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Method for preparing diagnostic hexokinase

A technology of hexokinase and bacterial strains, which is applied in the field of preparation of hexokinase for diagnosis, and can solve problems such as small market demand, slow progress in industrial production research, and complicated import routes

Pending Publication Date: 2017-03-08
河北省微生物研究所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At present, the research on hexokinase in China mainly focuses on the metabolic pathways of plants and human tumors, while the research on the industrial production of hexokinase for diagnosis is progressing slowly due to factors such as large application limitations and small market demand. As far as the applicant knows, there is currently no diagnostic hexokinase product with independent intellectual property rights
The raw materials HK of commercially available hexokinase kits are all imported, and there is no HK preparation for diagnosis with independent intellectual property rights in China.
Imported enzymes have brought a lot of inconvenience to kit manufacturers due to high prices, complicated import routes, and difficulty in solving quality problems.

Method used

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  • Method for preparing diagnostic hexokinase

Examples

Experimental program
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Effect test

Embodiment 1

[0089] The preparation method of hexokinase for diagnosis comprises the following process steps:

[0090] A. Purification and rejuvenation of strains

[0091] A 1 1. Dissolve Saccharomyces cerevisiae in sterile water and streak on Bengal red medium, culture at 30-35°C for 2-3 days, select a single colony with good growth and streak culture, continue culturing at 32-37°C, select The well-growing strains were fermented and tested for enzyme activity. Finally, the strain with higher enzyme activity was selected as the strain for subsequent fermentation.

[0092] Inoculate the single colony of Saccharomyces cerevisiae screened in the sterilized test tube culture medium, and cultivate it at a temperature of 28°C for 16 hours. The test tube culture medium is composed of the following raw materials in mass percentage:

[0093] Peptone 1%; yeast powder 0.5%; glucose 1%; the balance is water; pH=6.5;

[0094] Saccharomyces cerevisiae is produced by Angel Yeast Co., Ltd., and the pr...

Embodiment 2

[0121] The difference between this embodiment and embodiment 1 is:

[0122] The preparation method of hexokinase for diagnosis comprises the following process steps:

[0123] A. Purification and rejuvenation of strains

[0124] A 1 1. Inoculate the single colony of Saccharomyces cerevisiae screened in the sterilized test tube medium, and cultivate it for 12 hours at a temperature of 30°C. The test tube medium adopts the following raw materials in mass percentage:

[0125] Peptone 3%; yeast powder 1.5%; glucose 3%; the balance is water; pH=6.5-7.5;

[0126] A 2 , Purification and preservation of rejuvenated strains

[0127] According to the ratio of mass ratio of 1:3, step A 1 The cultured strains are stored in a sterile glycerol solution with a mass percentage of 50% at -20°C;

[0128] B, fermentation preparation contains the fermented liquid of hexokinase, comprises following process step:

[0129] B 1 1. Preparation of Shake Flask Seeds

[0130] Step A 2 After the ...

Embodiment 3

[0151] The difference between this embodiment and embodiment 1 is:

[0152] The preparation method of hexokinase for diagnosis comprises the following process steps:

[0153] A. Purification and rejuvenation of strains

[0154] A 1 1. Inoculate the single colony of Saccharomyces cerevisiae selected in the sterilized test tube culture medium, and cultivate it for 14 hours at a temperature of 29 ° C. The test tube culture medium adopts the following raw materials in mass percentage:

[0155] Peptone 2%; yeast powder 1%; glucose 2%; the balance is water; pH=7.0;

[0156] A 2 , Purification and preservation of rejuvenated strains

[0157] According to the ratio of mass ratio of 1:2, step A 1 The cultured strains were stored in a sterile glycerol solution with a mass percentage of 40% at -20°C;

[0158] B, fermentation preparation contains the fermented liquid of hexokinase, comprises following process steps:

[0159] B 1 1. Preparation of Shake Flask Seeds

[0160] Step A...

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Abstract

The invention belongs to production of enzymic preparations and particularly relates to a method for preparing diagnostic hexokinase. The method includes the process steps of purifying and rejuvenating a strain, preparing fermentation liquor containing hexokinase by fermentation and purifying the fermented crude hexokinase enzyme. The method for preparing the diagnostic hexokinase has the advantages that the problems that the diagnostic hexokinase with independent intellectual property is not available domestically, imported enzymic preparations are high in price and inconvenient to use and the like currently are solved, the method is reliable in strain source, feasible, suitable for industrialized production and capable of filling the blank that the diagnostic hexokinase with the independent intellectual property is not available domestically, and major performance indexes of the diagnostic hexokinase prepared by the method are superior to those of the imported products.

Description

technical field [0001] The invention belongs to the production of enzyme preparations, in particular to a preparation method of hexokinase for diagnosis. Background technique [0002] Hexokinase is a six-carbon sugar phosphorylase with hexose as the specific substrate, and plays an important role in the glycolysis process of organisms. Hexokinase is an allosteric enzyme that catalyzes glucose from a stable state to an active state. It has a small Km value, strong affinity for substrates, and low specificity. It can act on a variety of six-carbon sugars. Utilizing its action principle and characteristics, the enzyme is mainly used as a clinical diagnostic reagent. [0003] With the improvement of living standards, the number of diabetic patients is also rising sharply. At present, there are two clinical methods for detecting glucose in blood, which are glucose oxidase (GOD) method and hexokinase (HK) method. The GOD method is characterized by strong specificity of glucose ...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N1/18C12R1/865
CPCC12N9/1205C12N1/18C12Y207/01001
Inventor 刘春卯罗同阳孙国龙郑翔吴芳彤杨何宝曹倩荣秦艳梅马清河
Owner 河北省微生物研究所有限公司
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