CN108977429B - Marine microorganism YS2071 lipase stability protective agent - Google Patents

Marine microorganism YS2071 lipase stability protective agent Download PDF

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CN108977429B
CN108977429B CN201811071040.XA CN201811071040A CN108977429B CN 108977429 B CN108977429 B CN 108977429B CN 201811071040 A CN201811071040 A CN 201811071040A CN 108977429 B CN108977429 B CN 108977429B
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lipase
marine microorganism
protective agent
stability
enzyme
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CN108977429A (en
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郝建华
王伟
孙晶晶
刘均忠
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Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
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Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)

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Abstract

The invention relates to a marine microorganism YS2071 lipase stability protective agent, belonging to the technical field of biology, wherein the protective agent comprises the following components: 5-10% of glycerol, 5-15% of sorbitol, 5-20% of fructose, 1-2% of sodium benzoate, 5-20mM of calcium chloride, 0.1-1% of potassium sorbate and 8-10 of the most stable pH value. The protective agent greatly improves the stability of the marine microorganism YS2071 lipase, the enzyme activity retention rate of 91.29 percent when the marine microorganism YS2071 lipase is stored for 90 days at 25 ℃, the enzyme activity retention rate is improved by 70.1 percent compared with a control sample without the protective agent, the stability of the marine microorganism YS2071 lipase is greatly improved, and the protective agent has wide application prospect.

