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Pichia-pastoris engineering bacterium for producing glutathione

A technology of glutathione and recombinant bacteria, applied in the biological field, can solve the problems of high cost, environmental pollution, complicated operation, etc., and achieve the effect of high yield

Inactive Publication Date: 2013-05-29
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The production of GSH by chemical synthesis is relatively mature, but there are problems such as high cost, many reaction steps, long reaction time, complicated operation, optical resolution and environmental pollution; enzymatic production of glutathione needs to obtain related enzyme systems , need to consume ATP, need to add substances such as precursor amino acids, resulting in higher costs; production of GSH by fermentation is to select (or construct) microorganisms with strong GSH synthesis ability and high intracellular content, screen and optimize the medium formula, establish and Optimize fermentation control strategy, improve and enhance downstream process technology, etc., and ultimately improve the yield and quality of GSH

Method used

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  • Pichia-pastoris engineering bacterium for producing glutathione
  • Pichia-pastoris engineering bacterium for producing glutathione
  • Pichia-pastoris engineering bacterium for producing glutathione

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Obtaining of Pichia strains expressing bifunctional glutathione synthetase

[0036] 1. Construction of glutathione constitutive expression vector

[0037] 1. Construction of recombinant plasmid pGAPZαH

[0038] pGAPZαA (purchased from Invitrogen, catalog number V20520) was digested with BamHI and dephosphorylated to obtain the dephosphorylated linear plasmid pGAPZαA.

[0039] With Forward: 5'-AGATCTATGACATTTCCCTTGCTACCTGCA-3', Reverse:

[0040] 5'-GGATCCTTAA ATAAGTCCCAGTTTCTC-3' is a primer, and the pPIC9k plasmid (purchased from Invitrogen, catalog number V17520) is used as a template to amplify to obtain a 2535bp PCR product. After sequencing, the PCR product has the sequence 3 shown in the sequence table Nucleotides, namely his4;

[0041] The 2535 bp PCR product was digested with BamHI / BglII and purified with an Omega purification kit, and then directly ligated with the above-mentioned dephosphorylated linear plasmid pGAPZαA to obtain the recombinant pl...

Embodiment 2

[0066] Embodiment 2, recombinant strain produces glutathione

[0067] 1. Fermentation of recombinant bacteria

[0068] Inoculate the above-mentioned recombinant Pichia yeast strains GS115 / SagshF, GS115 / LmgshF, GS115 / LpgshF and control strain GS115 expressing the bifunctional glutathione synthase gene (gshF) from different sources in 25ml of sterilized YPD After cultivating in the liquid culture medium on a shaker at 30°C and 220r / min for 42h, collect the bacteria in the above-mentioned fermentation broth with equal OD, centrifuge at 15000g / min for 2min, discard the supernatant and wash twice with sterile water. The final cells were stored at -20°C for future use.

[0069] 2. Detection of glutathione content

[0070] Thaw the collected bacteria at room temperature (25°C), add an equal amount of finely crushed glass beads and an equal volume of Breaking buffer (50 mM potassium phosphate buffer solution pH7.4, 5% glycerol (v / v), 5 mM mercaptoethanol and 1 mM EDTA) followed by ...

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Abstract

The invention discloses a pichia-pastoris engineering bacterium for producing glutathione and provides a method for constructing a recombinant bacterium. The method comprises the step of guiding the coding gene of bifunctional glutathione synthetase into an original strain to obtain the recombinant bacterium. Experiments show that the recombinant bacterium prepared by adopting the method can be used for producing the glutathione, ensures the high yield and lays a foundation for the industrial production and the preparation of the glutathione.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Pichia pastoris engineering bacterium for producing glutathione. Background technique [0002] As an important small molecule tripeptide compound, glutathione widely exists in animal and plant tissues, cells and microbial cells. Glutathione was first discovered in baker's yeast and was officially named glutathione in 1921. The full name of glutathione is γ-L-glutamyl-L-cysteinyl-glycine (γ-L-glutamyl-L-cysteinyl-glycine, GSH), which is composed of glutamic acid (Glu), cysteine Amino acid (Cys) and glycine (Gly) are condensed through peptide bonds, and the structural formula is as follows: [0003] [0004] Usually GSH exists in two forms of reduced glutathione and oxidized glutathione, its molecular weight is 307.33, its melting point is 189-193°C, its crystal is colorless, transparent and elongated waxy, its isoelectric point is 5.93, easy Soluble in water, dilute alcohol, l...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P21/02C12R1/84
Inventor 朱泰承葛少林李寅
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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