentration of the saccharide and/or sugar alcohol and/or amino acid is 200 mM to 500mM.

Preferably, the concentration of the buffer solution is 30-50 mM, the concentration of the salt substance is 20-60 mM, and the concentration of Mg ²⁺ The concentration of (2) to (5) mM, the volume concentration of the surfactant is 0.3% to 0.5%, and the total concentration of the saccharide and/or sugar alcohol and/or amino acid is 350 mM to 400mM.

Specifically, the concentration of the buffer solution may be 20mM, 30mM, 40mM, 50mM, and the concentration of the salt substance may be 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, mg ²⁺ The concentration of (C) may be 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, the volume concentration of the surfactant may be 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, and the total concentration of the saccharide and/or sugar alcohol and/or amino acid may be 200mM, 300mM, 400mM, 500mM.

The concentration of the omnipotent nuclease liquid enzyme preparation of the present invention is not limited, and may be any concentration, for example, the concentration of the omnipotent nuclease may be 100 to 1000U/. Mu.L, and specifically may be 100U/. Mu.L, 200U/. Mu.L, 300U/. Mu.L, 400U/. Mu.L, 500U/. Mu.L, 600U/. Mu.L, 700U/. Mu.L, 800U/. Mu.L, 900U/. Mu.L, 1000U/. Mu.L.

The invention provides a full-functional nuclease liquid enzyme preparation, which comprises the following components: the comprehensive nuclease and the comprehensive nuclease stability protective agent can ensure that the comprehensive nuclease has the stability at 50-80 ℃ and pH 4.0-10.0, can keep better activity stability when being treated for 1h at 60-80 ℃, keep better activity stability when being treated for 12h at 50 ℃, keep better activity stability when being treated for 12h at pH 4.0 or 10.0, and can improve the storage stability of the enzyme, keep better activity stability when being stored for 30 days at normal temperature, and keep better activity stability when being stored for 1 year at 4 ℃.

Based on the same inventive concept, the invention also provides a preparation method of the above-mentioned full-functional nuclease liquid enzyme preparation, comprising the following steps:

Specifically, before adding the omnipotent nuclease into the protective agent mixed solution, the method further comprises the following steps: performing ultrafiltration displacement on the omnipotent nuclease through a buffer solution (namely, displacing the omnipotent nuclease in the buffer solution) to obtain omnipotent nuclease solution; and adding the omnipotent nuclease enzyme solution into the mixed solution of the protective agent to obtain the omnipotent nuclease enzyme preparation.

In some embodiments, the salt species, mg ²⁺ Adding surfactant, saccharide and/or sugar alcohol and/or amino acid into buffer solution (for regulating pH of the system to be consistent with that of the buffer solution), and filtering with a filter membrane to obtain a protective agent mixed solution; adding the omnipotent nuclease enzyme solution into the mixed solution of the protective agent to ensure that the concentration of each material is consistent with the formula, namely, the concentration of the buffer solution is 20-50 mM, the concentration of the salt substance is 20-100 mM and the concentration of Mg in the omnipotent nuclease liquid enzyme preparation ²⁺ The concentration of the surfactant is 2-10 mM, the volume concentration of the surfactant is 0.05-0.5%, and the total concentration of the sugar substance and/or sugar alcohol and/or amino acid is 200-500 mM, thus obtaining the omnipotent nuclease liquid enzyme preparation.

In some embodiments, the salt species, mg ²⁺ Adding surfactant, saccharide and/or sugar alcohol and/or amino acid into buffer solution to obtain mixed protecting agent solution, which is the comprehensive nuclease stability protecting agent, and the concentration of buffer solution in the comprehensive nuclease stability protecting agent is 20-100 mM, the concentration of salt material is 40-200 mM and Mg is used for preparing the comprehensive nuclease liquid enzyme preparation with target concentration ²⁺ The concentration of the surfactant is 4-20 mM, and the volume concentration of the surfactant is 01% -1% of total concentration of sugar substances and/or sugar alcohol and/or amino acid is 400-1000 mM.

