CN116064458A - Method for extracting glutamine transaminase by using polyethylene glycol - Google Patents

Abstract

The invention discloses a method for extracting glutamine transaminase by using polyethylene glycol, which comprises the following steps: step 1, preparing fermented liquid; step 2, filtering and concentrating; step 3, extracting glutamine transaminase; step 4, obtaining glutamine transaminase Transaminase crude product; step five, preparing finished product of transaminase; in step two, the activity of transaminase in the concentrate is about 100U/ml; in step three, the mass percentage concentration of polyethylene glycol is 5% -50%; in the step three, the standing time is 24h; in the step five, the mixed enzyme activity of the qualified product is 100u/g ± 10%; the present invention adopts polyethylene glycol to extract Streptomyces mobara fermentation broth The transglutaminase, which overcomes the defects of the organic solvent precipitation method and salting out method in the prior art, has a simpler and safer process, is environmentally friendly, has the advantages of high extraction yield and low production cost, and is more Widespread use offers advantages.

Description

A kind of method adopting polyethylene glycol to extract transglutaminase

technical field

The invention relates to the technical field of production of biological enzyme preparations, in particular to a method for extracting glutamine transaminase by using polyethylene glycol.

Background technique

Transglutaminase widely exists in animals (including humans), plants and microorganisms. It is an acyltransferase that catalyzes acyl transfer reactions between protein molecules and within molecules, thereby causing covalent cross-linking between proteins (or polypeptides). This kind of cross-linking has a significant impact on the properties, gel ability, thermal stability and water-holding capacity of proteins, thereby improving the foamability, plasticity and water-holding capacity of protein foods; and can catalyze the cross-linking of various essential amino acids and proteins , can improve the nutritional value of protein. Due to its high specificity, safety and excellent cross-linking properties, it is widely used in food processing industries such as meat products, aquatic products, soybean products, flour products, rice products and dairy products. In recent years, with the in-depth research on the application of this enzyme, it has been found that it has a wide range of applications in the fields of medicine, daily chemical industry, tanning, and textiles.

In the prior art, the extraction methods for transglutaminase include the following two types: one is the organic solvent precipitation method: that is, ethanol precipitation or acetone precipitation method is used. Although this method is simple, flammable and explosive substances are used in the production process, and there are serious safety hazards; the second is the salting-out method: that is, saturated ammonium sulfate, saturated sodium sulfate, saturated sodium chloride, etc. are used for salting-out. The obtained precipitate must be dialyzed and desalted with a large amount of pure water before it can be used in the food processing industry or other industries, and the process is relatively complicated.

Contents of the invention

The object of the present invention is to provide a method for extracting transglutaminase using polyethylene glycol, so as to solve the problems raised in the above-mentioned background technology.

To achieve the above object, the present invention provides the following technical solutions: a method for extracting transglutaminase using polyethylene glycol, comprising the following steps: Step 1, preparing fermentation broth; Step 2, filtering and concentrating; Step 3, extracting glutamine Amide transaminase; Step 4, obtaining the crude product of Transglutaminase; Step 5, preparing the finished product of Transglutaminase;

Wherein in the above-mentioned step 1, specifically include the following steps:

1.1 Preparation of seed liquid: inoculate the spores of Streptomyces mobara into the seed tank containing the seed medium, and cultivate for 24-48 hours to obtain the seed liquid;

1.2 Preparation of fermentation liquid: inoculate the seed liquid in a fermenter with a built-in fermentation medium, and cultivate for 40-60 hours to obtain a fermentation liquid;

Wherein in the above step 2, the fermented liquid prepared in step 1.2 is taken, filtered through a plate and frame to obtain a filtrate, and the filtrate is subjected to microfiltration and ultrafiltration, and concentrated 5-10 times to obtain a concentrated solution;

Among them, in the above step three, under the condition of 10-15°C, take the concentrated solution prepared in step two, add polyethylene glycol to it while stirring, and when the polyethylene glycol is completely dissolved, let it stand for a period of time , discard the supernatant and centrifuge to obtain a transglutaminase precipitate;

Wherein in the above step 4, the transglutaminase precipitate prepared in step 3 is taken, and after freeze-drying treatment, the crude product of transglutaminase is obtained;

Wherein in the above step 5, the crude transglutaminase prepared in the step 4 is taken, a certain amount of carrier is added according to the product requirements, the enzyme activity of the mixture is determined after fully mixing, and qualified products are packaged and stored.

Preferably, in the step 1.1, the culture conditions are: culture temperature 28-30°C; stirring speed 100-250 rpm; ventilation rate 1:0.3-0.6 (v/v).

Preferably, in the step 1.2, the culture conditions are: culture temperature 28-30° C.; stirring speed 100-200 rpm; ventilation rate 1:0.5-0.8 (v/v).

