Method for expressing xylose isomerase by applying pGAP regulation of Pichia pastoris

A technology of xylose isomerase and Pichia pastoris is applied in the field of applying pGAP of Pichia pastoris to regulate and express xylose isomerase, which can solve the problems of high cost and difficult purification of products, and achieves low production cost and short fermentation period. Effect

Inactive Publication Date: 2010-04-28
INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problem that the cost of producing xylose isomerase is higher and the product is difficult to purify by using the strains that naturally produce xylose isomerase, and provide a pGAP (glyceraldehyde triphosphate decapitation) using Pichia pastoris. Hydrogenase promoter) regulates and expresses the method for xylose isomerase

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] 1. First, amplify the pGAP promoter from Pichia pastoris;

[0021] 2. Fusing the His6 tag at the 3' end of the xylose isomerase sequence, constructing a Pichia pastoris expression vector that is regulated by pGAP to regulate the xylose isomerase gene, and transforming the vector into the Pichia pastoris genome;

[0022] 3. Screen high-copy recombinants as engineering strains according to the resistance genes carried on the vector;

[0023] 4. Inoculate the engineered bacterial strain in the liquid medium [the formula of the liquid medium (per liter): 25.7ml of 85% phosphoric acid, CaSO 4 2H 2 O 0.87g, K 2 SO 4 17.5g, MgSO 4 ·7H 2 O 13.9g, KOH 3.73g, Glycerin 40g, Peptone 19g, Yeast extract 9g, PTM4 trace element 3ml] [PTM4 formula (per liter): CuSO 4 ·5H 2 O 2.0g, NaI 2H 2 O 0.1g, MnSO 4 ·H 2 O 3.0g, Na 2 MoO 4 2H 2 O 0.2g, H 3 BO 3 0.02g, CoCl 2 ·6H 2 O 1.05g, ZnCl 2 7.0g, Fe 2 (SO 4 ) 3 ·7H 2 O 22g, biotin 0.2g, sulfuric acid 1ml], carry out fe...

Embodiment 2

[0025] 1. First, amplify the pGAP promoter from Pichia pastoris;

[0026] 2. Fusing the His6 tag at the 3' end of the xylose isomerase gene sequence, constructing the Pichia pastoris expression vector regulated by pGAP for the xylose isomerase gene, and transforming the vector into the Pichia pastoris genome;

[0027] 3. Screen high-copy recombinants as engineering strains according to the resistance genes carried on the vector;

[0028] 4. Inoculate the engineered bacterial strain in the liquid medium [the formula of the liquid medium (per liter): 26.7ml of 85% phosphoric acid, CaSO 4 2H 2 O 0.93g, K 2 SO 4 18.2g, MgSO 4 ·7H 2 O 14.9g, KOH 4.13g, oleic acid 20g, peptone 20g, Yeast extract 10g, PTM4 trace element 4ml] [PTM4 formula (per liter): CuSO 4 ·5H 2 O 2.0g, NaI 2H 2 O 0.1g, MnSO 4 ·H 2 O 3.0g, Na 2 MoO 4 2H 2 O 0.2g, H 3 BO 3 0.02g, CoCl 2 ·6H 2 O 1.05g, ZnCl 2 7.0g, Fe 2 (SO 4 ) 3 ·7H 2 O 22g, biotin 0.2g, sulfuric acid 1ml], carry out fermenta...

Embodiment 3

[0030] 1. First, amplify the pGAP promoter from Pichia pastoris;

[0031]2. Fusing the His6 tag at the 3' end of the xylose isomerase gene sequence, constructing the Pichia pastoris expression vector regulated by pGAP to regulate the xylose isomerase gene, and transforming the vector into the Pichia pastoris genome;

[0032] 3. Screen high-copy recombinants as engineering strains according to the resistance genes carried on the vector;

[0033] 4. Inoculate the engineering bacterial strain bacterial liquid in the liquid medium [the formula of the liquid medium (per liter): 27.2ml of 85% phosphoric acid, CaSO 4 2H 2 O 1.03g, K 2 SO 4 18.2g, MgSO 4 ·7H 2 O 15.2g, KOH 4.23g, sorbitol 35g, peptone 20g, Yeast extract 10g, PTM4 trace element 4ml] [PTM4 formula (per liter): CuSO 4 ·5H 2 O 2.0g, NaI 2H 2 O 0.1g, MnSO 4 ·H 2 O 3.0g, Na 2 MoO 4 2H 2 O 0.2g, H 3 BO 3 0.02g, CoCl 2 ·6H 2 O 1.05g, ZnCl 2 7.0g, Fe 2 (SO 4 ) 3 ·7H 2 O 22g, biotin 0.2g, sulfuric acid 1...

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Abstract

The invention relates to a method for expressing xylose isomerase by applying pGAP regulation of Pichia pastoris, belonging to the biotechnology field. Firstly, pGAP promoters are amplificated in Pichia pastoris, a Pichia pastoris expression vector of which xylose isomerase is regulated by the pGAP is built, and then the Pichia pastoris expression vector is transformed into Pichia pastoris genome. High copy recons are selected as engineering bacterial strain according to resistance genes on the Pichia pastoris expression vector, and the engineering bacterial strain is inoculated into liquid culture medium containing carbon source to ferment engineering bacteria for secreting and expressing the xylose isomerase. The invention has the advantages of low production cost and short fermenting period. The using of the pGAP regulation of Pichia pastoris to express the xylose isomerase is favorable to purifying the product, and the problems of high production cost and hard product purification caused by using natural bacterial strains produced xylose isomerase to produce xylose isomerase can be solved. The invention is favourable for mass production of xylose isomerase.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a method for regulating and producing biological protein by using the pGAP promoter of Pichia pastoris, in particular to a method for regulating and expressing xylose isomerase by using the pGAP of Pichia pastoris. Background technique [0002] Xylose Isomerase (Xylose Isomerase EC5.3.1.5) is an allosteric enzyme produced by a series of microorganisms. The main function of xylose isomerase enzyme is to catalyze the interconversion of D-xylose (aldepentose) and D-xylulose (ketopentose), and also act on D-glucose to obtain D-fructose. Xylose isomerase is mainly used in high fructose syrup industry and hemicellulose ethanol industry. Various microorganisms have the function of producing xylose isomerase. At present, in industrial production, xylose isomerase is mainly produced by fermentation of natural strains containing xylose isomerase gene. But, because the growth of natura...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/81C12R1/84
Inventor 张爱联易国辉罗进贤张添元尹慧祥
Owner INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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