It should be noted that, the concentration of each component in the omnipotent nuclease stability protective agent in the invention can be understood as the initial concentration, and the concentration of each component in the omnipotent nuclease liquid enzyme preparation can be understood as the final concentration, and the setting of the initial concentration of each component in the omnipotent nuclease stability protective agent is to prepare the omnipotent nuclease liquid enzyme preparation with the target final concentration more conveniently; the invention has no requirement on the initial concentration of each component of the omnipotent nuclease stability protective agent, as long as the initial concentration of each component of the omnipotent nuclease stability protective agent is larger than the final concentration of each component in the omnipotent nuclease liquid enzyme preparation.

The following examples are further illustrative of the universal nuclease stability protectant, liquid enzyme preparation and method of preparing the same of the present application. This section further illustrates the summary of the application in connection with specific embodiments, but should not be construed as limiting the application. The technical means employed in the examples are conventional means well known to those skilled in the art, unless specifically stated. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present application are those conventional in the art.

Technical terms:

purifying: reference herein to purification refers to the removal of impurities or contaminants from a sample containing a target component to obtain a target component having a higher absolute or relative concentration.

And (3) a protective agent: refers to the auxiliary materials which are added and maintain the biological activity and do not prevent the denaturation of the active components in the process of preserving the biological products. Enzyme stability refers to the ability of an enzyme to retain activity, as determined by measuring the enzyme activity before and after treatment to calculate the activity lost by the enzyme during treatment.

Preservation rate (%) of enzyme activity: refers to the percentage of enzyme activity remaining during the treatment. I.e., the ratio of the enzyme activity after the treatment to the enzyme activity before the treatment was measured after the treatment.

Example 1

Universal nuclease activity detection method

Definition of enzyme activity: the amount of enzyme used to reduce the DeltaA 260 value by 1.0 (corresponding to complete digestion of 37. Mu.g salmon sperm DNA into oligonucleotides) in 30 minutes at 37℃and pH 8.0 was defined as one activity unit (U).

The operation steps are as follows: preparation of 1mM MgCl ₂ Nuclease samples diluted with 0.1mg/mL BSA (bovine serum albumin), 50mM Tris-HCl and pH 8.0 buffer solution are prepared, salmon sperm DNA concentration is 50 mug/mL, 2.5mL salmon sperm DNA solution is added with 0.125mL diluted nuclease, the reaction is carried out in a water bath at 37 ℃ for 30 minutes, absorbance change at 260nm is measured, and enzyme activity is calculated after corresponding blank is subtracted.

Example 2

The embodiment of the application provides a omnipotent nuclease stability protective agent, which comprises Tris-HCl buffer solution with pH of 8.0, naCl and Mg ²⁺ (provided by magnesium chloride), tween 20, trehalose, arginine;

the embodiment of the application also provides a full-functional nuclease liquid enzyme preparation, which comprises the following components: omnipotent nuclease, tris-HCl buffer solution with pH of 8.0, naCl and Mg ²⁺ (provided by magnesium chloride), tween 20, trehalose, arginine;

wherein the activity concentration of the omnipotent nuclease is 400U/. Mu.L, the concentration of the Tris-HCl buffer solution is 20mM, the pH is 8.0, the concentration of NaCl is 100mM, and the concentration of Mg ²⁺ The concentration of (2) mM, the volume concentration of Tween 20 was 0.05%, the concentration of trehalose was 200mM, and the concentration of arginine was 150mM.

The embodiment of the application also provides a preparation method of the full-functional nuclease liquid enzyme preparation, which comprises the following steps:

s1, performing ultrafiltration and replacement on the omnipotent nuclease in a 20mM Tris-HCl buffer solution with the pH value of 8.0 according to the activity concentration of 800U/. Mu.L to obtain omnipotent nuclease enzyme solution;

s2, adding NaCl, magnesium chloride, tween 20, trehalose and arginine into 20mM Tris-HCl buffer solution with pH of 8.0, fully dissolving, and filtering by using a 0.22 mu m filter membrane to obtain a protective agent mixed solution;

s3, mixing the omnipotent nuclease enzyme solution in the S1 with the mixed solution of the protective agent in the S2, so that the final concentration of each material is consistent with the formula, and obtaining the omnipotent nuclease liquid enzyme preparation.

