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Method for modifying oxalate decarboxylase by means of ethylenediaminetetraacetic dianhydride

A technology of ethylenediaminetetraacetic acid dianhydride and oxalate decarboxylase is applied in the field of modification of oxalate decarboxylase, which can solve the problems of dilution and loss of enzyme protein, and achieve the effects of improving adsorption capacity, enhancing tolerance and simple operation.

Inactive Publication Date: 2016-10-26
南宁奕德环境科技有限公司
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, these studies did not solve the practical problem that the enzyme protein is easily diluted and lost when oxalate decarboxylase preparations are used to prevent and treat calcium oxalate stones

Method used

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  • Method for modifying oxalate decarboxylase by means of ethylenediaminetetraacetic dianhydride
  • Method for modifying oxalate decarboxylase by means of ethylenediaminetetraacetic dianhydride
  • Method for modifying oxalate decarboxylase by means of ethylenediaminetetraacetic dianhydride

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preparation example Construction

[0026] Preparation of oxalate decarboxylase: using "Lin Rihui, Xu Lili, Nong Mian, et al. Research on expression and enzymatic properties of recombinant oxalate decarboxylase [J]. Food and Fermentation Industry, 2011,37(2):57-61" prepared by the method. Specifically: culture and ferment the oxalate decarboxylase-producing genetically engineered bacteria E.coli BL21(DE3) / pET32a / YvrK, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.4mmol / L , MnCl 2 to 6mmol / L, induce the expression of oxalate decarboxylase, collect the cells by centrifugation, resuspend the cells in 50mmol / L pH 8.0 phosphate buffer, ultrasonically break, and collect the supernatant by centrifugation to obtain the crude enzyme solution. The plus fast low-pressure liquid chromatography system is used to purify the crude enzyme solution, and the obtained purified enzyme solution is transferred to a pre-treated dialysis bag (molecular weight cut-off 8000-14000), and placed in a phospha...

example 1

[0031] A method for modifying oxalate decarboxylase with ethylenediaminetetraacetic acid dianhydride, comprising dissolving ethylenediaminetetraacetic acid dianhydride and oxalate decarboxylase in a molar ratio of 0.5:1 into a phosphate buffer solution with a pH of 3.0 and a concentration of 20mmol / L , stirred and reacted for 1 hour; after the reaction, the reaction product was dialyzed with a dialysis bag with a molecular weight cut-off of 8000, and then centrifugally concentrated with an ultrafiltration tube at 2000rpm, 2°C, and 10min to obtain oxalic acid modified with ethylenediaminetetraacetic acid dianhydride decarboxylase.

[0032] The above-mentioned concentration step can also be concentrated by using a dialysis bag, and the dialysis bag is placed in a 20% (w / v) polyethylene glycol (PEG) 20000 solution for 2 hours at 2°C.

Embodiment 2

[0034] A method for modifying oxalate decarboxylase with ethylenediaminetetraacetic acid dianhydride, comprising dissolving ethylenediaminetetraacetic acid dianhydride and oxalate decarboxylase at a molar ratio of 20:1 into a phosphate buffer solution with a pH of 4.0 and a concentration of 100mmol / L , stirred and reacted for 5 hours; after the reaction, the reaction product was dialyzed with a dialysis bag with a molecular weight cut-off of 9000, and then centrifugally concentrated with an ultrafiltration tube at 3000rpm, 4°C, and 20min to obtain oxalic acid modified with ethylenediaminetetraacetic acid dianhydride decarboxylase.

[0035] The above concentration step can also be concentrated by using a dialysis bag, and the dialysis bag is placed in a 25% (w / v) polyethylene glycol (PEG) 20000 solution for 2.5 hours at 4°C.

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Abstract

The invention discloses a method for modifying oxalate decarboxylase by means of ethylenediaminetetraacetic dianhydride. The method comprises the steps of dissolving ethylenediaminetetraacetic dianhydride and oxalate decarboxylase in phosphate buffer, and conducting stirring reaction for 1-24 h; dialyzing the reaction product after reaction ends, and then conducting concentration to obtain ethylenediaminetetraacetic dianhydride modified oxalate decarboxylase. Chemical modification of oxalate decarboxylase is directly conducted with ethylenediaminetetraacetic dianhydride, conditions are mild, operation is easy, and cost is low; the heat stability, heat resistance, pH adaptability and trypsin tolerance of modified oxalate decarboxylase are improved; the adsorption capacity of oxalate decarboxylase on calcium oxalate crystals is improved especially; the problem that oxalate decarboxylase can be affected by gastric acid and pepsin in use to lose activity easily is solved; the practical problem that zymoprotein in enzymic preparations is diluted easily to disappear in practical use is also solved.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and in particular provides a method for modifying oxalate decarboxylase with ethylenediaminetetraacetic acid diacid anhydride, the obtained modified oxalate decarboxylase and its application. Background technique [0002] Oxalate decarboxylase (Oxalate decarboxylase, Oxdc, EC 4.1.1.2) catalyzes the cleavage of the C-C bond of oxalic acid, and can catalyze the conversion of oxalic acid into formic acid and CO2 without cofactors. It is the main catalytic enzyme for the metabolic degradation of oxalic acid in plants and microorganisms. Based on the high specificity of the substrate and the high efficiency of the enzymatic reaction, the enzyme has been widely used in the degradation of oxalate in the fields of agriculture, food, industrial production, and medical treatment. For calcium oxalate stone disease, there are some research reports on the prevention and treatment of calcium oxalate decarbo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/96C12N9/88
CPCC12N9/88C12Y401/01002
Inventor 林日辉贺俊斌黄文勤
Owner 南宁奕德环境科技有限公司
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