Method of preparing creatine phosphate sodium

A technology of sodium phosphate phosphate and creatine kinase, applied in the biological field, can solve the problems of lack of specific purification method, difficult removal of impurity proteins, high product cost, and achieve the effects of simple and easy separation, easy separation and purification, and low cost.

Inactive Publication Date: 2013-04-03
HUNAN BAOLISHI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The current biological tissue extraction method uses animal muscle tissue as raw material to extract creatine kinase. Due to the low content of the enzyme in muscle and the lack of specific purification methods, the product cost is too high and the removal of impurities in the product is difficult.
In addition, the large consumption of ATP and the way of one-time input during the enzyme-catalyzed reaction also increase the cost of the product
[0007] The current genetic engineering method is to use genetic engineering methods to recombine and construct Escherichia coli engineering strains containing creatine kinase. When synthesizing creatine phosphate sodium, additional ATP needs to be added and a one-time input is used, and the product cost is relatively high.

Method used

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  • Method of preparing creatine phosphate sodium
  • Method of preparing creatine phosphate sodium
  • Method of preparing creatine phosphate sodium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] (1) Construct expression strains of creatine kinase and acetate kinase respectively, induce expression and perform crushing treatment

[0061] (1) Chemically synthesize creatine kinase gene (ck-mm) and acetate kinase gene (ak): the nucleotide sequence of the creatine kinase gene (ck-mm) is shown in SEQ ID NO.1; the acetic acid The nucleotide sequence of the kinase gene (ak) is shown in SEQ ID NO.2;

[0062] (2) Digest the synthesized ck-mm gene, ak gene and plasmid pBluescriptII SK(-) with EcoRI and XhoI, recover the desired target fragments and connect them separately to obtain the ligated products, and transform the ligated products into Escherichia coli For Top10 competent cells, pick positive clones to extract recombinant plasmids, sequence and identify them, and obtain recombinant plasmids pBluescriptII SK(-)-ck-mm and pBluescriptII SK(-)-ak;

[0063] (3) Digest recombinant plasmids pBluescriptII SK(-)-ck-mm and pBluescriptII SK(-)-ak and expression vector pET28a(...

Embodiment 2

[0086] (1) Construct the expression strains of creatine kinase and acetate kinase respectively, and the steps of inducing expression and crushing treatment are the same as in Example 1.

[0087] (2) Preparation of high-purity creatine kinase and acetate kinase by specific metal chelation affinity purification method

[0088] Utilize the method of specific metal chelate affinity chromatography to purify the homogenized and broken Escherichia coli recombinant bacterial liquid, the chromatographic filler of the affinity chromatography column used is nickel chelated IDA resin, the Escherichia coli recombinant bacterial liquid is 45BV / h Pass through an affinity chromatography column at a pH of 9.0, 1mol / L imidazole solution at a flow rate of 50BV / h, and desalt the eluent by membrane dialysis to obtain purified creatine kinase and acetate kinase.

[0089] (3) Preparation of co-immobilized enzymes

[0090] Mix creatine kinase, acetate kinase and epoxy resin particles at a ratio of 5...

Embodiment 3

[0106] (1) Construct the expression strains of creatine kinase and acetate kinase respectively, and the steps of inducing expression and crushing treatment are the same as in Example 1.

[0107] (2) Preparation of high-purity creatine kinase and acetate kinase by specific metal chelation affinity purification method

[0108] Utilize the specific metal chelate affinity chromatography method to purify the Escherichia coli recombinant bacterial liquid of the homogenate broken, the chromatographic filler of the affinity chromatography column used is iron chelation IDA resin, the Escherichia coli recombinant bacterial liquid is 35BV / h Pass through an affinity chromatography column at a flow rate of 40BV / h with pH 8.5, 0.8mol / L imidazole solution, and desalt the eluate by membrane dialysis to obtain purified creatine kinase and acetate kinase .

[0109] (3) Preparation of co-immobilized enzymes

[0110] Mix creatine kinase, acetate kinase and epoxy resin particles at a ratio of 30...

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Abstract

The invention relates to a method of preparing creatine phosphate sodium by utilizing a gene engineering technology. The method comprises the steps: respectively constructing creatine kinase and acetate kinase expression strains, removing the limit of creatine kinase-containing animal source, specially purifying the creatine kinase and acetate kinase by utilizing an affinity purification technology, co-immobilizing the creatine kinase and acetate kinase onto vector particles to form co-immobilized enzyme, continuously regenerating ATP from the acetate kinase for sustained reaction while the creatine kinase catalyzes and generates creatine phosphate sodium, thereby finally providing low-cost, green and pollution-free creatine phosphate sodium.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing creatine phosphate sodium by using genetic engineering technology. Background technique [0002] Phosphocreatine is a high-energy phosphate compound composed of creatine and phosphoric acid in muscle or other excitatory tissues such as the brain and nerves. Its high-energy properties make it a sports supplement widely used by athletes. In addition, sodium creatine phosphate, as a cardiomyocyte protective agent and energy supply, is used in adjuvant therapy such as myocardial ischemia and heart failure, or in clinical surgery, with definite clinical efficacy and high safety. Sodium creatine phosphate was first developed by Ouhui Pharmaceutical Factory and launched in Italy in 1992, and imported to my country in 1995. [0003] At present, there are chemical method and biological method (enzymatic method) for domestic production of creatine phosphate sodium. The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/00C12N15/70C12N9/12C12N11/08C12R1/19
CPCY02P20/50
Inventor 许岗黄斌吴峰曾红宇
Owner HUNAN BAOLISHI BIOTECH
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