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Method for modifying oxalate decarboxylase with monomethoxypolyethylene glycol

A technology of oxalate decarboxylase and polyethylene glycol, applied in the direction of microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve the problems of denaturation and inactivation, and achieve low cost, enhanced tolerance, and mild conditions Effect

Inactive Publication Date: 2014-03-05
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

However, as a protein, enzymes also have the problem of being easily denatured and inactivated by environmental factors

Method used

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  • Method for modifying oxalate decarboxylase with monomethoxypolyethylene glycol
  • Method for modifying oxalate decarboxylase with monomethoxypolyethylene glycol
  • Method for modifying oxalate decarboxylase with monomethoxypolyethylene glycol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] (1) Preparation of oxalate decarboxylase

[0026] Fermentation and cultivation of genetically engineered bacteria E. coli BL21(DE3) / pET32a / YvrK, adding 0.4mmol / L IPTG to induce the expression of oxalate decarboxylase, harvesting the bacteria and extracting with 50mmol / L pH8.0 phosphate buffer to obtain crude enzyme liquid, Enzyme purification was performed using a fast protein liquid chromatography system AKTA-FPLC.

[0027] (2) Activation of mPEG

[0028] Weigh 1g of mPEG and dissolve it in 1.5mL of chloroform, add acetic anhydride at the ratio of acetic anhydride / mPEG molar ratio of 20:1, add 6mL of DMSO dried with calcium hydride at the same time, stir and react at room temperature for 9h, then add the mixed solution dropwise 4 times the volume of diethyl ether on ice, stirring at high speed. The precipitate was dissolved in 2 mL of chloroform, and the precipitation was repeated three times. The precipitate was vacuum-dried at room temperature for 24 h, and stored ...

Embodiment 2

[0041] (1) Preparation of oxalate decarboxylase

[0042] Same as (1) operation in the embodiment

[0043] (2) Activation of mPEG

[0044] Operation with (2) in Example 1.

[0045] (3) mPEG modified oxalate decarboxylase

[0046] Put mPEG5000-aldehyde and Oxdc enzyme protein at a molar ratio of 10:1 to 150:1 at 37°C and pH 5.0 and stir for 12 hours, then add 20-fold molar ratio of glycine to the enzyme protein to terminate the reaction. The reaction solution was put into a 8000-14000 dialysis bag, and dialyzed in PEG20000 for 12 hours. The final solution was centrifuged. The modification rate and enzyme activity recovery rate were determined by TNBS method as shown in the table below.

[0047]

[0048] Aldehyde / enzyme (molar ratio) 10:1 20:1 30:1 40:1 50:1 100:1 150:1 Modification rate (%) 7.54 11.96 18.17 25.30 39.12 25.13 10.25 Enzyme recovery 97.67 94.57 87.60 83.72 78.29 40.31 12.40

[0049] When the mPEG / enzyme molar rati...

Embodiment 3

[0051] (1) Preparation of oxalate decarboxylase

[0052] Same as (1) operation in the embodiment

[0053] (2) Activation of mPEG

[0054] Operation with (2) in Example 1.

[0055] (3) mPEG modified oxalate decarboxylase

[0056] Put mPEG5000-aldehyde and Oxdc enzyme protein at a molar ratio of 50:1 at 4-45°C and pH 5.0 and stir for 12 hours, then add 20-fold molar ratio of glycine to the enzyme protein to terminate the reaction. The reaction solution was put into a 8000-14000 dialysis bag, and dialyzed in PEG20000 for 12 hours. The final solution was centrifuged. The modification rate and enzyme activity recovery rate were determined by TNBS method as shown in the table below.

[0057]

[0058] temperature °C 4 25 37 45 Modification rate (%) 14.88 24.31 39.02 56.98 Enzyme recovery 92.71 89.77 67.21 35.12

[0059] The effect of mPEG-modified oxalate decarboxylase was compared at different temperatures, and the results are shown in the ...

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Abstract

The invention relates to a method for modifying oxalate decarboxylase, particularly a method for modifying oxalate decarboxylase with monomethoxypolyethylene glycol, belonging to the technical field of biochemical engineering. The method comprises the following steps: preparation of recombinant oxalate decarboxylase, activation of monomethoxypolyethylene glycol (mPEG), modification of oxalate decarboxylase and the like. The modified oxalate decarboxylase has higher heat stability, pH adaptability, heat resistance and trypsinase tolerance. The method solves the realistic problem that the oxalate decarboxylase in use can be influenced by digestion of gastric acid and proteinase and can be easily digested to lose activity at present. The method can be used for preparing an enzyme preparation for preventing and treating calculus caused by oxalate crystallization.

Description

technical field [0001] The invention belongs to the field of biochemical technology, and specifically relates to a method for modifying oxalate decarboxylase with monomethoxypolyethylene glycol, including the preparation of recombinant oxalate decarboxylase and the activation of monomethoxypolyethylene glycol (mPEG) , Modification of oxalate decarboxylase and other processes. Background technique [0002] Oxalic acid 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. Dong Tingting and others pointed out that urinary system stone disease seriously ...

Claims

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Application Information

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IPC IPC(8): C12N9/96C12N9/88C12R1/19
CPCC12N9/88C12Y401/01002
Inventor 林日辉韦成昱黄文勤
Owner 南宁奕德环境科技有限公司
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