Method for producing gamma-glutamyl transpeptidase using bacillus subtilis

A glutamyl transpeptidase, Bacillus subtilis technology, applied in microorganism-based methods, biochemical equipment and methods, transferase and other directions, can solve problems such as inability to separate, low γ-GTP content, and no commercial value , to achieve the effect of low cost

Inactive Publication Date: 2011-10-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since then, Suzuki et al. have used genetic engineering to construct γ-GTP producing bacteria of E. Coli, but due to the extremely low content of γ-GTP, they cannot be isolated and have no commercial value.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Bacillus subtilis SK11.004 was fermented under the following conditions:

[0022] Slope culture: medium components (g / L): peptone 10, beef extract 5, sodium chloride 5, agar 20, pH 7.0, sterilized at 121°C for 20 minutes, culture conditions: culture temperature 37°C, culture time 14 Hour;

[0023] Seed culture: medium components (g / L): glucose 20, yeast extract 10, corn steep liquor 10, MgSO 4 ·7H 2 O0.5,K 2 HPO 4 1. pH 7.0, sterilized at 121°C for 20 minutes, culture conditions: 250mL Erlenmeyer bottle liquid 30mL, shaker flask rotation speed 200rpm, culture temperature 37°C, culture time 12 hours; the first-grade seed medium is inserted at 2.5% by volume In 30 liters of secondary seed tanks (filling capacity 18 liters) liquid seed culture medium, wherein defoamer polyoxyethylene oxypropylene triglyceride 0.5 (mL / L), culture condition is: stirring speed 200rpm, control ventilation rate: 10L / min for 0-5 hours, 15L / min for 5-12 hours; control the culture temperature...

Embodiment 2

[0026] Preparation of enzyme powder:

[0027] The obtained fermented liquid is centrifuged (4000rpm) to remove thalline, and the fermented liquid supernatant is obtained, and the ultrafiltration membrane ultrafiltration with a cut-off molecular weight of 10,000 is used to remove salt and miscellaneous proteins, and then precipitated with 20% alcohol, and separated The precipitate was obtained, and finally the precipitate was freeze-dried to obtain γ-glutamyl transpeptidase powder with an enzyme activity of 3.8 U / mL.

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Abstract

A method for producing Gamma-glutamyltranspeptidase by using Bacillus subtilis belongs to the biotechnology field of foods. The method carries out the three-stage culture of the slope, the seeds and the fermentation of the producing bacteria Bacillus subtilis SK11.004 of the Gamma-glutamyltranspeptidase; and the Gamma-glutamyltranspeptidase powder is obtained by carrying out separation and purification of fermentation broth. The provided method for producing the Gamma-glutamyltranspeptidase by using the Bacillus subtilis SK 11.004 has small breeding workload, low cost and safe and reliable performances, the method can carry out the liquid fermentation production, the cost is low, the method can be used in large-scale production, and the enzyme activity of the Gamma-glutamyltranspeptidase which is produced by using the Bacillus subtilis can be up to more than 4U / mL. The fermented product is non-toxic and can be directly used for producing a plurality of valuable Gamma-glutamyl compounds, such as L-Gamma-glutamyl-L-dopa, Gamma-glutamyl taurine, Gamma-glutamyl tryptophan, glutathione, theanine, and the like.

Description

technical field [0001] The invention relates to a method for producing gamma-glutamyl transpeptidase by Bacillus subtilis, belonging to the field of food biotechnology. Background technique [0002] γ-glutamyltranspeptidase (γ-glutamyltranspeptidase, γ-GTP, EC2.3.2.2), that is, γ-glutamyltransferase, is one of the enzymes related to glutathione metabolism in organisms and also catalyzes the transfer of L-γ-glutamyl specific enzyme. γ-glutamyl transpeptidase can catalyze the following three types of reactions: [0003] (1) Transpeptide reaction: [0004] (2) Autorotation peptide reaction: [0005] (3) Hydrolysis reaction: [0006] It is widely distributed in organisms, from bacteria to higher mammals. The activity of γ-glutamyl transpeptidase in serum is a very sensitive indicator of hepatobiliary diseases. It has been used clinically as an indicator for the detection of liver cell carcinogenesis and liver cancer formation. Its role in the process of other tissue l...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N1/20C12R1/125
Inventor 江波张涛帅玉英
Owner JIANGNAN UNIV
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