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Culture medium used for culturing microbial expression heparanase

A culture medium and heparinase technology, applied in the direction of microorganism-based methods, microorganisms, biochemical equipment and methods, etc., can solve the problems of unfavorable experimental research, high cost, and large changes in product component content, so as to reduce production costs, Economically Significant Effects

Inactive Publication Date: 2009-12-02
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, tryptone and yeast extract in LB medium are components with complex components, and the content of each batch of products varies greatly, which is not conducive to the development of experimental research on bacterial growth, protein expression, separation and purification, etc. Moreover, the cost of LB medium is relatively high, so it is extremely important to explore a medium that can increase the soluble expression of heparanase I and reduce the cost of enzyme production.

Method used

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  • Culture medium used for culturing microbial expression heparanase
  • Culture medium used for culturing microbial expression heparanase
  • Culture medium used for culturing microbial expression heparanase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1. Orthogonal optimization experiment for the components of the culture medium for culturing microorganisms expressing heparanase

[0021] In order to determine the final composition of the culture medium for culturing microorganisms expressing heparanase, the present invention optimizes the components of the culture medium through orthogonal experiments.

[0022] 1. Determination of influencing factors

[0023] The first thing to be determined in the optimization work of orthogonal experiments on the medium used to culture microorganisms expressing heparanase is to determine the influencing factors.

[0024]The factors that affect the growth of bacteria are the amount of carbon source and nitrogen source, and the ratio between them. If the carbon-to-nitrogen ratio is too small, the bacteria will grow vigorously, causing premature aging and autolysis of the bacteria; if the carbon-to-nitrogen ratio is too high, the number of bacteria will be reduced, and the im...

Embodiment 2

[0088] Example 2. Verification of the fermentation effect of the culture medium for culturing microorganisms expressing heparanase

[0089] The optimal medium for culturing microorganisms expressing heparanase screened in Example 1 was used to carry out the fermenter experiment.

[0090] The total volume of the fermenter used was 5L, and the liquid filling capacity was 2L. Considering that the optimum pH for the growth of engineering Escherichia coli is in the range of 6.8 to 7.2 in general, 3M NaOH solution is used for feeding control to adjust the pH value at 7.00 during the fermentation process, and 3M hydrochloric acid solution is used for pH adjustment when necessary. In addition, in the later stage of growth, nutrient consumption can be supplemented by adding 20g / ml glucose solution artificially to supplement the carbon source, and 10× the medium with the above optimal formula can supplement the nitrogen source, but the concentrations of yeast extract, glucose and calciu...

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Abstract

The invention discloses a culture medium used for culturing microbial expression heparanase. The culture medium contains 5 to 20g / L of yeast extract, 0.5 to 20g / L of glucose and 0.05mM to 5.0mM of calcium ion. The culture medium also contains 10.0 to 25.0g / L of Na2HPO.12H2O, 1.0 to 5.0g / L of KH2PO4, 0.1 to 1.0g / L of NaC1 and 0.5 to 5.0g / L of NH4C1. Recombinant heparanase can be expressed in a high yield manner in the culture medium, and the maximal enzyme activity of the heparanase reaches 20652.63IU / L of fermentation liquid and is far higher than the expression effect of a common LB culture medium. The culture medium greatly reduces the production cost of the heparanase and has a very obvious economic meaning for the application of the heparanase, in particular to heparin which is prepared by an enzymatic method and has low molecular weight.

Description

technical field [0001] The invention relates to a medium for cultivating microorganisms expressing heparanase, in particular to a medium for cultivating microorganisms expressing heparanase fusion protein. Background technique [0002] Heparinase (heparinase) is a class of polysaccharide lyase that acts on heparin, found in many kinds of microorganisms, including Corynebacterium sp. 39:64-67), Sphingobacterium sp. (Gao Ningguo et al., Production of Sphingobacterium heparanase, Acta Microbiology 2003 Vol.43:813-816), Bacillus subtilis (Wang Zhongyan et al., Screening of heparinase-producing bacteria and research on its crude enzyme properties, Sichuan University Journal (Natural Science Edition) 2002 Vol.39: 777-779), Bacillus circulans (Yasutaka Tahara et al., Purification and characterization of heparinase that degrades both heparin and heparin sulfate from Bacillus circulans BioSci.Biotechnol.Biochem.2002Vol.66:1181-1184), Bacteroides heparinus Prevotella heparinolytica (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N9/24C07K19/00C12R1/19
Inventor 邢新会叶逢春陈银
Owner TSINGHUA UNIV
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