Example 3

The embodiment of the application provides a omnipotent nuclease stability protective agent, which comprises HEPES buffer solution with pH of 8.0 and NaCl, KCl, mg ²⁺ (provided by magnesium chloride), octylphenoxy polyethanol, sucrose, lysine;

the embodiment of the application also provides a full-functional nuclease liquid enzyme preparation, which comprises the following components: HEPES buffer, naCl, KCl, mg, pH 8.0, totipotent nuclease ²⁺ (provided by magnesium chloride), octylphenoxy polyethanol, sucrose, lysine;

wherein the activity concentration of the omnipotent nuclease is 400U/. Mu.L, the concentration of HEPES buffer solution is 50mM, the pH is 8.0, the concentration of NaCl is 20mM, the concentration of KCl is 50mM, and the concentration of Mg ²⁺ The concentration of (2) mM, the volume concentration of octylphenoxy polyethanol was 0.5%, the concentration of sucrose was 300mM, and the concentration of lysine was 100mM.

s1, performing ultrafiltration and displacement on the omnipotent nuclease in 50mM HEPES buffer solution with pH of 8.0 at the activity concentration of 800U/. Mu.L to obtain omnipotent nuclease solution;

s2, adding NaCl, KCl, magnesium chloride, octyl phenoxy polyethanol, sucrose and lysine into 50mM HEPES buffer solution with pH of 8.0, fully dissolving, and filtering by using a 0.22 mu m filter membrane to obtain a protective agent mixed solution;

Example 4

The embodiment of the application provides a omnipotent nuclease stability protective agent, which comprises disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH of 8.0, KCl and Mg ²⁺ (provided by magnesium chloride), tween 80, trehalose, sorbitol, arginine;

the embodiment of the application also provides a full-functional nuclease liquid enzyme preparation, which comprises the following components: all-aroundNuclease-capable sodium phosphate buffer solution with pH of 8.0, KCl and Mg ²⁺ (provided by magnesium chloride), tween 80, trehalose, sorbitol, arginine;

wherein the activity concentration of the omnipotent nuclease is 400U/. Mu.L, the concentration of the sodium phosphate buffer solution is 50mM, the pH is 8.0, the concentration of KCl is 100mM, and the concentration of Mg ²⁺ The concentration of (2) was 5mM, the volume concentration of Tween 80 was 0.3%, the concentration of trehalose was 200mM, the concentration of sorbitol was 100mM, and the concentration of arginine was 100mM.

s1, performing ultrafiltration and replacement on the omnipotent nuclease in 50mM sodium phosphate buffer solution with pH value of 8.0 at the activity concentration of 800U/. Mu.L to obtain omnipotent nuclease liquid;

s2, adding KCl, magnesium chloride, tween 80, trehalose, sorbitol and arginine into 50mM sodium phosphate buffer solution with pH of 8.0, fully dissolving, and filtering by using a 0.22 mu m filter membrane to obtain a protective agent mixed solution;

Example 5

The embodiment of the application provides a omnipotent nuclease stability protective agent, which comprises Tris-HCl buffer solution with pH of 8.0, naCl and Mg ²⁺ (provided by magnesium chloride), triton X-100, mannitol, lysine;

the embodiment of the application also provides a full-functional nuclease liquid enzyme preparation, which comprises the following components: omnipotent nuclease, tris-HCl buffer solution with pH of 8.0, naCl and Mg ²⁺ (provided by magnesium chloride), triton X-100, mannitol, lysine;

wherein the activity concentration of the omnipotent nuclease is 400U/. Mu.L, the concentration of the Tris-HCl buffer solution is 30mM, the pH is 8.0, the concentration of NaCl is 50mM, and the concentration of Mg ²⁺ The concentration of (2) was 10mM, the volume concentration of triton X-100 was 0.2%, the concentration of mannitol was 200mM, and the concentration of lysine was 200mM.

s1, performing ultrafiltration and replacement on the omnipotent nuclease in 30mM Tris-HCl buffer solution with pH of 8.0 at the activity concentration of 800U/. Mu.L to obtain omnipotent nuclease liquid;

s2, adding NaCl, magnesium chloride, triton X-100, mannitol and lysine into 30mM Tris-HCl buffer solution with pH of 8.0, fully dissolving, and filtering by using a 0.22 mu m filter membrane to obtain a protective agent mixed solution;

