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High-density industrial fermentation method for recombinant pichia pastoris phytase

A fermentation method and phytase technology, applied in the field of high-density industrial fermentation of recombinant Pichia phytase, can solve the problem of low effect critical value and achieve high-efficiency expression

Active Publication Date: 2011-07-06
GUANGDONG VTR BIO TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(2) Crabtree effect exists, and the critical value of the effect is very low, generally around 0.1-1g / L

Method used

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  • High-density industrial fermentation method for recombinant pichia pastoris phytase
  • High-density industrial fermentation method for recombinant pichia pastoris phytase
  • High-density industrial fermentation method for recombinant pichia pastoris phytase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1. Working seed cultivation

[0023] Pick a single colony from the YPD streaked plate and culture it in the primary seed medium (50mlYPD in 250ml triangular bottle) for about 28 hours until the bacterial concentration reaches 60g / L to obtain the primary seed culture solution; % of the inoculum was inserted into the secondary seed medium (100mlYPD in a 500ml Erlenmeyer bottle) and cultivated for 26 hours to a bacterial concentration of 64g / L to obtain a working seed solution for fermentation.

[0024] 2. Preparation before inoculation

[0025] Use pH6.86 and pH4.00 to calibrate the pH meter probe, and use saturated anhydrous sodium sulfite to calibrate the DO (dissolved oxygen) electrode to 1%, and then pour 15L of the fermenter culture medium (BSM) that has been weighed into the fermenter , autoclave at 121°C for 30 minutes.

[0026] 3. Vaccination

[0027] After cooling to 30°C, use a sterile method to dissolve PTM with a concentration of 4.4mL / L 1 Add 66ml into th...

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Abstract

The invention relates to the field of fermentation, in particular to a high-density industrial fermentation method for recombinant pichia pastoris phytase. The high-density industrial fermentation method for the recombinant pichia pastoris phytase comprises the following steps of: 1) inoculating fermentation work seed liquid to a fermentation tank; 2) supplementing and feeding glucose; 3) starving; and 4) performing methanol-induced expression on phytase. The method has the advantages that: according to the growth rule of thalli, by combining a dissolved-oxygen-stat (DO-stat) and substrate concentration feedback supplementing method, an expensive glucose electrode is not required to be used, and the supplementing speed of 50 percent glucose solution is adjusted according to the growth speed and concentration of the thalli after inoculation, so that high-density fermentation is realized, and the phytase is efficiently expressed by adjusting the supplementing speed of methanol.

Description

technical field [0001] The invention relates to the field of fermentation, in particular to a high-density industrial fermentation method of recombinant Pichia pastoris phytase. Background technique [0002] The Pichia pastoris expression system is a eukaryotic expression system developed in the past 20 years. It was used to produce single-cell proteins in the early days. It was not until 1987 that Cgata et al. used Pichia pastoris for the first time to express foreign genes Expression (JM Regg, et al. 1987). [0003] Methanotrophic Pichia pastoris is a widely studied and used exogenous protein expression system, and more than 400 proteins have been expressed so far. The advantages of this system include: (1) high expression of foreign proteins; (2) high efficiency of foreign protein secretion; (3) high efficiency of post-transcriptional modification, such as glycosylation; (4) high-density culture can be achieved. [0004] Alcohol oxidase (alcohol oxidase, AOX) is a key e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12R1/84
Inventor 胡爱红刘金山史宝军
Owner GUANGDONG VTR BIO TECH
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