阅读说明：本技术 酶组合物及其制备方法 (Enzyme composition and preparation method thereof ) 是由 张小蒙 张莉 杨恒月 周敏 于 2019-11-06 设计创作，主要内容包括：本发明属于酶活性技术领域,涉及一种酶组合物及其制备方法。该酶组合物包括：酶以及蝉花活性物质。本发明提供的酶组合物能够提高酶的热稳定性,并且不影响酶的活性。(The invention belongs to the technical field of enzyme activity, and relates to an enzyme composition and a preparation method thereof. The enzyme composition comprises: enzymes and cicada fungus active substances. The enzyme composition provided by the invention can improve the thermal stability of the enzyme and does not influence the activity of the enzyme.)

1. An enzyme composition, comprising: enzymes and cicada fungus active substances.

2. The enzyme composition according to claim 1, wherein the cordyceps sobolifera active substance is a fermentation broth of cordyceps sobolifera mycelium.

3. The enzyme composition according to claim 2, wherein the fermentation broth of cordyceps sobolifera mycelium is prepared by a process comprising the steps of:

(a) obtaining the cordyceps sobolifera mycelium;

(b) sequentially carrying out purification culture and activation culture on the cordyceps sobolifera mycelia;

(c) inoculating the activated cordyceps sobolifera mycelium into a fermentation tank, and fermenting to obtain the fermentation liquor of the cordyceps sobolifera mycelium.

4. The enzyme composition according to claim 3, wherein the step (b) of sequentially performing the purification culture and the activation culture on the cordyceps sobolifera mycelium comprises the following steps:

(b1) inoculating the cordyceps sobolifera mycelium onto a plate culture medium, and culturing for 15-20 days at the temperature of 23-28 ℃;

(b2) inoculating the cordyceps sobolifera mycelium obtained by the culture in the step (b1) into a liquid culture medium, and culturing for 15-20 days at the temperature of 23-28 ℃.

5. The enzyme composition according to claim 1, wherein in step (c), the pressure in the fermenter is 0.7 to 0.9kg/cm²The aeration rate is 0.8-1.5 VVM.

6. The enzyme composition according to claim 5, wherein in step (c), the temperature of the fermentation is 23-28 ℃ and the time of the fermentation is 15-20 days.

7. The enzyme composition according to claim 1, wherein the mass ratio of the enzyme to the cordyceps sobolifera active substance is 1: 1-1: 500.

8. The enzyme composition according to any one of claims 1 to 7, wherein the enzyme comprises at least one of phytase, cellulase and α -amylase.

9. The process for preparing an enzyme composition according to any one of claims 1 to 8, wherein the enzyme composition is obtained by mixing the enzyme with the cordyceps sobolifera active substance.

10. The method of claim 9, wherein the mixing of the enzyme and the cordyceps sobolifera active substance is carried out by dissolving the enzyme and the cordyceps sobolifera active substance in a buffer capable of keeping the enzyme in a configuration.

Technical Field

The invention belongs to the technical field of enzyme activity, and particularly relates to an enzyme composition and a preparation method thereof.

Background

Enzymes are widely used as catalysts in various production processes. In the application process, the catalyst is usually accompanied with high-temperature treatment, especially in industrial manufacturing, but the high-temperature treatment can destroy the structure of the enzyme, so that the enzyme loses catalytic activity, for example, in the feed preparation process, when the temperature reaches 90 ℃, the enzyme is inactivated for 2-5 minutes.

The existing method for improving the thermal stability of enzyme mainly comprises the following steps: the first is to screen enzyme with natural heat stability; secondly, the thermal stability of the enzyme is improved by using a molecular technology method; thirdly, additives are used.

The inventors found in the course of carrying out the present invention that the first method of improving the thermostability of an enzyme had the disadvantages of: naturally occurring thermostable enzymes are often not produced in large scale and are not economically viable. The second method of improving the thermostability of the enzyme has the disadvantages that: the enzyme improved by molecular biotechnology means needs to mutate itself, and then the mutated enzyme is screened in large quantity to obtain the enzyme with high thermal stability. Furthermore, mutations can affect the specificity of the enzyme. The third method of improving the thermostability of the enzyme has the disadvantages that: the existing additives are generally high in salt concentration and influence the activity of enzyme. In addition, the thermal stability of the enzyme is improved by the immobilization method, which also affects the activity of the enzyme.

Therefore, a method for improving the thermostability of an enzyme without affecting the activity of the enzyme is required.

