Creatine kinase mutant applicable to phosphocreatine enzymic method production process

A technology of creatine kinase and creatine phosphate, which is used in the introduction of foreign genetic material, enzymes, and applications using vectors, can solve the problem of high production costs, lack of systematic research on the optimization of creatine kinase performance, and inability to meet the requirements of phosphocreatine kinase. It can reduce the production cost and improve the efficiency of the enzyme catalyzed reaction.

Inactive Publication Date: 2015-02-18
杭州清科生物科技有限公司
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

However, no systematic studies have been conducted on the performance optimization of creatine kinase itself
At present, only relying on enzyme immobilization technology is still unable to meet the needs of efficient production of creatine phosphate, and the production cost of biological enzymatic technology is still high

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] 1. Synthetic mutant creatine kinase gene. The creatine kinase gene is derived from Genebank NW_003159633.1 (25315-33102), and the mutation site is GLY268, which is replaced by ASN or SER.

[0016] 2. The synthetic mutant rabbit creatine kinase gene and plasmid pUC19 were double-digested with restriction endonucleases EcoRI and XhoI, the target fragments were recovered and ligated, and the obtained ligated products were transformed into Escherichia coli Top10, and positive clones were picked And sequence identification, get the recombinant plasmid.

[0017] 3. Digest the recombinant plasmid and the expression vector pET21+ with restriction endonucleases EcoRI and XhoI, recover the target fragments obtained by digestion and connect them to obtain the recombinant expression vector plasmid.

[0018] 4. Transform the recombinant expression vector plasmid into Escherichia coli BL21DE3, pick positive clones, and obtain the creatine kinase-expressing Escherichia coli recombina...

Embodiment 2

[0024] 1. Synthetic mutant creatine kinase gene. The creatine kinase gene is derived from Genebank NW_003159633.1 (25315-33102), the mutation sites are GLY268 and CYS146, and two of them are replaced by ASN, THR or SER.

[0025] 2. The rest of the steps are consistent with Example 1.

[0026] Compared with the wild enzyme, the specific activity of the mutant enzyme increased by 2 to 3 times, and its optimum pH also shifted 1.2 pH units to the alkaline direction, and the activity in the range of pH 8.0 to 10.0 was also higher than that of the wild type. 1% to 10% higher. The stability in an alkaline environment is also greatly improved compared with the wild-type enzyme. In addition, the enzyme activity of the mutant enzyme in the range of 15°C to 45°C is 10% to 20% higher than that of the wild type, and its thermal stability is also greatly improved.

Embodiment 3

[0028] 1. Chemically synthesized mutant creatine kinase gene. The creatine kinase gene was derived from Genebank NW_003159633.1 (25315-33102), the mutation sites were GLY268, His295 and CYS146, and three of them were replaced by ASN, THR, ALA or SER.

[0029] 2. The rest of the steps are consistent with Example 1.

[0030] Compared with the wild enzyme, the specific activity of the mutant enzyme is increased by 2 to 3 times, and the activity in the range of pH 8.0 to 10.0 is also 1% to 15% higher than that of the wild type. Its optimum pH also shifted by 1 pH unit to the alkaline direction, and its stability in an alkaline environment was also greatly improved compared with the wild-type enzyme. In addition, the optimum reaction temperature of the mutant enzyme was also increased by about 10°C, and the enzyme activity in the range of 15°C to 50°C was 10% to 20% higher than that of the wild type, and its thermal stability was also greatly improved.

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Abstract

The invention relates to a method for improving the property of creatine kinase as a reaction catalytic enzyme in phosphokinase production by mutating and transforming rabbit muscle creatine kinase by using a gene engineering technique. According to the method, the gene engineering technique is adopted to perform multi-point mutation on rabbit muscle creatine kinase, so that the properties, such as the enzymatic property, the substrate affinity, the acid/alkali resistance and the thermal stability, of creatine kinase in phosphokinase production are improved. The method has the advantages that the catalysis property of creatine kinase in a phosphokinase enzymic method production process environment with the pH value of 8-10 and the temperature of 25-45 DEG C is greatly improved through enzyme mutation transformation, the production efficiency is improved, the production cost is lowered, and great significance is achieved for developing the enzymic method phosphocreatine industry.

Description

technical field [0001] The invention belongs to the technical field of molecular bioengineering, and in particular relates to modifying rabbit creatine kinase through a gene mutation method so as to be suitable for the enzyme requirement for enzymatic production of creatine phosphate. Background technique [0002] Creatine kinase (EC 2.7.3.2) is usually found in the cytoplasm and mitochondria of animal heart, muscle, and brain tissues. It is an important kinase that is directly related to intracellular energy operation, muscle contraction, and ATP regeneration. It is reversible It catalyzes the transphosphoryl reaction between creatine and ATP. In the creatine phosphate synthesis pathway, it can reversibly catalyze the formation of creatine phosphate from creatine. During the positive reaction, ATP is required to provide a phosphoryl group. Creatine phosphate contains high-energy bonds and has an energy storage function, so it can compensate the concentration of ATP in the b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/70
CPCC12N9/1223C12N15/70C12Y207/03002
Inventor 蔺宗吴学强周海梦姚玲
Owner 杭州清科生物科技有限公司
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