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Biological enzyme for catalyzing synthesis of glutathione and preparation and extraction methods of biological enzyme

A technology of glutathione and extraction method, applied in the biological field, can solve the problems of heavy separation and purification workload, affecting activity, complicated operation, etc.

Inactive Publication Date: 2016-06-22
JIANGSU CHENGXIN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Biological fermentation method: there are problems such as low GSH content in the production strain, difficult extraction, high cost and unstable yield
[0006] Chemical synthesis method: relatively complex, many reaction steps, long reaction time, high cost, complicated operation, racemization may occur and affect activity, environmental pollution and other unfavorable factors
[0007] Chemical-enzymatic synthesis, that is, firstly synthesize S-benzylglycylcysteine ​​by chemical method, and then generate GSH with glutamic acid under the action of biocatalytic enzyme glutamyl transpeptidase: S-benzyl- The yield of glutathione is seriously affected by the purity of the enzyme, and the separation and purification of the enzyme is very labor-intensive and costly

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Construction of genetically engineered bacteria for biological enzymes

[0044] The biological enzyme gene fragment synthesized from the whole gene (sequence shown in SEQ ID NO: 1, synthesized by Nanjing GenScript Biotechnology Co., Ltd.) was subjected to restriction enzymes NdeI and HindIII (purchased from NewEngland Biolabs, and operated according to the instructions) enzymes After cutting, it was recombined into the corresponding site of the Escherichia coli expression vector pET29a to obtain the recombinant plasmid pET29b-GSH.

[0045]The constructed plasmid pET29b-GSH was transformed into Escherichia coli expression host strain BL21(DE3) by calcium chloride method to obtain the genetically engineered strain BL21(DE3) / pET29b-GSH.

Embodiment 2

[0047] Preparation of biological enzymes

[0048] Prepare LB solid medium: Weigh 1g of bio-grade peptone, 0.5g of yeast powder, and 1g of industrial-grade NaCl with an electronic scale, put them in a 200ml beaker, add drinking water to make up to 100ml, stir and mix until completely dissolved. Weigh 2 g of biological-grade agar powder with an electronic scale and add it to the beaker, stir and mix well, and add sterile ampicillin after disinfection.

[0049] Plate culture: take the original strains and streak on LB solid medium containing ampicillin, and incubate in a constant temperature incubator at a constant temperature of 37°C for 15-17 hours.

[0050] Test tube culture: pick a single colony from the plate (diameter 1-2mm, plump, smooth, without wrinkles on the edge) into 5-10ml liquid medium containing ampicillin (1g peptone, 0.5g yeast powder, 1g NaCl, dilute to 100ml , Ampicillin 10mg) 37±1°C, rotation speed 200rpm, shake culture at constant temperature for 5.5~6.5h, ...

Embodiment 3

[0058] Extraction of biological enzymes

[0059] Cooling: By increasing the tank pressure of the fermenter, the fermentation hydraulic pressure is sent to the storage container, and the temperature of the fermented liquid is lowered to 0-4°C.

[0060] Centrifugation: By high-speed centrifugation, solid-liquid separation is carried out to collect bacteria sludge.

[0061] Buffer solution configuration: Sodium phosphate buffer solution preparation: 0.04416% NaH2PO4·H2O and 0.60144% Na2HPO4·12H2O mixed solution, weighing needs to use sodium phosphate salt, mix and stir in the buffer preparation container until completely dissolved, and set aside.

[0062] Sludge mixing: Buffer mass = Sludge weight × 3. Add buffer, stir and mix until there is no solid matter, and cool down the system to 0-4°C during the process of stirring and mixing.

[0063] High-pressure homogenization: add water to the feeding cup of the homogenizer, adjust the homogenization pressure to 75-80 MPa by adjusti...

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PUM

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Abstract

The invention provides a biological enzyme that catalyzes the synthesis of glutathione and its preparation and extraction method. The biological enzyme is the amino acid sequence shown in SEQ ID NO: 2; the biological enzyme is derived from a genetically engineered strain constructed by in vitro recombination The nucleotide sequence is shown in SEQ ID NO: 1; the preparation method of the biological enzyme that catalyzes glutathione synthesis comprises the steps of: constructing a genetically engineered strain of glutathione biosynthetic enzyme, The nucleotide sequence is recombined into the expression vector by enzyme cutting, and then transferred into the host cell. The enzyme obtained by the invention has high catalytic efficiency, low production cost, environmental protection, high economic value and market competitiveness.

Description

technical field [0001] The invention relates to a biological enzyme that catalyzes the synthesis of glutathione and a preparation and extraction method thereof, belonging to the field of biological technology. Background technique [0002] Glutathione (GSH) is a tripeptide combined with glutamic acid, cysteine ​​and glycine, which can promote the metabolism of sugar, fat and protein, accelerate the excretion of free radicals, protect the synthesis of liver, detoxify, eliminate Active hormones and other functions. [0003] In 1888, GSH was first isolated from yeast. At present, the main production methods of GSH are: extraction method, biological fermentation method, chemical synthesis method, and chemical-enzymatic method. [0004] Extraction method: Due to the extremely low content of GSH in tissues, few available raw materials, and low preparation purity and yield, it is not widely used in actual production. [0005] Biological fermentation method: there are problems suc...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N15/75
CPCC12N9/1044C12Y203/02013
Inventor 邢将军祝俊余玉奎徐飞吴锋
Owner JIANGSU CHENGXIN PHARMA
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