Preferably, in the second step, the activity of transglutaminase in the concentrate is about 100 U/ml.

Preferably, in the step 3, the mass percent concentration of polyethylene glycol is 5%-50%.

Preferably, in said step 3, the standing time is 24h.

Preferably, in the step five, the mixed enzyme activity of the qualified product is 100u/g±10%.

Preferably, in said step five, the packaging specification is 1kg/bag.

Preferably, in the fifth step, the carrier is starch and dextrin.

Compared with the prior art, the beneficial effects of the present invention are: the present invention uses polyethylene glycol to extract the transglutaminase in the Streptomyces mobara fermentation broth, which overcomes the limitations of the organic solvent precipitation method and the salting-out method in the prior art. Defects, the process is simpler and safer, and it is environmentally friendly. It has the advantages of high extraction yield and low production cost, providing favorable conditions for its wider use.

Description of drawings

Fig. 1 is a flow chart of the method of the present invention.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Please refer to Fig. 1, a kind of technical scheme provided by the present invention:

Example 1

A method for extracting transglutaminase by using polyethylene glycol, comprising the following steps: step 1, preparing fermentation broth; step 2, filtering and concentrating; step 3, extracting transglutaminase; step 4, obtaining crude transglutaminase ; Step 5, preparing the finished product of transglutaminase;

Wherein in the above-mentioned step 1, specifically include the following steps:

1.1 Preparation of seed liquid: Inoculate the spores of Streptomyces mobara into the seed tank containing the seed medium, and cultivate for 48 hours to obtain the seed liquid; wherein, the culture conditions are: culture temperature 28-30°C;

Stirring speed 100-250 rpm; ventilation volume 1:0.3-0.6(v/v);

1.2 Preparation of fermentation liquid: inoculate the seed liquid into a fermenter with a built-in fermentation medium, and cultivate for 60 hours to obtain a fermentation liquid; wherein, the culture conditions are: culture temperature 28-30°C; stirring speed 100-200 rpm; ventilation Volume 1: 0.5-0.8(v/v);

Wherein in the above step 2, the fermented liquid prepared in step 1.2 is taken, and after plate and frame filtration, the filtrate is obtained, and the filtrate is processed by microfiltration and ultrafiltration, and concentrated 5-10 times to obtain a concentrated solution, in which glutamine The transaminase activity is about 100U/ml;

Wherein in the above step three, under the condition of 10-15°C, take 10L of the concentrated solution prepared in step two, and add 1kg of food-grade polyethylene glycol 400 to it while stirring, when the food-grade polyethylene glycol 400 After completely dissolving, let it stand for 24 hours, discard the supernatant and centrifuge to obtain the transglutaminase precipitate;

Wherein in the above step 4, the transglutaminase precipitate prepared in step 3 is taken, and after freeze-drying treatment, the crude product of transglutaminase is obtained;

Wherein in above-mentioned step 5, get the transglutaminase crude product prepared in step 4, add a certain amount of starch and dextrin according to product requirements, measure the enzymatic activity of mixture after fully mixing, qualified product is packaged and stored; Wherein, the mixture of qualified product The enzyme activity is 100u/g±10%, and the packaging specification is 1kg/bag.

Example 2

The extraction method of transglutaminase was the same as in Example 1, except that the amount of food-grade polyethylene glycol 400 added in step 3 was changed to 2kg.

Example 3

The extraction method of transglutaminase was the same as in Example 1, except that the amount of food-grade polyethylene glycol 400 added in step 3 was changed to 3kg.

Example 4

The extraction method of transglutaminase was the same as in Example 1, except that the food-grade polyethylene glycol 400 in step 3 was changed to food-grade polyethylene glycol 2000.

Example 5

The extraction method of transglutaminase was the same as that in Example 1, except that the food-grade polyethylene glycol 400 in step 3 was changed to food-grade polyethylene glycol 2000, and the addition amount was changed to 2kg.

Example 6

The extraction method of transglutaminase was the same as that in Example 1, except that the food-grade polyethylene glycol 400 in step 3 was changed to food-grade polyethylene glycol 2000, and the addition amount was changed to 3kg.

Example 7

The extraction method of transglutaminase was the same as in Example 1, except that the food-grade polyethylene glycol 400 in step 3 was changed to food-grade polyethylene glycol 4000.

Example 8

The extraction method of transglutaminase was the same as that in Example 1, except that the food-grade polyethylene glycol 400 in step 3 was changed to food-grade polyethylene glycol 4000, and the addition amount was changed to 2kg.

Example 9

The extraction method of transglutaminase was the same as that in Example 1, except that the food-grade polyethylene glycol 400 in step 3 was changed to food-grade polyethylene glycol 4000, and the addition amount was changed to 3kg.