Disclosure of Invention

The invention aims to provide an enzyme composition and a preparation method thereof, which improve the thermal stability of enzyme and do not influence the activity of the enzyme.

In order to achieve the above object, the first aspect of the present invention provides an enzyme composition comprising: enzymes and cicada fungus active substances.

In a preferred embodiment of the present invention, the cordyceps sobolifera active substance is a fermentation broth of cordyceps sobolifera mycelium.

Specifically, the fermentation broth of the cordyceps sobolifera mycelium is prepared by a method comprising the following steps of:

(a) obtaining the cordyceps sobolifera mycelium;

(b) sequentially carrying out purification culture and activation culture on the cordyceps sobolifera mycelia;

(c) inoculating the activated cordyceps sobolifera mycelium into a fermentation tank, and fermenting to obtain the fermentation liquor of the cordyceps sobolifera mycelium.

More specifically, the step (b) of sequentially performing the purification culture and the activation culture on the cordyceps sobolifera mycelium comprises the following steps of:

(b1) inoculating the cordyceps sobolifera mycelium onto a plate culture medium, and culturing for 15-20 days at the temperature of 23-28 ℃;

(b2) inoculating the cordyceps sobolifera mycelium obtained by the culture in the step (b1) into a liquid culture medium, and culturing for 15-20 days at the temperature of 23-28 ℃.

The plate culture medium and the liquid culture medium for inoculating the cordyceps sobolifera mycelium are not particularly limited, as long as the cordyceps sobolifera mycelium can quickly grow and reproduce on the cordyceps sobolifera mycelium. For example, the plating medium may be Potato Dextrose Agar (PDA) medium, the formulation of which is detailed in table 1; the liquid medium may be modified potato dextrose agar medium, and the formulation is detailed in table 2.

TABLE 1 Potato Dextrose Agar (PDA) Medium composition

TABLE 2 modified Potato dextrose agar Medium composition

More specifically, in step (c), the pressure in the fermenter is 0.7 to 0.9kg/cm²The aeration rate is 0.8-1.5 VVM.

More specifically, in step (c), the temperature of the fermentation is 23-28 ℃, and the time of the fermentation is 15-20 days.

In the invention, the enzyme and the cordyceps sobolifera active substance in the enzyme composition can be mixed according to any proportion. As can be understood by those skilled in the art, as the active substance of the cordyceps sobolifera increases, the temperature resistance of the enzyme is correspondingly improved, and when a certain proportion is reached, the temperature resistance of the enzyme is almost unchanged. For the cordyceps sobolifera active substance, the mass ratio of the enzyme to the cordyceps sobolifera active substance is 1: 1-1: 500; for example, 1: 5-300, 1: 5-200, 1: 5-100, 1: 5-50, 1: 5-30, 1: 5-20, 1: 5-10, or 1: 10.

In the course of experiments, the inventors found that the cordyceps sobolifera active substance has the effect of improving the thermostability of a plurality of existing enzymes, such as at least one of phytase, cellulase and α -amylase.

In a second aspect of the present invention, the enzyme composition may be obtained by mixing the enzyme with the cordyceps sobolifera active substance.

Specifically, the enzyme and the cordyceps sobolifera active substance are mixed, the enzyme and the cordyceps sobolifera active substance are dissolved in a buffer solution capable of keeping the enzyme in a configuration, the selection of the buffer solution for keeping the enzyme in the configuration can be determined according to the activity of the enzyme, the pH value of the buffer solution is further determined, the buffer solution required for keeping the enzyme in the configuration can be different for different enzymes, for example, phytase, cellulase and α -amylase can be dissolved by using an acetate buffer solution or a phosphate buffer solution, the acetate buffer solution can be a sodium acetate buffer solution or a potassium acetate buffer solution, the concentration of the acetate buffer solution can be 20-100mmol/L, and the phosphate buffer solution can be a sodium phosphate buffer solution or a potassium phosphate buffer solution, and the concentration of the phosphate buffer solution can be 50-150 mmol/L.

The enzyme composition provided by the invention can improve the thermal stability of the enzyme and does not influence the activity of the enzyme.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.

FIG. 1 shows a graph of the results of the thermostability of the phytase in example 2.

FIG. 2 is a graph showing the results of thermal stability of cellulase in example 3.

FIG. 3 is a graph showing the results of the thermal stability of α -amylase in example 4.

Detailed Description

Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.