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Preparation method of cross-linked oxalate decarboxylase aggregates (CLEAs)

A technology of oxalate decarboxylase and aggregates, which is applied in the field of preparation of cross-linked oxalate decarboxylase aggregates, can solve the problems of high cost and difficult operation, and achieve the effect of high separation efficiency, high purity and simple operation

Inactive Publication Date: 2012-06-13
南宁奕德环境科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For stone disease caused by oxalate crystallization, there have been some reports on the prevention and treatment of oxalate decarboxylase, among which the more practical value is to crystallize and immobilize pure oxalate decarboxylase to prepare cross-linked oxalate decarboxylation Enzyme crystal preparation (oxdc-CLECs) (crystallized oxalate decarboxylase and method of use, Chinese Patent Publication No.: CN 101583375A), but the preparation of oxdc-CLECs must first go through the crystallization operation of enzyme protein, the required degree of enzyme purification and The crystallization conditions are very high, the operation is difficult, and the cost is high

Method used

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  • Preparation method of cross-linked oxalate decarboxylase aggregates (CLEAs)
  • Preparation method of cross-linked oxalate decarboxylase aggregates (CLEAs)
  • Preparation method of cross-linked oxalate decarboxylase aggregates (CLEAs)

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0028] (1) Induced expression of recombinant oxalate decarboxylase

[0029] 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 250r / min. Bacteria grow to OD 600 When it is 0.8, add 6mmol / L of MnCl 2 , 0.4mmol / L IPTG, induced expression at 30°C for 8h, centrifuged to collect the bacterial pellet, added 10mL phosphate buffer (50mmol / L, pH 8.0) to resuspend the bacteria per gram of wet weight of the bacteria, and ultrasonically disrupted the bacteria , centrifuged to collect the supernatant as crude enzyme solution.

[0030] (2) Precipitation and separation of oxalate decarboxylase

[0031] Add 5ml of crude enzyme solution to a 10ml test tube, add pre-cooled absolute ethanol drop by drop until the ethanol concentration is 10% to 50%, put it in a refrigerator at 4°C overnight, collect the precipitate by centrifugation, and wash with 50mmol / L pH8.0 phosphate The buffer was rediss...

example 2

[0037] (1) Induced expression of recombinant oxalate decarboxylase is the same as example 1

[0038] (2) Precipitation and separation of oxalate decarboxylase

[0039] Add 5ml of crude enzyme solution to a 10ml test tube, add pre-cooled acetone drop by drop to a concentration of 10% to 50%, put it in a refrigerator at 4°C overnight, centrifuge to collect the precipitate, and redissolve it with 50mmol / L pH8.0 phosphate buffer , to obtain purified oxalate decarboxylase.

[0040] Enzyme activity detection and protein concentration analysis showed that acetone precipitation could remove 73.6% of the protein in the crude enzyme solution, the enzyme activity recovery reached 79.2%, and the purification factor was 2.84 times.

[0041] (3) Preparation of cross-linked oxalate decarboxylase aggregates

[0042] Add 5ml of purified enzyme solution to a 10ml centrifuge tube, add absolute ethanol drop by drop until the ethanol concentration is 20% to 50%, put it in a refrigerator at 4°C o...

example 3

[0045] (1) Induced expression of recombinant oxalate decarboxylase is the same as example 1

[0046] (2) Precipitation and separation of oxalate decarboxylase

[0047] Add 5ml of crude enzyme solution to a 10ml test tube, add pre-cooled saturated ammonium sulfate drop by drop to a concentration of 20% to 80%, put it in a refrigerator at 4°C overnight, collect the precipitate by centrifugation, and wash with 50mmol / L pH8.0 phosphate buffer Redissolved to obtain depurified acid decarboxylase.

[0048] (3) Preparation of cross-linked oxalate decarboxylase aggregates

[0049] Add 5ml of purified enzyme solution to a 10ml centrifuge tube, add absolute ethanol drop by drop until the ethanol concentration is 10% to 50%, put it in a refrigerator at 4°C overnight, add BSA to 1.0mg / L~1.5mg / L, drop by drop Add glutaraldehyde solution to 0.01%~0.08%, cross-linking reaction at pH 3~9, 4°C for 0.5~3.5h, centrifuge, discard supernatant, wash with 50mmol / L pH8.0 phosphate buffer until the s...

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Abstract

The invention provides a method for cross-linking and fixing oxalate decarboxylase by cross-linked oxalate decarboxylase aggregates (CLEAs), belonging to the technical field of biochemical engineering. The method comprises the following steps: induced expression of recombinant oxalate decarboxylase, separation and purification of oxalate decarboxylase, and preparation of CLEAs. The invention is characterized in that ethanol, acetone or ammonium sulfate are utilized to carry out precipitation separation on oxalate decarboxylase in a crude enzyme liquid; ethanol is utilized to prepare the oxalate decarboxylase aggregates; 0.01-0.15% glutaric dialdehyde is utilized to carry out cross-linking reaction for 0.5-3.5 hours under the conditions of adding 0.1-1.5mg / L BSA (bovine serum albumin), pH value 3-9 and 4 DEG C, thereby preparing the CLEAs; and the enzyme activity recycling rate of the cross-linked immobilized enzyme is up to more than 90%. The invention has the advantages of simple experimental operations and low cost; the obtained CLEAs have obviously higher stability than free oxalate decarboxylase; and thus, the invention can be used for developing enzyme preparations for oxalate calculus disease.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and specifically provides a preparation method of cross-linked oxalate decarboxylase aggregates (oxdc-CLEAs). Background technique [0002] Oxalic acid (HOOC-COOH) is widely distributed in organisms including plants, microorganisms and humans in the form of oxalate. There is no enzyme related to degrading oxalate in the human body. When people eat certain foods with high oxalate content, such as spinach, amaranth, strawberry, orange, plum, peanut butter, sorghum and tea, it will easily cause oxalate to accumulate in the human body, thus Cause various pathological states such as urinary stones, kidney stones, hyperoxaluria, and hypocalcemia. Urolithiasis seriously affects human health. According to different geographical environments, its incidence rate is 3% to 15%, and it is also on the rise. Stone disease is easy to relapse after treatment, and the recurrence rate after curing is as high as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/00C12N9/88C12R1/19
Inventor 林日辉梁跃黄文勤
Owner 南宁奕德环境科技有限公司
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