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Method for producing phosphocreatine by microbial enzyme method

A microbial enzyme and creatine phosphate technology, which is applied in the field of biotechnology to produce creatine phosphate, can solve the problems of affecting the yield of creatine phosphate, difficulty in separation and extraction process, and inability to guarantee optimal conditions, etc., and achieve simple process steps and safe operation , the effect of good industrial application prospects

Inactive Publication Date: 2012-12-05
天津启仁医药科技有限公司
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Problems solved by technology

[0008] However, the creatine kinase used in traditional enzymatic methods is generally extracted from animal muscle. This kind of crude enzyme system prepared from animal muscle cell plasma is bound to introduce more miscellaneous proteins into the reaction system, which will give the converted The separation and extraction process brings difficulties; there are also inventions using 3-phosphoglyceric acid as the starting substrate, and a composite enzyme system including creatine kinase, but because there are many enzymes participating in the reaction, the most suitable enzymes required by various enzymes The conditions are different, so it is not guaranteed to meet the optimal conditions for all enzymes at the same time, which ultimately affects the yield of creatine phosphate

Method used

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  • Method for producing phosphocreatine by microbial enzyme method
  • Method for producing phosphocreatine by microbial enzyme method
  • Method for producing phosphocreatine by microbial enzyme method

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Embodiment 1

[0050] The construction of embodiment 1 genetically engineered bacterium Escherichia coli and the expression of recombinant creatine kinase

[0051] According to the creatine kinase gene sequence of mouse muscle included in the NCBI database, the upstream primer CPK1: 5'-GGGAAT TCC ATA TGC CGT TCG GCA ACA CCC ACA AC-3' (SEQ ID No.1) and the downstream primer CPK2: 5 were designed '-CCG CTC GAG CTT CTG CGC GGG GAT CAT GTC GTC G-3' (SEQ ID No. 2). Total RNA was extracted from mouse skeletal muscle, and cDNA was prepared by reverse transcription. Using mouse skeletal muscle cDNA as a template, the creatine kinase gene was amplified by PCR. The recombinant expression plasmid pET-21a(+)-CPK was constructed by genetic engineering method. The recombinant expression vector pET-21a(+)-CPK was transformed into E.coli BL21(DE3) to obtain a genetically engineered strain.

[0052] The positive recombinant strains were inoculated into 4 mL of LB medium containing 100 μg / mL ampicillin for...

Embodiment 2

[0054] Embodiment 2 Fermentation of recombinant creatine kinase genetically engineered recombinant bacteria

[0055] The recombinant Escherichia coli in Example 1 was inoculated in 4 mL of LB medium containing 100 μg / mL ampicillin and cultivated overnight as a primary seed liquid; then inserted into 100 mL of LB culture containing 100 μg / mL ampicillin with 1% transfer amount culture medium at 37°C, 200rpm for 8-12h as the secondary seed liquid; the secondary seed liquid was transferred to a 100L fermenter with 1% inoculum, the fermentation medium was fresh LB medium, and the culture temperature was 37°C. The initial rotation speed is 300rpm, and the initial pH is 7.2. Samples were taken regularly, and when the OD600nm of the bacterial solution reached 1.0-1.2, IPTG induction solution with a final concentration of 1 mM was added for induction. The fermentation liquid in the lower tank was centrifuged at 3,000 rpm for 15 minutes at 4°C to collect the bacterial cells, and the fe...

Embodiment 3

[0056] Example 3 Preparation of Lysate of Recombinant Creatine Kinase Genetic Engineering Recombinant Bacteria

[0057] According to Example 2, ferment recombinant creatine kinase genetically engineered recombinant bacteria to obtain fermented cells, collect the cells and perform ultrasonic crushing to obtain crude enzyme liquid of creatine kinase, and measure its enzyme activity according to method 1, and the enzyme activity is about 6 U / mL.

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Abstract

The invention discloses a method for producing phosphocreatine by converting creatine and triphosadenine by a microbial enzyme method. According to the method, a genetic engineering method is adopted; recombinant escherichia coli BL21(DE3)-pET21a(+)-CPK which expresses creatine kinase efficiently is constructed; and the reaction of the creatine and the triphosadenine is catalyzed to produce the phosphocreatine by taking fermentation bacteria or lysate as an enzyme source. The invention provides a new green production process method for the phosphocreatine, which has the advantages of mild conditions, short period, few impurities in an enzymatic system, convenience in separation and extraction of products, simple in process steps, safety in operation and the like; and the method has a very good industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of producing creatine phosphate by biotechnology, and relates to a method for producing creatine phosphate by transforming creatine and adenosine triphosphate through microbial enzymatic method. Background technique [0002] Creatine phosphate (CP or Phosphocreatine, PCr or PC), also known as creatine phosphate, is an important derivative of creatine, which was isolated from mammalian muscle in 1927. In cells with high energy conversion, creatine phosphate has at least two functions: one is as an energy carrier in cells, and the other is as an energy buffer. As a high-energy storage substance in the human body, creatine phosphate has been widely used as a preventive drug for various heart diseases, and can also be taken directly as a nutritional product. At the same time, it has been widely used in clinical diagnosis as a biochemical agent. [0003] The preparation method of creatine phosphate mainly contai...

Claims

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Application Information

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IPC IPC(8): C12P13/00C12R1/19
Inventor 高智慧任丽梅刘磊姚瑞娟余琼林王文芳刘阳
Owner 天津启仁医药科技有限公司
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