Description

Marine microorganism YS2071 lipase stability protective agent
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a marine microorganism YS2071 lipase stability protective agent.
Background
The inactivation of enzyme is an important factor for limiting the industrial production and application of enzyme preparations, because the storage stability and thermal stability of enzyme are the key to the large-scale production and commercialization of enzyme preparations, so how to improve the storage stability and reduce the inactivation of enzyme is a hot spot of research in the field of biochemical engineering in recent years. The storage stability of the enzyme is of practical significance for the application of the enzyme. At present, there are 3 main methods for stabilizing enzymes, namely, adding a protective agent, chemical modification and immobilization treatment. Due to the various reasons of simple method, low cost, easy operation and the like, the addition of the protective agent is the most important means for improving the stability of the enzyme in the industry at present.
Lipases are an important class of hydrolases acting on glycerol acylesters, the basic function of which is to catalyze the hydrolysis of glycerol esters to glycerol, diacylglycerol, monoacylglycerol and fatty acids. In addition, lipases can also catalyze the hydrolysis, ester synthesis, acidolysis, transesterification, alcoholysis, ammonolysis, and other reactions of glycerides and water-insoluble esters, which usually have a certain stereoisomeric specificity and chemoselectivity. Compared with solid enzyme, the liquid enzyme greatly saves energy and raw materials in the extraction process, shortens the process, reduces the cost, reduces the environmental pollution and improves the labor conditions of workers. However, liquid lipases are unstable and easily inactivated when the water content is high, and it is important to obtain a protective agent that can stably store the liquid lipase.
Due to its unique living environment, the secreted lipase may have special properties and potential uses. The bacteria YS2071 are separated from the sea water of yellow sea in China. Researches show that the lipase secreted by YS2071 has stronger hydrolytic capability on long-chain fatty acid ester, particularly has better decomposition effect on nitrophenyl laurate and glycerol trilaurate, and has good application potential in catalyzing the hydrolysis of ethyl ester type fish oil into fatty acid and the conversion of biodiesel. As a result of long-term adaptation to ocean low-temperature environment, the enzyme has poor thermal stability and is easy to inactivate at a lower temperature, which seriously influences the development and application of the enzyme. Therefore, the composite protective agent of the lipase secreted by YS2071 lays a foundation for the application and development of the lipase.
Disclosure of Invention
The invention aims to provide a marine microorganism YS2071 lipase stability protective agent.
The invention is realized by the following technical scheme:
a marine microorganism YS2071 lipase stability protective agent comprises the following components in percentage by mass: 5-10% of glycerol, 5-15% of sorbitol, 5-20% of fructose, 1-2% of sodium benzoate, 5-20mM of calcium chloride, 0.1-1% of potassium sorbate and 8-10% of most stable pH; the marine microorganism YS2071 lipase is produced by marine microorganism YS 2071.
The invention also provides marine microorganism YS2071 lipase enzyme liquid, which is prepared by adding the marine microorganism YS2071 lipase into the protective agent, wherein the mass concentration of the marine microorganism YS2071 lipase in the protective agent is 10-15 mg/ml.
The preparation method of the marine microorganism YS2071 lipase stability protective agent comprises the following steps: adding glycerol, sorbitol, fructose, sodium benzoate, calcium chloride and potassium sorbate into a solution of 50mM Tris-HCl and pH 8.0-10.0 in proportion, uniformly stirring, standing at 20-30 ℃ for 1-2 hours until the solute is completely dissolved, and performing constant volume by using the solution of 50mM Tris-HCl and pH 8.0-10.0 to prepare the marine microorganism YS2071 lipase stability protective agent;
Compared with the prior art, the invention has the beneficial effects that:
by adopting the stability protective agent provided by the invention, the thermal stability of the marine microorganism YS2071 lipase is greatly improved, the enzyme activity retention rate is 91.29% after the marine microorganism YS2071 lipase is stored for 90 days at 25 ℃, the enzyme activity retention rate is improved by 70.1% compared with a control sample without the protective agent, and the stability protective agent has wide application prospect.
Drawings
FIG. 1: influence of alcohols with different concentrations on the enzyme activity stability of the marine microorganism YS2071 lipase;
FIG. 2: influence of saccharides with different concentrations on the enzyme activity stability of the marine microorganism YS2071 lipase;
FIG. 3: the influence of sodium benzoate on the enzyme activity stability of the marine microorganism YS2071 lipase;
FIG. 4: influence of potassium sorbate on enzyme activity stability of marine microorganism YS2071 lipase.
Detailed Description
The technical solutions of the present invention are further illustrated below by way of examples, but the scope of the present invention is not limited by the examples in any way, and is not limited to the following examples:
example 1 Effect of alcohols on the enzymatic Activity stability of the Marine microorganism YS2071 Lipase
Preparing equal-volume glycerol, propylene glycol, ethanol and sorbitol solutions with the content of 1.5, 3, 4.5, 6, 7.5, 9 and 10.5 percent respectively, adding marine microorganism YS2071 lipase enzyme powder into the solutions respectively to enable the final concentration of the enzyme solution to be 2mg/mL, adding no reagent into a control group, placing each group of enzyme solutions in an incubator at 40 ℃ for 48 hours, and measuring the residual enzyme activity of each group of enzyme solutions after 48 hours.
The polyhydric alcohols (glycerin, propylene glycol, ethylene glycol, polyhydric alcohol-D) and the like, the polyethylene glycol (PEG), the thickening agent-B, the sodium alginate and the like can be combined with water molecules, the activity of water is reduced, the influence of the water molecules on enzyme protein is reduced, and after the substances are added, the viscosity of the enzyme liquid is increased, so that the enzyme liquid system is more uniform and stable, therefore, the proper selection is adopted, and the stability of the liquid enzyme can be improved. The influence of alcohols with different concentrations on the enzymatic activity stability of the marine microorganism YS2071 lipase is shown in figure 1, and it can be seen that the stability effect of glycerol and sorbitol on the marine microorganism YS2071 lipase is good, the highest enzyme activity retention rates are 75.10% and 65.85% respectively, and propylene glycol and ethanol have different degrees of inhibition effects on the enzyme activity.
