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Method for improving pH stability of creatinase

A technology of creatinase and stability, applied in the field of enzyme engineering, can solve the problems such as rarely seen creatinase, and achieve the effect of being suitable for industrial application, reducing enzyme inactivation, and convenient application

Active Publication Date: 2017-09-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports about improving the pH stability of creatinase

Method used

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  • Method for improving pH stability of creatinase
  • Method for improving pH stability of creatinase
  • Method for improving pH stability of creatinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Analysis of acidic amino acids on the molecular surface of creatinase

[0024] The covalent modification of polylysine depends on the number of acidic amino acids on the surface of the enzyme. According to the analysis of the amino acid sequence and structure of creatinase (PDB ID: 1CHM), creatinase derived from Pseudomonasputida contains 108 acidic amino acids, of which 76 An acidic amino acid is located on the surface of the enzyme molecule, and the side chain functional group is exposed on the surface of the enzyme, which can participate in the modification of the poly-lysine on the surface of the enzyme molecule.

[0025] Embodiment: 2: Polylysine modified creatinase

[0026] The molar ratios of carboxyl groups and poly-lysine amino groups on the surface of creatinase were 1:20, 1:50, 1:100, 1:150 and 1:200, respectively.

[0027] The molar ratio of CRE-COOH to EDC was 1:10, the reaction pH was 7.0, and the mixture was mixed slowly at 4°C overnight, and ...

Embodiment 3

[0030] Embodiment 3: Polylysine modified creatinase

[0031] The molar ratios of carboxyl groups and poly-lysine amino groups on the surface of creatinase were 1:100, the molar ratios of CRE-COOH and EDC were 1:2, 1:5, 1:10, 1:15 and 1:20, and the reaction pH was 7.0, slowly rotate and mix overnight at 4°C, and calculate the relative enzyme activity; ultrafilter the modified enzymes obtained in different molar ratios to remove unbound small molecule modifiers, and place them in buffer solutions with different pH values ​​(pH 4.0, 5.0, 6.0 , 7.0, 8.0, 9.0 and 10.0), placed at 25°C for 16 hours, measured the enzyme activity, calculated the relative activity with the enzyme activity under the optimum pH condition of the free enzyme or modified enzyme as 100%, and determined the pH stability of the enzyme. The results are shown in Table 2.

[0032] Table 2 The pH stability of enzymes modified by the ratio of carboxyl groups and EDC on the surface of different creatinases

[0033...

Embodiment 4

[0034] Embodiment 4: Polylysine modified creatinase

[0035] The molar ratio of carboxyl groups and poly-lysine amino groups on the surface of creatinase is 1:100, the molar ratio of CRE-COOH and EDC is 1:10, the reaction pH is 5.5, 6.5, 7.0, 7.5 and 8.5, and the mixture is mixed slowly at 4°C Overnight, the relative enzyme activity was calculated; the modified enzymes obtained at different pHs were ultrafiltered to remove unbound small molecule modifiers, and placed in buffer solutions of different pH values ​​(pH4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 ) at 25°C for 16 hours to measure the enzyme activity, and calculate the relative activity by taking the enzyme activity of the free enzyme or the modified enzyme under the optimum pH condition as 100% to calculate the relative activity to determine the pH stability of the enzyme. The results are shown in Table 3.

[0036] Table 3 The pH stability of different reaction pH conditions modifying enzymes

[0037]

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Abstract

The invention discloses a method for improving pH stability of creatinase, and belongs to the field of enzyme engineering. According to the method, creatinase of which the pH stability is improved and the kcat / Km is only reduced by 24 percent is obtained by modifying free creatinase homodimer through polylysine. The method is mild in conditions and easy to operate, and is more suitable for industrial application; convenience is provided for the application of the creatinase to the fields, such as medical diagnosis. Moreover, the method provided by the invention also provides a feasible way for stability transformation of other multi-subunit enzymes.

Description

technical field [0001] The invention relates to a method for improving the pH stability of creatinase, belonging to the field of enzyme engineering. Background technique [0002] Creatinase (creatinase; EC 3.5.3.3; CRE) belongs to the hydrolytic enzyme class, which catalyzes the hydrolysis of creatine to produce urea and sarcosine, and is a key enzyme in creatinine metabolism. At present, creatinase is found in many kinds of bacteria. Except for a few sources of creatinase, which exist in the form of single subunit, most creatinases are homodimers. Among them, creatine from Pseudomonasputida Enzymes are the most studied. [0003] The creatinine content in serum and urine is an important indicator for diagnosing renal function, and creatinase is the key enzyme for enzymatic detection of creatinine content. However, due to the poor stability of creatinase, especially in the environment of low pH of urine, creatinase has poorer stability, resulting in serious loss of enzyme a...

Claims

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Application Information

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IPC IPC(8): C12N9/96C12N9/78C12Q1/34
CPCC12N9/78C12N9/96C12Q1/34C12Y305/03003G01N2333/978
Inventor 张玲杨海麟高亚楠辛瑜王武
Owner JIANGNAN UNIV
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