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Method for improving heat stability of creatinase

A thermal stability, creatinase technology, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of poor stability, large amount of creatinase, no chemical bond formation, etc., and achieves convenient application, improved thermal stability, and thermal stability. Sex-enhancing effect

Active Publication Date: 2017-02-15
JIANGNAN UNIV
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Problems solved by technology

[0005] The thermal stability of creatinase from various sources is not ideal, and the amount of creatinase in the current clinical application of enzyme assay kits is relatively large, the reason is that the stability of the enzyme is poor
Schumann et al. studied the stabilizer of Pseudomonas putida creatinase, and found that adding DTE, BSA or glycerol to the enzyme solution can improve the stability of the enzyme, and further modified the molecular structure of the enzyme by random mutation, and obtained Five mutants, A109V, V355M, V182I, A109V+V355M and A109V+V355M+V182I3, had improved stability, but no new identifiable chemical bond formation was found

Method used

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  • Method for improving heat stability of creatinase
  • Method for improving heat stability of creatinase
  • Method for improving heat stability of creatinase

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Embodiment 1: the construction of mutant strain

[0026] (1) Chemically synthesize the CRE gene fragment, whose sequence is shown in GenBank accession number A10619.1 (creatinase derived from Pseudomonasputida), connect the gene fragment to pET28a(+), transform E.coli, and obtain the expression wild enzyme Recombinant E. coli. Extract the recombinant E.coli and the plasmid, and verify that the correct plasmid is the recombinant plasmid pET28a(+)-CRE.

[0027] (2) Using pET28a(+)-CRE as a template, carry out site-directed mutagenesis by overlapping extension PCR technology, and then digest and connect to pET28a(+). The mutant plasmid pET28a(+)-H125C / L130C with amino acid substitutions at positions 125 and 130 of cholesterol oxidase in GenBank: CAA00921.1 was obtained.

[0028] (3) Transform pET28a(+)-H125C / L130C into Escherichia coli E.coli BL21(DE3), verify the recombinant transformants, and verify that the correct one is the mutant strain E.coli-H125C / L130C. Creatin...

Embodiment 2

[0029] Embodiment 2: Purification of enzyme produced by mutant strain fermentation

[0030] (1) Preparation and purification of crude enzyme solution

[0031] The culture conditions of the seed liquid: use 250mL shake flask culture, the filling liquid is 20% LB medium, and add filter-sterilized 100mg·mL in the medium -1 Take 50 μL of kanamycin sulfate, take a single colony into the culture medium, and culture overnight at 37°C and 200 rpm.

[0032] Fermentation broth culture conditions: cultured in 500mL shake flasks, filled with 20% LB medium, and added filter-sterilized 100mg·mL -1 Add 100 μL of kanamycin sulfate, add 5% seed solution, cultivate at 37°C, 200 rpm until the OD reaches 0.6-0.8, add IPTG with a final concentration of 1 mM, induce culture for 16 hours at 16°C, 200 rpm.

[0033] Collection of bacteria and obtaining crude enzyme solution: centrifuge the fermentation broth at 8000rpm for 5min, weigh the wet weight, add 20mL pH7.5, 50mmol·mL according to 1g of wet ...

Embodiment 3

[0035] Example 3: Activity determination of CRE and thermal stability comparison of creatinase before and after mutation

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Abstract

The invention discloses a method for improving the heat stability of creatinase, and belongs to the fields of genetic engineering and enzyme engineering. According to the method, a mutant is obtained by carrying out amino-acid substitution and mutation on the basis of the creatinase from a Pseudomonas putida source. By comparing the mutant H125C / L130C provided by the invention with wild-type creatinase, the Tm value is increased by 8.2 DEG C, and the heat stability is obviously improved. Through the method provided by the invention, the heat stability of the creatinase is effectively improved.

Description

technical field [0001] The invention relates to a method for improving the thermal stability of creatinase, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] Creatinase (creatinase; E 3.5.3.3; CRE; classification name creatine aminohydrolase, creatinaseamidinohydrolase) belongs to hydrolase, catalyzes the hydrolysis of creatine to produce urea and sarcosine. Creatine hydrolase is the key enzyme in the creatine content determination method by sarcosine oxidase method. [0003] As one of the indicators for clinical renal function monitoring, creatinine has always attracted much attention. With the development of science, its determination method has evolved from the original alkaline picric acid method (Jeffe's reaction) to the enzymatic method, which can be said to be a huge progress, because the latter can overcome some inherent shortcomings of the former and is widely used . The three tool enzymes involved in creatine de...

Claims

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Application Information

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