Example 2 Effect of different saccharides on the stability of the Lipase of the Marine microorganism YS2071
Respectively adding glucose, alpha-lactose, sucrose, fructose, xylan and maltose solutions into 2mg/mL isometric marine microorganism YS2071 lipase solutions, respectively preparing 2%, 4%, 6%, 8%, 10% and 12% isometric glucose, alpha-lactose, sucrose, fructose, xylan and maltose solutions, respectively adding enzyme powder into the solutions to enable the final concentration of the enzyme solution to be 2mg/mL, adding no reagent into a control group, placing each group of enzyme solutions in an incubator at 40 ℃ for 48 hours, and measuring the residual enzyme activity of each group of enzyme solutions after 48 hours.
The influence of the saccharides with different concentrations on the enzymatic activity stability of the marine microorganism YS2071 lipase is shown in figure 2, and it can be seen that fructose has a certain protection effect on the enzymatic activity of the marine microorganism YS2071 lipase, and the highest enzyme activity retention rates are 65.12 percent respectively; and other saccharides have different degrees of inhibition on enzyme activity.
Example 3 Effect of preservatives on the enzymatic Activity stability of the Lipase of the Marine microorganism YS2071
Adding preservatives such as sodium benzoate and potassium sorbate into 2mg/ml of marine microorganism YS2071 lipase liquid with the same volume respectively to enable the content of the additive to be 0.4%, not adding any additive into a control group, placing each group of the lipase liquid in an incubator at 40 ℃ for 48h, and measuring the residual enzyme activity of each group of the enzyme liquid after 48 h.
The influence of sodium benzoate on the enzyme activity stability is shown in fig. 3, and the result shows that when the content of sodium benzoate is 1.6%, the enzyme activity retention rate reaches the highest value, namely 65.00%, and the stabilization effect is good. The influence of potassium sorbate on the enzyme activity stability is shown in fig. 4, when the potassium sorbate content is 0.4%, the enzyme activity retention rate reaches the maximum, 65.58%, and the stabilization effect is good. Example 4
A marine microorganism YS2071 lipase stability protective agent comprises the following components in percentage by mass: 5% of glycerol, 5% of sorbitol, 5% of fructose, 1% of sodium benzoate, 5mM of calcium chloride and 0.1% of potassium sorbate, wherein the most stable pH is 8;
The preparation method of the marine microorganism YS2071 lipase stability protective agent comprises the following steps: adding glycerol, sorbitol, fructose, sodium benzoate, calcium chloride and potassium sorbate into a solution of 50mM Tris-HCl and pH8.0 according to a certain proportion, uniformly stirring, standing at 20 ℃ for 1 hour until solute is completely dissolved, and performing constant volume by using a solution of 50mM Tris-HCl and pH8.0 to prepare the marine microorganism YS2071 lipase stability protective agent.
Example 5: a marine microorganism YS2071 lipase stability protective agent comprises the following components in percentage by mass: 6% of glycerol, 9% of sorbitol, 9% of fructose, 1.7% of sodium benzoate, 15mM of calcium chloride and 0.5% of potassium sorbate, wherein the most stable pH is 9;
the preparation method of the marine microorganism YS2071 lipase stability protective agent comprises the following steps: adding glycerol, sorbitol, fructose, sodium benzoate, calcium chloride and potassium sorbate into a solution of 50mM Tris-HCl and pH10.0 according to a certain proportion, uniformly stirring, standing at 20-30 ℃ for 2 hours until solute is completely dissolved, and fixing the volume by using a solution of 50mM Tris-HCl and pH10.0 to obtain the marine microorganism YS2071 lipase stability protective agent;
example 6
A marine microorganism YS2071 lipase stability protective agent comprises the following components in percentage by mass: 8% of glycerol, 10% of sorbitol, 15% of fructose, 1.5% of sodium benzoate, 10mM of calcium chloride and 0.61% of potassium sorbate, wherein the most stable pH is 9;
The preparation method of the marine microorganism YS2071 lipase stability protective agent comprises the following steps: adding glycerol, sorbitol, fructose, sodium benzoate, calcium chloride and potassium sorbate into a solution of 50mM Tris-HCl and pH9.0 according to a certain proportion, uniformly stirring, standing at 25 ℃ for 1.5 hours until solute is completely dissolved, and fixing the volume by using a solution of 50mM Tris-HCl and pH9.0 to obtain the marine microorganism YS2071 lipase stability protective agent;
comparative test
Test 1: the marine microorganism YS2071 lipase stability protective agent prepared in the embodiment 4 plus the marine microorganism YS2071 lipase;
test 2: the marine microorganism YS2071 lipase stability protective agent prepared in the example 5 plus the marine microorganism YS2071 lipase;
test 3: the marine microorganism YS2071 lipase stability protective agent prepared in the embodiment 6 plus the marine microorganism YS2071 lipase;
control group: water and marine microorganism YS2071 lipase
Adding the lipase produced by the marine microorganism YS2071 into the protective agent of the embodiment 4, the embodiment 5 and the embodiment 6, wherein the mass concentration of the lipase in the protective agent is 10 mg/ml;
the enzyme activity is measured after the sample is stably stored for 90 days at 25 ℃, the enzyme activity retention rate of the test 1, the test 2 and the test 3 is still 91.29 percent, which is 70.1 percent higher than that of the control group sample without the protective agent.
The method for measuring the lipase activity used in this example: reaction system: solution A: 83.3mg of pNP-lauate was dissolved in isopropanol to a volume of 25 mL. And B, liquid B: 0.1mol/L phosphate buffer solution with pH of 8.0 is added with 0.4 percent TritonX-100, and the volume is adjusted to 1L. Mixing 0.1mL of solution A and 1.5mL of solution B, preheating in a 40 ℃ constant temperature water bath for 4min, adding 0.1mL of enzyme solution to be tested (the control group is the enzyme solution which is equivalently boiled and inactivated for 8 min), continuously incubating for 6min, taking out, adding 2mL of ethanol, stopping the reaction in an ice bath, and measuring the light absorption value at 410 nm. The enzyme activity unit (U) is defined as the amount of enzyme required by the enzyme to catalyze the hydrolysis of a substrate to produce 1.0. mu. mol/L pNP per minute at 40 ℃ and pH 8.0.