Example 6

The embodiment of the application provides a omnipotent nuclease stability protective agent, which comprises HEPES buffer solution with pH of 8.0, KCl and Mg ²⁺ (provided by magnesium chloride), triton X-100, xylitol, arginine;

the embodiment of the application also provides a full-functional nuclease liquid enzyme preparation, which comprises the following components: omnipotent nuclease, HEPES buffer solution with pH of 8.0, KCl and Mg ²⁺ (provided by magnesium chloride), triton X-100, xylitol, arginine;

wherein the activity concentration of the omnipotent nuclease is 400U/. Mu.L, the concentration of HEPES buffer solution is 50mM, the pH is 8.0, the concentration of KCl is 60mM, and the concentration of Mg ²⁺ The concentration of (2) was 5mM, the volume concentration of triton X-100 was 0.2%, the concentration of xylitol was 100mM, and the concentration of arginine was 300mM.

S2, adding KCl, magnesium chloride, triton X-100, xylitol and arginine into 50mM HEPES buffer solution with pH of 8.0, fully dissolving, and filtering by using a 0.22 mu m filter membrane to obtain a protective agent mixed solution;

Example 7

The present example provides a liquid enzyme preparation of a holoendose, which is the same as example 2, except that arginine is not added, specifically, the liquid enzyme preparation of a holoendose comprises: omnipotent nuclease, tris-HCl buffer solution with pH of 8.0, naCl and Mg ²⁺ (provided by magnesium chloride), tween 20, trehalose;

wherein the activity concentration of the omnipotent nuclease is 400U/. Mu.L, the concentration of the Tris-HCl buffer solution is 20mM, the pH is 8.0, the concentration of NaCl is 100mM, and the concentration of Mg ²⁺ The concentration of (2) mM, the volume concentration of Tween 20 was 0.05%, and the concentration of trehalose was 200mM.

The embodiment also provides a preparation method of the above-mentioned all-purpose nuclease liquid enzyme preparation, which comprises the following steps:

S2, adding NaCl, magnesium chloride, tween 20 and trehalose into 20mM Tris-HCl buffer solution with pH of 8.0, fully dissolving, and filtering by using a 0.22 mu m filter membrane to obtain a protective agent mixed solution;

Example 8

The present example provides a liquid enzyme preparation of a holohydronuclease, which is different from example 2 in that trehalose is not added, and specifically comprises holohydronuclease, tris-HCl buffer with pH of 8.0, naCl, mg ²⁺ (provided by magnesium chloride), tween 20, arginine;

wherein the activity concentration of the omnipotent nuclease is 400U/. Mu.L, the concentration of the Tris-HCl buffer solution is 20mM, the pH is 8.0, the concentration of NaCl is 100mM, and the concentration of Mg ²⁺ Concentration of (2)2mM, tween 20 at a volume concentration of 0.05% and arginine at a concentration of 150mM.

S2, adding NaCl, magnesium chloride, tween 20 and arginine into 20mM Tris-HCl buffer solution with pH of 8.0, fully dissolving, and filtering by using a 0.22 mu m filter membrane to obtain a protective agent mixed solution;

Example 9

The present example provides a liquid enzyme preparation of a holoendose, which is the same as example 5, except that no lysine is added, specifically, the liquid enzyme preparation of a holoendose comprises: omnipotent nuclease, tris-HCl buffer solution with pH of 8.0, naCl and Mg ²⁺ (provided by magnesium chloride), triton X-100, mannitol;

wherein the activity concentration of the omnipotent nuclease is 400U/. Mu.L, the concentration of the Tris-HCl buffer solution is 30mM, the pH is 8.0, the concentration of NaCl is 50mM, and the concentration of Mg ²⁺ The concentration of (2) was 10mM, the volume concentration of triton X-100 was 0.2%, and the concentration of mannitol was 200mM.

