Batch supplying fermentation technique for producing acetonic acid oxidase high-effectively by using recombinant bacillus coli

A technology of recombinant Escherichia coli and pyruvate oxidase, applied in the field of bioengineering, can solve the problems of low fermentation level and high market price of pyruvate oxidase

Inactive Publication Date: 2008-08-27
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently commercially produced pyruvate oxidase is mainly derived from wild-type Lactobacillus plantarum and Chlorococcus v

Method used

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  • Batch supplying fermentation technique for producing acetonic acid oxidase high-effectively by using recombinant bacillus coli
  • Batch supplying fermentation technique for producing acetonic acid oxidase high-effectively by using recombinant bacillus coli
  • Batch supplying fermentation technique for producing acetonic acid oxidase high-effectively by using recombinant bacillus coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 Fermentation to the fermentation process of gradient cooling at the end of the initial growth stage

[0080] The E.coli DH5d / pSMLPyOD seeds stored in glycerol tubes were activated in LB medium for 12 hours, and then transferred to a fermenter at a 10% inoculum size. The parameters of the fermenter are set as follows: the temperature is 37° C., the initial ventilation rate is 5 L / min, the initial stirring speed is 300 rpm, and the initial dissolved oxygen (DO) is 100%. The dissolved oxygen level in the fermentation process is controlled between 30-40% by adjusting the stirring speed and the ventilation rate, and the ammonia water controls the pH to 7.

[0081]After about 11-12 hours of fermentation, the dissolved oxygen starts to rise. When it rises to 70-80%, it indicates that the initial growth stage is over. At this time, the temperature is adjusted to 35°C and feeding is started. The feeding rate is 0.2ml / min / L. After 4 hours, the temperature was adjusted ...

Embodiment 2

[0083] Fermentation process of one-time cooling when embodiment 2 fermented to the end of the initial growth stage

[0084] The culture medium and the initial fermentation stage are the same as in Example 1.

[0085] After 11-12 hours of fermentation, the dissolved oxygen starts to rise, and when it rises to 70-80%, it indicates that the initial growth stage is over, and the fermentation temperature is adjusted to 32° C. at this time. Reduce the stirring speed to 450RPM, and add a small amount of feeding medium (5-10ml). After 2-3 hours, the cells start to grow and metabolize, and the dissolved oxygen decreases. When the dissolved oxygen rises again, start feeding, and the feeding rate is 0.1mL / min / L, when the dissolved oxygen drops below 30%, control it to 30-40% by increasing the speed and ventilation. Ammonia water controls the pH to 6.7±0.1. If the pH rises to 7 spontaneously, it indicates that the feeding rate is too low. At this time, the feeding rate should be increas...

Embodiment 3

[0087] All adopt the fermentation process of 37 ℃ in the fermentation process of embodiment 3

[0088] The culture medium is the same as in Example 1. Ferment for 11-12 hours, the initial growth stage is over, and the dissolved oxygen starts to rise. Keep the fermentation temperature at 37°C. When the dissolved oxygen rises above 60%, start feeding. The feeding rate is 1ml / min, and the feeding rate is constant. , until the end of fermentation. The dissolved oxygen level in the fermentation process is controlled between 30-40% by adjusting the stirring speed and the ventilation rate, and the pH of the ammonia water is controlled to be 6.7±0.2.

[0089] Adopt this fermentation technique, after 24 hours, bacterial concentration is no longer increasing, and fermentation finishes, and bacterial cell concentration (OD 600 ) reached 60, and the output of pyruvate oxidase reached the highest 1200U / L in 12 hours, and then decreased again. For the fermentation process curve, see im...

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Abstract

The invention discloses a fed-batch fermentation process for producing pyruvate oxidase through fermentation, which comprises: culturing recombinant escherichia coli which contains a pyruvate oxidase expression cassette under the culture temperature about 37DEG C, thereby expressing the pyruvate oxidase, lowering the temperature from about 37DEG C to about 32 DEG C after the initial growth stage of the recombinant escherichia coli is finished, beginning feeding, controlling the dissolved oxygen level between 30% and 40%, then, lowing the temperature for 1DEG C each four hours, and keeping the temperature constant after the temperature is lowered to 32DEG C until the fermentation is finished. The method of the invention increases the output of the pyruvate oxidase under the condition that the cost is not changed, the fermentation process is easily controlled, and thereby the method of the invention has extremely good industry application value.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a batch-type fed-batch fermentation process for efficiently expressing pyruvate oxidase in recombinant Escherichia coli. Background technique [0002] Pyruvate oxidase (Pyruvate oxidase, EC 1.2.3.3) is a very important enzyme for clinical diagnosis, and its most important application is for alanine aminotransferase (ALT) and aspartate aminotransferase in blood Enzyme (AST) activity determination; in addition, it can also be used for pyruvate determination, pyruvate kinase activity determination, etc. [0003] Currently commercially produced pyruvate oxidase is mainly derived from wild-type Lactobacillus plantarum and Chlorococcus viridans, the enzyme production is only 120U / L, and the fermentation level is low, resulting in high market price of pyruvate oxidase. [0004] Since pyruvate oxidase has a wide range of applications, it is very meaningful to further increase i...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N1/21C12R1/19
Inventor 张嗣良赵劼王永红储炬庄英萍袁中一
Owner EAST CHINA UNIV OF SCI & TECH
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