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Bioengineering method for synthesizing sodium phosphocreatine

A creatine phosphate and bioengineering technology is applied in the field of preparation of creatine kinase to achieve the effects of low process cost and improved controllability

Inactive Publication Date: 2012-07-04
BIOTRAND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The invention provides a method for synthesizing creatine phosphate sodium, aiming to solve the defects of chemical synthesis and biological extraction enzymatic synthesis
[0009] This project uses recombinant creatine kinase, which solves the problem of enzyme source guarantee, retains the advantages of enzymatic synthesis, and significantly improves the controllability of the reaction and purification process

Method used

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  • Bioengineering method for synthesizing sodium phosphocreatine
  • Bioengineering method for synthesizing sodium phosphocreatine
  • Bioengineering method for synthesizing sodium phosphocreatine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] RNA was extracted from human brain samples by conventional methods, and human brain cDNA was obtained by reverse transcription. Using the primers described in claim 6 as primers, the CKBB gene was fished from human brain cDNA by PCR method. The specific PCR conditions are

[0013] 95℃, 5min;

[0014]

[0015] 72℃, 10min

[0016] The product was detected by agarose gel electrophoresis, and the size was 1109bp, which was consistent with the prediction.

[0017] The above CKBB gene was digested with restriction endonucleases NdeI and XhoI, and after recovery, it was ligated with the vector pET28a digested with NdeI and XhoI, transformed into Escherichia coli, and the transformant with kanamycin resistance was screened. After plasmid extraction and enzyme digestion identification, it was proved that the recombinant protein CKBB gene had been cloned into pET28a.

[0018] The pET28a-CKBB was transformed into BL21(DE3) by CaCl2 method, the transformants were screened on...

Embodiment 2

[0020] The recombinant engineered bacterium gained in embodiment 1 inserts culture medium according to 2% inoculum (containing tryptone 10g / L, yeast extract 10g / L, K 2 HPO 4 3H 2 O 15g / L, NaH 2 PO 4 2H 2 O 10g / L, 1ml defoamer. Sterilize at 121°C for 20 minutes, add kanamycin to a final concentration of 50 mg / L before inoculation, and sterile MgSO 4 To a final concentration of 0.8g / L, sterile glucose to a final concentration of 2g / L, a sterile trace element concentrate 10ml / L, and the pH value adjusted to 6.55) in a 5L fermenter for fed-batch high-density fermentation, the pH value Controlled at 6.55±0.05, after 5 hours of incubation at 37°C, the working concentration of IPTG induction was 0.1mM. After 20 hours of induction, the bacteria were collected by centrifugation and stored in a -20°C refrigerator for later use.

Embodiment 3

[0022] Weigh 10 g of the bacterium obtained in Example 2, dilute to 100 g / L with phosphate buffer, place in an ice-water bath, use an ultrasonic cell disruptor, set the ultrasonic power to 30%, work for 4 seconds and stop for 6 seconds, and circulate for 1 hour. Obtain 100ml of bacteriostasis solution. It was centrifuged at 4° C. and 12000 rpm for 1 hour, and the centrifuged supernatant was collected. Load the sample to a nickel affinity chromatography column (2.6 / 10cm) that has been equilibrated with 50mM phosphate buffer, equilibrate the column with the same buffer, and elute the impurity on the column with a buffer containing 20mM imidazole , and finally the target protein on the column was eluted with a buffer containing 300 mM imidazole, the eluate was collected, dialyzed with 20 mM phosphate buffer, and freeze-dried to obtain a recombinant creatine kinase preparation. Measure the concentration and specific activity of the enzyme preparation, and store it in a -20°C refr...

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Abstract

The invention provides a bioengineering method for synthesizing sodium phosphocreatine. The bioengineering method comprises the following steps of: recombining an escherichia coli engineering strain containing creatine kinase by using a genetic engineering method; carrying out large-scale high-density culture and separate purification by using the escherichia coli engineering strain to obtain high-activity creatine kinase; and carrying out enzyme-method catalytic synthesis by using the creatine kinase under the appropriate condition to obtain the phosphocreatine. The bioengineering method disclosed by the invention has the advantages of high substrate utilization rate, high product recovery rate, mild reaction conditions, short reaction time, little pollution to environment, low cost and the like and is suitable for industrial large-scale production.

Description

technical field [0001] The invention relates to a preparation method of creatine kinase, and the enzyme catalyzes the synthesis of creatine sodium phosphate in vitro. The method can be used for large-scale production and preparation of creatine phosphate sodium. Background technique [0002] Phosphocreatine is a substance that exists in the human body, mainly in cardiac muscle, skeletal muscle, brain and other organs and tissues. Its physiological functions mainly include: 1. It acts as a buffer for the energy substance ATP, and the large amount of ATP consumed by the above-mentioned organs and tissues in the process of life activities is supplemented by it to avoid fluctuations in ATP supply; 2. It is energy The carrier for the transmission of high-energy phosphate groups in the substance ATP molecule. The high-energy phosphate group of the ATP molecule generated by the mitochondria is transmitted to the energy-consuming part in the cytoplasm through the phosphocreatine in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/00C12N15/54C12N15/70C12N1/21C12N15/11C12N9/12C12R1/19
Inventor 郑春杨冯俊杰王永宏王琳
Owner BIOTRAND
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