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-phosp

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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

Examples

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Example Embodiment

[0050] Example 1 Construction of genetically engineered bacteria Escherichia coli and expression of recombinant creatine kinase

[0051] According to the mouse muscle creatine kinase gene sequence 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). The total RNA of mouse skeletal muscle was extracted, and cDNA was prepared by reverse transcription. Using mouse skeletal muscle cDNA as a template, the creatine kinase gene was amplified by PCR. Using genetic engineering methods, the recombinant expression plasmid pET-21a(+)-CPK was constructed. The recombinant expression vector pET-21a(+)-CPK was transformed into E.coli BL21(DE3) to obtain genetically engineered strains.

[0052] The positive recombinant strain was inoculated into 4mL of LB medium containing 100μg / mL of ampicillin for overnight cul...

Example Embodiment

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

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

Example Embodiment

[0056] Example 3 Preparation of lysate of recombinant creatine kinase genetically engineered recombinant bacteria

[0057] According to Example 2, the recombinant creatine kinase genetically engineered bacteria was fermented to obtain fermented cells, the cells were collected and ultrasonically broken to obtain the crude enzyme solution of creatine kinase, and the enzyme activity was measured according to method 1, and the enzyme activity was 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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