S2, adding NaCl, magnesium chloride, triton X-100 and mannitol into 30mM Tris-HCl buffer solution with pH of 8.0, fully dissolving, and filtering by using a 0.22 mu m filter membrane to obtain a protective agent mixed solution;

Example 10

The present example provides a liquid enzyme preparation of a holo-nuclease, which is different from example 5 in that mannitol is not added, specifically, the liquid enzyme preparation of a holo-nuclease comprises: omnipotent nuclease, tris-HCl buffer solution with pH of 8.0, naCl and Mg ²⁺ (provided by magnesium chloride), triton X-100, lysine;

wherein the activity concentration of the omnipotent nuclease is 400U/. Mu.L, the concentration of the Tris-HCl buffer solution is 30mM, the pH is 8.0, the concentration of NaCl is 50mM, and the concentration of Mg ²⁺ The concentration of (2) was 10mM, the volume concentration of triton X-100 was 0.2%, and the concentration of lysine was 200mM.

S2, adding NaCl, magnesium chloride, triton X-100 and lysine into 30mM Tris-HCl buffer solution with pH of 8.0, fully dissolving, and filtering by using a 0.22 mu m filter membrane to obtain a protective agent mixed solution;

Comparative example 1

The present comparative example provides a liquid enzyme preparation of a holoenological nuclease, which is the same as example 2 except that trehalose and arginine are not added, specifically, the liquid enzyme preparation of a holoenological nuclease comprises: omnipotent nuclease, tris-HCl buffer solution with pH of 8.0, naCl and Mg ²⁺ (provided by magnesium chloride), tween 20;

wherein the activity concentration of the omnipotent nuclease is 400U/. Mu.L, the concentration of the Tris-HCl buffer solution is 20mM, the pH is 8.0, the concentration of NaCl is 100mM, and the concentration of Mg ²⁺ Is 2mM and Tween 20 is 0.05% by volume.

The comparative example also provides a preparation method of the above-mentioned all-purpose nuclease liquid enzyme preparation, which comprises the following steps:

S2, adding NaCl, magnesium chloride and Tween 20 into 20mM Tris-HCl buffer solution with pH of 8.0, fully dissolving, and filtering by using a 0.22 mu m filter membrane to obtain a protective agent mixed solution;

Comparative example 2

The present comparative example provides a liquid enzyme preparation of a holoase, which is the same as in example 5 except that mannitol and lysine are not added, and specifically, the liquid enzyme preparation of a holoase comprises: omnipotent nuclease, tris-HCl buffer solution with pH of 8.0, naCl and Mg ²⁺ (provided by magnesium chloride), triton X-100;

wherein the activity concentration of the omnipotent nuclease is 400U/. Mu.L, the concentration of the Tris-HCl buffer solution is 30mM, the pH is 8.0, the concentration of NaCl is 50mM, and the concentration of Mg ²⁺ Is 10mM, and the volume concentration of the triton X-100 is 0.2%.

S2, adding NaCl, magnesium chloride and triton X-100 into 30mM Tris-HCl buffer solution with pH of 8.0, fully dissolving, and filtering by using a 0.22 mu m filter membrane to obtain a protective agent mixed solution;

Performance testing

The omnipotent nuclease liquid enzyme preparations obtained in examples 2 to 6 were respectively added to a pH 4.0 acetic acid-sodium acetate buffer solution and a pH 10.0 glycine-sodium hydroxide buffer solution, and treated at room temperature for 12 hours, the activities before and after the omnipotent nuclease treatment were measured according to the method mentioned in example 1, the activities before the treatment were recorded as 100%, and the results are shown in Table 1.

TABLE 1 Activity stability of liquid enzyme preparation of full-functional nuclease at pH 4.0 and pH 10.0

	Example 2	Example 3	Example 4	Example 5	Example 6
						pH 4.0-12h	93.5％	92.8％	94.5％	94.1％	93.8％
pH 10.0-12h	94.2％	91.5％	92.6％	95.2％	93.5％

According to the experimental results shown in Table 1, the protective agent formulations provided in examples 2 to 6 can ensure the stability of the totipotent nuclease at pH 4.0 and pH 10.0, and can maintain the activity of more than 90% in the treatment time of 12 hours.

The omnipotent nuclease liquid enzyme preparations obtained in examples 2 to 6 were treated in water baths at 50℃and 60℃and 70℃and 80℃respectively, and the activities before and after the omnipotent nuclease treatment were measured according to the method mentioned in example 1, and the activities before the treatment were recorded as 100%.

