Production method of glucose dehydrogenase

A technology for glucose dehydrogenase and production method, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as source limitation of glucose dehydrogenase, difficulty in meeting market demands, etc., and avoid substrate inhibition. Effect

Inactive Publication Date: 2012-07-25
ENZYMEWORKS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The technical problem to be solved by the present invention is to overcome the problem that the source of glucose dehydrogenase in the pr

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] This embodiment provides a method for producing glucose dehydrogenase without adopting fed-batch fed-batch technology in the fermentation process, and the specific process is as follows:

[0037] The recombinant Escherichia coli seeds preserved in glycerol tubes were activated by the seed medium for 15 hours, and then transferred to the fermenter at a 2% inoculation amount. Fermentation tank parameter settings: temperature 37°C, initial ventilation rate 5L / min, initial stirring speed 200rpm, initial dissolved oxygen (DO) 100%. The dissolved oxygen level in the fermentation process is controlled between 30% and 40% by adjusting the stirring speed and ventilation, and the pH is controlled by ammonia water to 7.2±0.1. Ferment for about 3 hours, when OD 600 At 4 o'clock, start to cool down to 28°C, add 0.03% IPTG accounting for the total amount of fermentation broth, continue to cultivate for 12 hours until the dissolved oxygen rises to 100%, and the fermentation e...

Embodiment 2

[0040] This embodiment provides a method for producing glucose dehydrogenase by using a constant-velocity flow-feeding technology in the fermentation process, and the specific process is as follows:

[0041] The recombinant Escherichia coli seeds preserved in glycerol tubes were activated by the seed medium for 15 hours, and then transferred to the fermenter at a 2% inoculation amount. Fermentation tank parameter settings: temperature 37°C, initial ventilation rate 5L / min, initial stirring speed 200rpm, initial dissolved oxygen (DO) 100%. The dissolved oxygen level in the fermentation process is controlled between 30% and 40% by adjusting the stirring speed and ventilation, and the pH is controlled by ammonia water to 7.2±0.1. Ferment for about 3 hours, when OD 600 When 4 was reached, the addition of feed medium to the broth was started at a constant flow rate of 40 g / l / h. About 14 hours, OD 600 When it reaches 20°C, start to cool down to 28°C, add 0.03% of IPTG...

Embodiment 3

[0044] This embodiment provides a method for producing glucose dehydrogenase using the gradient fed-batch feeding technology of the present invention in the fermentation process. The specific process is as follows:

[0045] The recombinant Escherichia coli seeds preserved in glycerol tubes were activated by the seed medium for 15 hours, and then transferred to the fermenter at a 2% inoculation amount. Fermentation tank parameter settings: temperature 37°C, initial ventilation rate 5L / min, initial stirring speed 200rpm, initial dissolved oxygen (DO) 100%. The dissolved oxygen level in the fermentation process is controlled between 30% and 40% by adjusting the stirring speed and ventilation, and the pH is controlled by ammonia water to 7.2±0.1. Ferment for about 3 hours, when OD 600 When it reaches 4, start adding feed medium to the fermentation broth at a flow rate of 8 g / l / h; around 5 hours, when OD 600 When it reaches 8, adjust the feeding speed to 16g / l / h; abou...

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Abstract

The invention relates to a production method of glucose dehydrogenase, which mainly uses flow feeding technology in microbial fermentation and genetically engineered microorganisms to ferment to produce glucose dehydrogenase. The production method specifically includes: culturing recombinant escherichia coli containing glucose dehydrogenase gene at the culture temperature of 37 DEG C so as to express glucose dehydrogenase; inoculating the recombinant escherichia coli to culture medium for fermentation, adding supplementary medium into fermented broth containing the recombinant escherichia coli during fermentation by the flow feeding technology, and increasingly the flow feeding speed in a gradient form with prolonging of the fermentation time; and adding inducer when initial growth phase of the bacterium is over, reducing the temperature from 37 DEG C to about 28 DEG C, and maintaining constant temperature until fermentation is over. By the method, fermentation conditions for fermentation process meet actual needs of the bacterium as far as possible, the density of the bacterium can be increased to a high level, the yield of glucose dehydrogenase is increased, and great industrialapplication value is achieved.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for producing glucose dehydrogenase by fermenting genetically engineered bacteria, which is mainly used in the fed-batch feeding technology of microbial fermentation. Background technique [0002] Glucose dehydrogenase (GlcDH for short), is a member of the short-chain alcohol dehydrogenase family, in the coenzyme NAD(P) + When present, it can catalyze the conversion of β-D-glucose into gluconic acid. Glucose dehydrogenase is widely used to catalyze the preparation of chiral alcohols, hydroxy acids, amino acids, etc. These enzyme-catalyzed reactions generally require the participation of the coenzyme NAD(P)H, and these coenzymes are expensive, so the regeneration of NAD(P)H It is very necessary to cut costs. In the large-scale production of (s)-4-chloro-3-hydroxybutyric acid methyl ester synthesized by the asymmetric reduction of 4-chloro-3-carbonyl butyric ...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12R1/19
Inventor 陈令伟周强强武涛刘毅
Owner ENZYMEWORKS
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