Method for preparing pyruvic oxidase

A technology of pyruvate oxidase and solution, which is applied in the field of bioengineering, can solve the problems of enzyme activity decrease and increase, activity decrease, enzyme activity recovery rate decrease, etc., and achieve the effect of high recovery rate, short production cycle and mild operating conditions

Inactive Publication Date: 2013-09-04
QILU UNIV OF TECH
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Problems solved by technology

However, since the molecular weight of polyols is much smaller than that of polymers (such as PEG), when polyols and inorganic salts form an aqueous two-phase system, at least one of them needs to have a higher concentration in the system (compared to polyethylene glycol) Alcohol / inorganic salt system), polyol as an organic solvent, when its concentration is high, the activity of enzyme in its solution will decrease, and with the increase of its concentration, the decrease of enzyme activity will also increase; when the concentration of inorganic salt is higher When the concentration is high, the enzyme protein will precipitate due to salting out, resulting in a decrease in the recovery rate of the enzyme activity

Method used

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  • Method for preparing pyruvic oxidase

Examples

Experimental program
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Embodiment 1

[0060] The strain used in this example is Lactobacillus plantarum (Lactobacillus plantarum) ATCC8014, which was purchased from the American Type Culture Collection (ATCC).

[0061] The pyruvate oxidase-producing strain Lactobacillus plantarum (Lactobacillus plantarum) ATCC8014 was inoculated into a conical flask containing 20 mL of seed medium, and after standing at 30°C for 24 hours, liquid seeds were prepared; then the inoculation amount was 10% ( V / V) was inoculated in 200mL of fermentation medium, cultured on a shaker at 37°C and 250r / min for 18h, and a bacterial cell culture solution containing pyruvate oxidase was obtained. It has been tested that the enzyme production activity can reach 1562U / L.

[0062] Seed medium (g / L): peptone 10, beef extract 10, yeast extract 5, glucose 20, sodium acetate 5, diammonium citrate 2, Tween-801, K 2 HPO 4 2. MgSO 4 1. MnSO 4 0.5, pH 6.5.

[0063] Fermentation medium (g / L): peptone 10, beef extract 10, yeast extract 5, glucose 20, ...

Embodiment 2

[0076] As described in Example 1, the method for preparing pyruvate oxidase using a two-phase extraction system differs in that:

[0077] In step (2), 200 mL of sodium sulfate aqueous solution was prepared, and polyethylene glycol (PEG1500) with a molecular weight of 1500 was added to the solution so that the mass percentage concentration of sodium sulfate in the total system was 16 wt%, and polyethylene glycol in the total The mass percent concentration in the system was 26 wt%, and the mixture was uniformly mixed to prepare a polyethylene glycol / sodium sulfate two-phase aqueous system solution.

[0078] The recovery rate of the enzyme activity of pyruvate oxidase reached 92.1%, and the purification factor reached about 7.3.

Embodiment 3

[0080] As described in Example 1, the method for preparing pyruvate oxidase using a two-phase extraction system differs in that:

[0081] In step (2), 200 mL of potassium sodium tartrate aqueous solution is prepared, and polyethylene glycol (PEG1000) with a molecular weight of 1000 is added to the solution so that the mass percentage concentration of potassium sodium tartrate in the total system is 20 wt%. The mass percentage concentration in the total system is 20wt%, and they are mixed uniformly to prepare a polyethylene glycol / potassium sodium tartrate two-phase aqueous system solution.

[0082] The recovery rate of the enzyme activity of pyruvate oxidase reached 90.6%, and the purification factor reached about 7.1.

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Abstract

The invention relates to a method for preparing pyruvic oxidase. The method comprises the steps as follows: (1), cultivation of microbial cells containing the pyruvic oxidase, centrifugal collection of bacterial cells, and cell disruption; (2), preparation of a polyethylene glycol / inorganic salt two-aqueous phase extraction system solution; (3), three times of extraction separation by the aid of a two-aqueous phase extraction system, and obtaining of an inorganic salt solution of the pyruvic oxidase; and (4), flirtation and concentration of an ultrafiltration membrane, freezing and drying of a trapped fluid, and preparation of a pyruvate oxidase product. The method can prepare the pyruvic oxidase continuously in a large scale, the preparation technology of the oxidase is simple and convenient, the production period is short, the operation condition is mild, the recovery rate of the oxidase activity is high, and the production cost of the pyruvic oxidase is lower.

Description

technical field [0001] The invention relates to a method for preparing pyruvate oxidase, in particular to a method for preparing pyruvate oxidase by using a two-phase extraction technique, and belongs to the technical field of bioengineering. Background technique [0002] Pyruvate oxidase (Pyruvate oxidase, EC1.2.3.3) is a widely used enzyme preparation for clinical diagnosis, mainly used for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in blood ) activity determination, in addition, it is also used for the determination of pyruvate kinase activity and the determination of pyruvate, inorganic phosphate, urea and other compounds. [0003] The preparation of pyruvate oxidase adopts microbial fermentation method, that is, it is obtained by cultivating microbial cells with high activity of pyruvate oxidase, such as Lactobacillus plantarum, Aerococcus viridans, etc., and the preparation process includes the fermentation of microbial cells Cultivation and s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02
Inventor 董永胜
Owner QILU UNIV OF TECH
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