Specifically, the omnipotent nuclease liquid enzyme preparations obtained in examples 2 to 6 were treated at 50℃for 4 hours, 8 hours and 12 hours respectively for activity stability, and the results are shown in Table 2 below.

TABLE 2 Activity stability of liquid enzyme preparation of full-functional nuclease at 50 ℃and method for preparing the same

Specifically, the omnipotent nuclease liquid enzyme preparations obtained in examples 2 to 6 were respectively placed in water baths at 60℃and 70℃and 80℃for treatment for 1 hour, and the activity stability was tested, and the results are shown in Table 3 below.

TABLE 3 Activity stability of omnipotent nucleases at 60 ℃,70 ℃,80 ℃and methods of use

Treatment conditions	Example 2	Example 3	Example 4	Example 5	Example 6
						60℃-1h	90.2％	91.3％	91.5％	90.8％	91.5％
70℃-1h	85.5％	86.1％	84.5％	84.2％	85.0％
						80℃-1h	76.5％	77.0％	76.8％	75.2％	76.2％

According to the experimental results shown in tables 2 and 3, the formulations of the protective agents provided in examples 2 to 6 can ensure the stability of the totipotent nuclease at 50 to 80 ℃, can maintain more than 90% of activity after being treated at 50 ℃ for 12 hours, can maintain more than 90% of activity after being treated at 60 ℃ for 1 hour, can maintain more than 85% of activity after being treated at 70 ℃ for 1 hour, and can maintain more than 75% of activity after being treated at 80 ℃ for 1 hour.

The omnipotent nuclease liquid enzyme preparations obtained in examples 2 to 6 were stored at room temperature (25 ℃) and 4℃respectively, and the activities before and after the storage of the omnipotent nuclease were measured according to the method mentioned in example 1, and the initial activities were recorded as 100%. The activity stability of the extract was tested for 7 days, 15 days and 30 days at room temperature, and the results are shown in Table 4; the activity stability was measured at normal temperature for 4 months, 8 months and 1 year, and the results are shown in Table 5.

TABLE 4 Activity stability of liquid nuclease preparations at Normal temperature

Storage time	Example 2	Example 3	Example 4	Example 5	Example 6
						Initial vitality	100％	100％	100％	100％	100％
For 7 days	98.5％	97.8％	97.3％	97.1％	97.8％
						For 15 days	95.5％	94.9％	95.5％	94.8％	94.0％
For 30 days	92.3％	91.0％	90.5％	92.5％	91.8％

TABLE 5 Activity stability of liquid enzyme preparation of full-functional nuclease at 4℃in storage

According to the experimental results shown in tables 4 and 5, the formulations of the protective agents provided in examples 2 to 6 can ensure the stability of the omnipotent nuclease at normal temperature and 4 ℃, the activity is stable when stored for 30 days at normal temperature, and the activity is stable when stored for 1 year at 4 ℃.

According to the method, the omnipotent nuclease liquid enzyme preparations obtained in examples 7-10 and comparative examples 1-2 are respectively added into a pH 4.0 acetic acid-sodium acetate buffer solution and a pH 10.0 glycine-sodium hydroxide buffer solution, treated for 12 hours at normal temperature, and the activity stability of the omnipotent nuclease liquid enzyme preparations in examples 7-10 and comparative examples 1-2 is tested; the omnipotent nuclease liquid enzyme preparations obtained in examples 7 to 10 and comparative examples 1 to 2 were respectively placed in water baths at 50 ℃ and 60 ℃, 70 ℃ and 80 ℃ for treatment, wherein the treatment is carried out for 12 hours at 50 ℃, the treatment is carried out for 1 hour at 60 ℃, the treatment is carried out for 1 hour at 70 ℃ and the treatment is carried out for 1 hour at 80 ℃, and the activity stability of the omnipotent nuclease liquid enzyme preparations in examples 7 to 10 and comparative examples 1 to 2 is tested; the omnipotent nuclease liquid enzyme preparations obtained in examples 7 to 10 and comparative examples 1 to 2 are respectively stored at normal temperature (25 ℃) and 4 ℃, wherein the normal temperature is stored for 30 days, the 4 ℃ is stored for 1 year, and the activity and the stability of the omnipotent nuclease liquid enzyme preparations in examples 7 to 10 and comparative examples 1 to 2 are tested; the preservation rate of the enzyme activity before and after the treatment under each condition of the omnipotent nuclease was measured according to the method mentioned in example 1, and the initial activity was recorded as 100%, and the results are shown in FIG. 1.