Claims (3)

1. A marine microorganism YS2071 lipase stability protective agent is characterized by comprising the following components: 5-10% of glycerol, 5-15% of sorbitol, 5-20% of fructose, 1-2% of sodium benzoate, 5-20mM of calcium chloride, 0.1-1% of potassium sorbate and 8-10% of most stable pH; the marine microorganism YS2071 lipase is produced by marine microorganism YS2071, and the percentages are mass fractions.
2. A marine microorganism YS2071 lipase liquid is characterized in that the enzyme liquid is prepared by adding marine microorganism YS2071 lipase into the protective agent of claim 1, and the mass concentration of the marine microorganism YS2071 lipase in the protective agent is 10-15 mg/ml.
3. A method for preparing a stabilizing protective agent for the lipase of marine microorganism YS2071 as claimed in claim 1, which comprises the following steps: adding glycerol, sorbitol, fructose, sodium benzoate, calcium chloride and potassium sorbate into a solution of 50mM Tris-HCl and pH 8.0-10.0 according to a certain proportion, uniformly stirring, standing at 20-30 ℃ for 1-2 hours until solute is completely dissolved, and fixing the volume by using a solution of 50mM Tris-HCl and pH 8.0-10.0.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108497442A (en) * 2018-04-24 2018-09-07 东莞市翔实信息科技有限公司 A kind of cream zymolyte and preparation method thereof

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EP2079445B1 (en) * 2007-02-20 2015-11-04 Aptalis Pharma Limited Stable digestive enzyme compositions

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108497442A (en) * 2018-04-24 2018-09-07 东莞市翔实信息科技有限公司 A kind of cream zymolyte and preparation method thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
壳聚糖固定化海洋微生物YS2071脂肪酶及其酶学性质;张权 等;《渔业科学进展》;20151231;第36卷(第6期);100-106页 *
海洋脂肪酶YS2071的固定化及应用研究;姜峻颖;《中国优秀硕士学位论文全文数据库基础科学辑》;20180215(第2期);A006-391 *

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