Example 10

The extraction method of transglutaminase was the same as in Example 1, except that the food-grade polyethylene glycol 400 in step 3 was changed to food-grade polyethylene glycol 8000.

Example 11

The extraction method of transglutaminase was the same as that in Example 1, except that the food-grade polyethylene glycol 400 in step 3 was changed to food-grade polyethylene glycol 8000, and the addition amount was changed to 2 kg.

Example 12

The extraction method of transglutaminase was the same as that in Example 1, except that the food-grade polyethylene glycol 400 in step 3 was changed to food-grade polyethylene glycol 8000, and the addition amount was changed to 3kg.

Based on the above, the advantage of the present invention is that the invention adds a certain concentration of polyethylene glycol to the concentrated solution of Streptomyces mobara fermentation broth, so that the transglutaminase in the concentrated solution is precipitated, and then solid-state transglutaminase is obtained by liquid-solid separation. Transglutaminase; this process does not use organic solvents such as ethanol and acetone; does not use salting out such as saturated ammonium sulfate, sodium sulfide, sodium chloride; and does not use organic acids, so the production process is simple, safe and environmentally friendly.

It will be apparent to those skilled in the art that the invention is not limited to the details of the above-described exemplary embodiments, but that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Accordingly, the embodiments should be regarded in all points of view as exemplary and not restrictive, the scope of the invention being defined by the appended claims rather than the foregoing description, and it is therefore intended that the scope of the invention be defined by the appended claims rather than by the foregoing description. All changes within the meaning and range of equivalents of the elements are embraced in the present invention. Any reference sign in a claim should not be construed as limiting the claim concerned.

Claims (9)

1. A method for extracting glutamine transaminase by using polyethylene glycol, which comprises the following steps: step one, preparing fermentation liquor; step two, filtering and concentrating; step three, extracting glutamine transaminase; step four, obtaining a glutamine transaminase crude product; step five, preparing a glutamine transaminase finished product; the method is characterized in that:

the first step specifically comprises the following steps:

1.1 preparing seed liquid: inoculating Streptomyces mobaraensis spores into a seed tank containing a seed culture medium, and culturing for 24-48 hours to obtain seed liquid;

1.2 preparation of fermentation broth: inoculating the seed liquid into a fermentation tank filled with a fermentation medium, and culturing for 40-60 hours to obtain fermentation liquid;

in the second step, the fermentation broth prepared in the step 1.2 is filtered by a plate frame to obtain filtrate, and the filtrate is subjected to microfiltration and ultrafiltration treatment and concentrated 5-10 times to obtain concentrated solution;

wherein in the third step, under the condition of 10-15 ℃, the concentrated solution prepared in the second step is taken, polyethylene glycol is added into the concentrated solution while stirring, after the polyethylene glycol is completely dissolved, the concentrated solution is stood for a period of time, and the supernatant is removed and centrifuged to obtain glutamine transaminase precipitate;

in the fourth step, the glutamine transaminase precipitate prepared in the third step is taken and subjected to freeze drying treatment to obtain a glutamine transaminase crude product;

in the fifth step, the crude glutamine transaminase product prepared in the fourth step is taken, a certain amount of carrier is added according to the product requirement, the enzyme activity of the mixture is measured after the mixture is fully mixed, and the qualified product is packaged and stored.

2. The method for extracting glutamine transaminase from polyethylene glycol according to claim 1, wherein: in the step 1.1, the culture conditions are as follows: the culture temperature is 28-30 ℃; stirring at 100-250 rpm; ventilation is 1:0.3-0.6 (v/v).

3. The method for extracting glutamine transaminase from polyethylene glycol according to claim 1, wherein: in the step 1.2, the culture conditions are as follows: the culture temperature is 28-30 ℃; stirring at 100-200 rpm; ventilation is 1:0.5-0.8 (v/v).

4. The method for extracting glutamine transaminase from polyethylene glycol according to claim 1, wherein: in the second step, the glutamine transaminase activity in the concentrated solution is about 100U/ml.

5. The method for extracting glutamine transaminase from polyethylene glycol according to claim 1, wherein: in the third step, the mass percentage concentration of polyethylene glycol is 5% -50%.

6. The method for extracting glutamine transaminase from polyethylene glycol according to claim 1, wherein: in the third step, the standing time is 24 hours.

7. The method for extracting glutamine transaminase from polyethylene glycol according to claim 1, wherein: in the fifth step, the enzyme activity of the mixture of qualified products is 100 u/g+/-10%.

8. The method for extracting glutamine transaminase from polyethylene glycol according to claim 1, wherein: in the fifth step, the packaging specification is 1 kg/bag.

9. The method for extracting glutamine transaminase from polyethylene glycol according to claim 1, wherein: in the fifth step, the carrier is starch and dextrin.

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