From the experimental results shown in fig. 1, comparative examples 1 and 2 were greatly reduced and examples 7 to 10 were slightly reduced as compared with the stability under each condition of examples 2 and 5 of the preferable formulation. The enzyme activity preservation rate of examples 2 and 5 is more than 90%, the enzyme activity preservation rate of comparative examples 1 and 2 is about 65% and the enzyme activity preservation rate of examples 7 to 10 is 80% -85% after the treatment for 12 hours at pH 4.0 and pH 10.0. The enzyme activity preservation rate of examples 2 and 5 is more than 90%, the enzyme activity preservation rate of comparative examples 1 and 2 is about 60%, and the enzyme activity preservation rate of examples 7 to 10 is about 82% after the treatment at 50 ℃ for 12 hours. The enzyme activity retention rates of examples 2 and 5 were about 90%, the enzyme activity retention rates of comparative examples 1 and 2 were about 62%, and the enzyme activity retention rates of examples 7 to 10 were about 82% after treatment at 60℃for 1 hour. The enzyme activity retention rates of examples 2 and 5 were about 85%, the enzyme activity retention rates of comparative examples 1 and 2 were about 50% and the enzyme activity retention rates of examples 7 to 10 were about 82% after treatment at 70℃for 1 hour. The enzyme activity retention rates of examples 2 and 5 were about 75%, the enzyme activity retention rates of comparative examples 1 and 2 were about 30% and the enzyme activity retention rates of examples 7 to 10 were about 62% after treatment at 80℃for 1 hour. The enzyme activity preservation rate of examples 2 and 5 is 90% or more, the enzyme activity preservation rate of comparative examples 1 and 2 is about 70%, and the enzyme activity preservation rate of examples 7 to 10 is about 85% after storage for 30 days at normal temperature. The enzyme activity retention rates of examples 2 and 5 were 90% or more, the enzyme activity retention rates of comparative examples 1 and 2 were about 72%, and the enzyme activity retention rates of examples 7 to 10 were about 86% when stored at 4℃for 1 year.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims

1. A protective agent for the stability of omnipotent nuclease is characterized by comprising at least one of saccharide substances, sugar alcohol and amino acid.

2. The omnipotent nuclease stability protectant of claim 1, wherein the saccharide comprises a disaccharide and/or a polysaccharide, and the amino acid is a basic amino acid.

3. The omnipotent nuclease stability protectant of claim 2, wherein the disaccharide substance comprises at least one of trehalose and sucrose;

the polysaccharide substance comprises dextran;

the basic amino acid comprises at least one of lysine and arginine.

4. The omnipotent nuclease stability protectant of claim 1, wherein the sugar alcohol comprises at least one of mannitol, sorbitol, xylitol.

5. The omnipotent nuclease stability protectant of any one of claims 1 to 4, further comprising a buffer, a salt substance, mg ²⁺ And a surfactant.

6. The omnipotent nuclease stability protectant of claim 5, wherein the buffer is a buffer with a buffer capacity at pH of 4.0-10.0;

and/or the salt substance comprises at least one of NaCl and KCl;

and/or the Mg ²⁺ Provided by Mg salts;

and/or the surfactant is a nonionic surfactant.

7. The omnipotent nuclease stability protectant of claim 6, wherein the buffer comprises at least one of acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, disodium hydrogen phosphate-citric acid buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, glycine-sodium hydroxide buffer, tris-hydrochloric acid buffer, HEPES buffer;

8. A liquid enzyme preparation of a holoendose comprising: a omnipotent nuclease and a omnipotent nuclease stability protectant as claimed in any one of claims 1 to 7.

9. The liquid enzyme preparation of the holonuclease according to claim 8, which comprises at least one of saccharides, sugar alcohols and amino acids, holonuclease, buffer solution, salt substances and Mg ²⁺ A surfactant;

10. A method of preparing a liquid enzyme preparation of a total energy nuclease according to any one of claims 8 to 9, comprising the steps of: