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Method for promoting Eupergit C 250 to immobilize oxalate decarboxylase by using ammonium sulfate

A technology of oxalate decarboxylase and immobilized enzyme carrier, which is applied in the direction of being immobilized on/in an organic carrier, can solve the problems of increasing the cost of immobilization reaction, and achieves the benefits of recycling and reuse, improving stability, and improving immobilization. effect of efficiency

Inactive Publication Date: 2011-09-07
GUANGXI UNIV FOR NATITIES
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  • Summary
  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, some buffer pairs are relatively expensive, and the method of increasing the buffer concentration will greatly increase the cost of the immobilization reaction.
In addition, the use of Eupergit C immobilized oxalate decarboxylase for the degradation of oxalate and oxalate in fluid has not been reported so far

Method used

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  • Method for promoting Eupergit C 250 to immobilize oxalate decarboxylase by using ammonium sulfate

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0020] (1) Induced expression of recombinant oxalate decarboxylase

[0021] The genetically engineered bacteria E.coli BL21(DE3) / pET32a / YvrK were inoculated in LB medium containing ampicillin (0.1mg / ml) at 37°C and cultured at 200r / min. When the bacteria grow to an OD600 of 0.4, heat shock at 42°C for 5 minutes, lower the temperature back to 37°C, and add MnCl 2 The final concentration of IPTG was 5mmol / L, and the final concentration of IPTG was 0.4mmol / L. After inducing expression for 4 hours, the bacterial pellet was collected by centrifugation, and 10mL of phosphate buffer (50mmol / L, pH8.0) was added for each gram of wet weight of the bacteria. Suspended bacteria, after the bacteria were ultrasonically disrupted, the supernatant was collected by centrifugation as a crude enzyme solution.

[0022] (2) Separation and purification of oxalate decarboxylase by affinity chromatography

[0023] Enzyme purification was performed using the AKTA-FPLC system. First, equilibrate the...

example 2

[0028] (1) Induced expression of recombinant oxalate decarboxylase is the same as example 1

[0029] (2) Affinity chromatography separation and purification of oxalate decarboxylase with example 1

[0030] (3) Immobilization of oxalate decarboxylase

[0031]EupergitC is first soaked and swollen with phosphate buffer solution, added purified oxalate decarboxylase, and ammonium sulfate to make the concentration reach 0.1-2mol / L, at pH7-10, 20-30℃, shake and immobilize at a speed of 30-200rpm 4-72h, centrifuge to discard the supernatant, add 5% mercaptoethanol to block the remaining epoxy groups, filter after 12h reaction, wash with buffer, rinse mercaptoethanol and unfixed free enzyme until there is no enzyme in the eluate Activity, that is, immobilized oxalate decarboxylase.

[0032] The recovery rate of protein during immobilization process, the recovery rate of immobilized enzyme activity and the apparent specific activity of immobilized enzyme are shown in Table 1.

[003...

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Abstract

The invention provides a method for promoting Eupergit C 250 to immobilize oxalate decarboxylase by using ammonium sulfate, belonging to the technical field of biochemical engineering. The method comprises the steps of inducible expression of recombinant oxalate decarboxylase, separation and purification of the oxalate decarboxylase by means of affinity chromatography, and immobilization of the oxalate decarboxylase. The method is characterized in that the Eupergit C 250 containing active epoxy groups is used as an immobilized vector, and 0.1-2mol / L neutral salt of the ammonium sulfate is added in the immobilizing process to promote the adsorption and covalency connection reaction of the vector to the oxalate decarboxylase. The method provided by the invention is simple to operate, and enzymatic activity recovery rate reaches more than 84%; and the used vector material is moderate in price and has a stable materialization performance, and the prepared immobilized enzyme has a better operating characteristic, is easily recovered and reused, and can be applied to oxalate degradation in food making or papermaking and other industrial processes.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular provides a method for ammonium sulfate to promote Eupergit C 250 immobilized oxalate decarboxylase. Background technique [0002] Oxalate decarboxylase (EC 4.1.1.2) catalyzes the conversion of oxalic acid into formic acid and CO2. It is one of the three enzymes that catalyze the degradation of oxalic acid and oxalate in organisms. It has strong specificity for the substrate oxalic acid. Oxalate decarboxylase has been used in clinical medical detection of hyperoxaluria and urolithiasis, and it also has broad application prospects in food, industry and other fields. The application of oxalate decarboxylase in the beer industry can control and degrade beer stones rich in oxalate; it can be used in the alumina smelting industry to efficiently process oxalic acid waste in the production process; The scale risk of oxalate on equipment such as roads, filters or heat exchangers (C...

Claims

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Application Information

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IPC IPC(8): C12N11/08
Inventor 林日辉
Owner GUANGXI UNIV FOR NATITIES
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