Process for preparing phytase by efficiently fermenting recombinant pichiapastoris

A technology of Pichia pastoris and phytase, applied in the direction of hydrolase, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of high preparation cost of phytase, large amount of methanol added, etc., and achieve high-efficiency fermentation production Effect

Inactive Publication Date: 2010-11-24
WENZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

On the whole, there are defects in the salt composition in the medium and the addition

Method used

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  • Process for preparing phytase by efficiently fermenting recombinant pichiapastoris

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Experimental program
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Effect test

Embodiment 1

[0020] The strain used was Pichia pastoris 602, provided by Wenzhou Conch Challenge Bioengineering Co., Ltd.

[0021] Pick a single colony from the plate and insert it into YPD seed medium (500mL Erlenmeyer flask liquid volume: 125mL) (1L contains: yeast powder 10g, peptone 20g, glucose 20g) at 30°C for 20h at 220r / min. Then connect in the 25L fermentation tank that 12L batch fermentation culture medium is housed by 10% inoculum size (1L contains: glucose 30g, ammonium sulfate 15g, magnesium sulfate 8g, potassium sulfate 7.5g, potassium dihydrogen phosphate 3.5g, calcium sulfate 1.0g, potassium hydroxide 1.2g, PTM 14mL), adjust the pH to 4.5 with ammonia water before inoculation. Maintain the dissolved oxygen concentration above 25% by controlling the air flow and speed. The batch culture ends after the carbon source is metabolized, and then the feed medium (1 L containing: 250 g of glucose) is added to make the bacteria continue to grow. During the 24 hours of fermentation. ...

Embodiment 2

[0024] The strain used was Pichia pastoris 602, provided by Wenzhou Conch Challenge Bioengineering Co., Ltd.

[0025] Pick a single colony from the plate and insert it into YPD seed medium (500mL Erlenmeyer flask liquid volume 125mL) (1L contains: yeast powder 10g, peptone 20g, glucose 20g) at 30°C for 20h at 220r / min. Then insert in the 25L fermentation tank that 12L batch fermentation culture medium is housed by 10% inoculum size (1L contains: glucose 50g, ammonium sulfate 20g, magnesium sulfate 9g, potassium sulfate 5g, potassium dihydrogenphosphate 2g, calcium sulfate 1.0g , potassium hydroxide 1.2g, PTM 14mL), adjust the pH to 4.7 with ammonia water before inoculation. Maintain the dissolved oxygen concentration above 25% by controlling the air flow and speed. The batch culture ends after the carbon source is metabolized, and the feed medium (1L containing: glucose 250g) is added to continue the growth of the bacteria, and the carbon source (glucose) is stopped after 24 ...

Embodiment 3

[0028] The strain used was Pichia pastoris 602, provided by Wenzhou Conch Challenge Bioengineering Co., Ltd.

[0029] Pick a single colony from the plate and insert it into YPD seed medium (500mL Erlenmeyer flask liquid volume 125mL) (1L contains: yeast powder 10g, peptone 20g, glucose 20g) at 30°C for 20h at 220r / min. Then connect in the 25L fermentation tank that 12L batch fermentation medium is housed by 10% inoculum size (1L contains: glucose 60g, ammonium sulfate 25g, magnesium sulfate 9g, potassium sulfate 9.5g, potassium dihydrogen phosphate 3.5g, calcium sulfate 0.8g, potassium hydroxide 1.2g, PTM 14mL), adjust the pH to 5.0 with ammonia water before inoculation. Maintain the dissolved oxygen concentration above 25% by controlling the air flow and speed. The batch culture ends after the carbon source is metabolized, and the feed medium (1L containing: glucose 250g) is added to continue the growth of the bacteria, and the carbon source (glucose) is stopped after 24 hou...

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Abstract

The invention discloses a process for preparing phytase by efficiently fermenting recombinant pichiapastoris and a special batch fermentation culture medium thereof. The process comprises a thalli growth stage and an exogenous gene induction expression stage and relates to promoters such as an alcohol oxidase promoter, a methanol promoter and the like. The culture medium in a fed-batch fermentation stage adopts a low-salt formula which comprises the following components in percentage by weight: 3 to 6 percent of carbon source, 1.5 to 3 percent of nitrogen source, 0.8 to 1.0 percent of magnesium sulfate, 0.5 to 1.0 percent of potassium sulfate and 0.3 to 0.4 percent of monopotassium phosphate, wherein fermentation temperature is between 27 and 30 DEG C; and fermentation pH is between 4.5 and 5.5. Methanol induction in the exogenous gene induction expression stage is performed in five stages. The preparation process of the invention has the advantages of lower cost and higher phytase fermentation activity.

Description

technical field [0001] The invention belongs to the technical field of biological fermentation, in particular to a process for preparing phytase by fermenting recombinant Pichia pastoris. Background technique [0002] Phytic acid widely exists in plant tissues and corresponding grain products, and 40%-70% of phosphorus contained in grain feed and its processed products is phytate phosphorus. All animal production is based on plant-based diets, and plant-based diets contain a large amount of phytic acid. The content of phytic acid in common feed ingredients is shown in Table 1. [0003] Table 1 Contents of phytic acid in common feed ingredients [0004] [0005] Note: The numbers in the table are based on dry matter and are taken from literature (W.Eeckhout and M.De Paepe.Total phosphorus, phytate phosphorus and phytase activity in plant feed stuffs.Anim Feed Sci Technol, 1994, 47:19-29.) [0006] Phytic acid (or phytate) in plant-based feeds is the primary storage form ...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12R1/84
Inventor 杨海龙吴明江林格郝名慧姜小伟
Owner WENZHOU UNIVERSITY
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