Saccharomyces cerevisiae mutant bacterial strain and use thereof in glutathion production by fermentation

A technology of mutant strains, Saccharomyces cerevisiae, applied in fermentation, microorganism-based methods, enzymes, etc., can solve the problems of limited biomass accumulation, reduced microbial fermentation capacity, unstable yeast quality, etc. Good pH tolerance, beneficial to large-scale production

Inactive Publication Date: 2011-06-15
北京赫芙德食品科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The ubiquitous problem of Saccharomyces cerevisiae bacterial strain in the prior art is, one, relatively poor to acid tolerance, cause the pH value of fermented liquid system to reduce because of GSH production in the fermentation production process again, make bacterial strain growth relatively poor, The accumulation of biomass is limited, so that the fermentation ability of microorganisms is greatly reduced; second, the contamination of bacteria is one of the important reasons for the unstable quality of yeast, which is manifested by the small number of yeast cells and low germination rate, which makes the pH of the culture medium hinder yeast growth

Method used

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  • Saccharomyces cerevisiae mutant bacterial strain and use thereof in glutathion production by fermentation
  • Saccharomyces cerevisiae mutant bacterial strain and use thereof in glutathion production by fermentation
  • Saccharomyces cerevisiae mutant bacterial strain and use thereof in glutathion production by fermentation

Examples

Experimental program
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Effect test

Embodiment 1

[0024] This example illustrates a method for measuring glutathione (GSH), a fermentation product of Saccharomyces cerevisiae, and uses this method as an evaluation basis for screening high-yielding Saccharomyces cerevisiae mutant strains.

[0025] GSH reacts with alloxan under test conditions to produce the highest absorption peak at 305nm, so this method is also called alloxan 305 method. The sulfhydryl group of cysteine ​​can also react with alloxan, but its absorption peak is at 275nm, which will not interfere with the determination of GSH.

[0026] Draw a standard curve:

[0027] (1) Accurately weigh 0.006g of GSH standard substance, dissolve it with 40% ethanol, and set the volume to 100mL to obtain a GSH standard solution with a concentration of 200umol / L;

[0028] (2) Take 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9mL of the above-mentioned GSH standard solution in a test tube, add deionized water to 1.0mL, and prepare the concentration to be 0, 20 , 40, 60, 80, 10...

Embodiment 2

[0032] This example illustrates the mutagenesis screening method for S. cerevisiae Y518.

[0033] 1. Preparation of bacterial cell suspension

[0034] Inoculate and activate the active dry Saccharomyces cerevisiae purchased from Hubei Angel Yeast Co., Ltd., pick a ring of bacteria from the activated slant and inoculate it into 20mL basal medium. Insert 200 mL of basal medium into a 500 mL Erlenmeyer flask at 30°C, shake at 180 rpm, take out 10 mL of culture solution, centrifuge at 6000 rpm for 15 min and wash twice with normal saline, then add 0.1 mol / L phosphate buffer at pH 6.0, Hemocytometer adjusted cell concentration to 106cfu / mL.

[0035] 2. Nitrosoguanidine (NTG) mutagenesis and determination of optimal mutagen concentration

[0036] Mix the bacterial suspension with 10 mg / mL NTG according to a certain ratio, so that the final concentrations of NTG are 0, 0.2, 0.4, 0.6, 0.8, 1.0 mg / mL, treat at 30 ° C for 30 min, add 2% Na 2 S 2 o 3 After the reaction was terminate...

Embodiment 3

[0063] This example illustrates the isolation and cloning procedure of the γ-glutamylcysteine ​​synthetase gene and the glutathione synthetase gene in Saccharomyces cerevisiae Y518.

[0064] Wherein, the extraction of genomic DNA of Saccharomyces cerevisiae Y518 strain adopts phenol-chloroform extraction method.

[0065] 1. Isolation and cloning of γ-glutamylcysteine ​​synthetase gene:

[0066] Get the genomic DNA of Saccharomyces cerevisiae Y518 strain as a template, and use the following nucleotide sequences as primers to amplify the γ-glutamylcysteine ​​synthetase gene:

[0067] Primer 1: 5'-ATGGGACTCTTAGCTTTGG-3'

[0068] Primer 2: 5'-TTAACATTTGCTTTCTATTGAAG-3'

[0069]PCR reaction conditions: 95°C for 5min, one cycle; 95°C for 30s, 60°C for 30s, 72°C for 2min, 35 cycles; finally 72°C for 10min.

[0070] The PCR product was recovered and ligated with the pMD18-T vector (purchased from TAKARA Company) to obtain the recombinant plasmid vector pMD18-T-gsh1. The recombinan...

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Abstract

The invention belongs to the technical field of biological engineering, and relates to a new saccharomyces cerevisiae strain mutant with high yield of glutathione and the class name of Saccharomyces cerevisiae Y518, and application of Gamma-glutamyl cysteine synthetase gene and glutathione synthetase gene that are generated by the strain, and the strain application for glutathione preparation. Anoriginal NTG saccharomyces cerevisiae strain with continuous induction is inductively processed for 4 times, thus obtaining the saccharomyces cerevisiae strain mutant Y518. As Gamma-glutamyl cysteinesynthetase and glutathione synthetase that are generated by the saccharomyces cerevisiae strain Y518 experience point mutation, compared with an original strain, the saccharomyces cerevisiae Y518 hasstronger acid resistance, stronger pH tolerance, good stability and remarkably raised yield of glutathione. The glutathione preparation that utilizes the saccharomyces cerevisiae Y518 is characterized by simple operation, stability, good repeatability, high yield and the like, generates stable and cheap glutathione, and is favorable for scaled glutathione production.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a mutant strain Saccharomycescerevisiae Y518 of Saccharomyces cerevisiae, which simultaneously produces gamma-glutamylcysteine ​​synthetase and glutathione synthetase, and the genes of the two enzymes All have point mutations, and the present invention also relates to the application of the mutant strain Saccharomyces cerevisiae Y518 in the production of glutathione by fermentation. Background technique [0002] Glutathione (GSH) is a biologically active tripeptide compound formed by condensation of L-glutamic acid, L-cysteine ​​and glycine through peptide bonds, and is a natural antioxidant. GSH was first isolated in 1888 and officially named in 1921. GSH widely exists in organisms such as animals, plants and microorganisms. There are many documents (Tateishi N., 1974.J.B.75:93~103; Issels R., 1988.Biochem Pharmacol.37:881~888; Meister.A., 1983.Ann.Rev.Biochem.52:711 ~...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N9/00C12N15/52C12P21/02C12R1/865
Inventor 王立梅梅艳珍齐斌郑丽雪许华伟
Owner 北京赫芙德食品科技有限公司
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