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Method for regulating and expressing L-arabinose isomerase by using Pichia methanolica pGAP

A technology of Pichia pastoris and arabinose, applied in the biological field, can solve the problems of difficult purification and high cost of products, and achieve the effects of low production cost and short fermentation period

Inactive Publication Date: 2010-09-15
INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 bacterial strain that produ

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] 1. First, amplify the pGAP promoter from Pichia pastoris;

[0020] 2. Fuse the His6 tag at the 3' end of the L-arabinose isomerase sequence to construct a Pichia pastoris expression vector regulated by pGAP for the L-arabinose isomerase gene, transform the vector into the Pichia pastoris genome, and transform the transformed product Spread on the G418-YPD plate containing G418≥300μg / mL;

[0021] 3. Select the Pichia recombinant from the G418-YPD plate in step 2 as the engineering strain;

[0022] 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, Glycerin 40g, 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 ...

Embodiment 2

[0025] Implementation of steps 1, 2, and 3 is the same as in Embodiment 1.

[0026] 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(22g, biotin 0.2g, sulfuric acid 1ml), ferment and express L-arabinose isomerase in bioreactor. The fermentation parameters are: temperature 28-30°C, pH 5.0, stirring speed 200-900r / min. After 1-2 days of fermentation, the supernatant was harvested by centrifugation and purified with the Ni-Agarose His-tagged protein purification kit for L-arabinose isomerase, and by protein electrophoresis (the molecular weight of...

Embodiment 3

[0028] Implementation of steps 1, 2, and 3 is the same as in Embodiment 1.

[0029] 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, sorbitol 35g, peptone 20g, Yeast extract 10g, PTM4 trace elements 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 (22g, biotin 0.2g, sulfuric acid 1ml), ferment and express L-arabinose isomerase in bioreactor. The fermentation parameters are: temperature 28-30°C, pH 5.0, stirring speed 200-900r / min. After 1-2 days of fermentation, the supernatant was harvested by centrifugation and purified with the Ni-Agarose His-tagged protein purification kit for L-arabinose isomerase, and by protein electrophoresis (the molecular weight of...

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Abstract

The invention discloses a method for regulating and expressing L-arabinose isomerase by using Pichia methanolica pGAP, belonging to the field of biotechnology. The method comprises the following steps of: amplifying a pGAP promoter in Pichia methanolica; consturcting a Pichia methanolica expression vector of an L-arabinose isomerase gene regulated by the pGAP and converting the vector into a Pichia methanolica genome; screening a high-copy recombinant as an engineering bacterial strain according to a resistance gene carried on the vector; inoculating the engineering bacterial strain into a liquid nutrient medium containing a carbon source, fermenting engineering bacteria and carrying out secretory expression to obtain the L-arabinose isomerase. The invention has low production cost and short fermentation cycle; and the L-arabinose isomerase regulated and expressed by utilizing the Pichia methanolica pGAP is beneficial to the purification of the product, solves the problems that the L-arabinose isomerase produced by utilizing bacterial strains of naturally-produced L-arabinose isomerase has higher cost, is difficult to purify, and the like, and is good for large-scale production of the L-arabinose isomerase.

Description

technical field [0001] The invention belongs to the field of biotechnology, 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 L-arabinose isomerase by using the pGAP of Pichia pastoris. Background technique [0002] L-arabinose isomerase (L-arabinose isomerase EC 5.3.1.4) is an allosteric enzyme produced by a series of microorganisms. L-arabinose isomerase can catalyze L-arabinose to L-ribulose; due to the structural similarity between L-arabinose and D-galactose, L-arabinose isomerase can also catalyze D-galactose Isomerized to D-tagatose. D-tagatose is a sweetener with low-energy, blood sugar-lowering and anti-caries properties. Although a variety of microorganisms have the function of producing L-arabinose isomerase, however, due to the slow growth of the natural L-arabinose isomerase-producing strains, high nutritional requirements, or the format...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/81C12N1/19C12R1/84
Inventor 张爱联崔艳艳罗进贤张添元
Owner INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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