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Method for producing D-amino acid oxidase through fermentation

An amino acid and oxidase technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, redox enzymes, etc., can solve the problem of low biomass, inability to obtain a large number of bacteria with high enzyme activity, low enzyme activity of bacteria, etc. question

Active Publication Date: 2021-05-28
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In the prior art, due to the inhibition of cell growth by the expression of DAAO in the fermentation process, the bacterial strains with high expression of DAAO have low biomass from fermentation. , can not obtain the deficiency of a large amount of high enzyme activity thallus, the purpose of the present invention is to obtain high biomass and express the D-amino acid oxidase of high enzyme activity by optimizing the fermentation method

Method used

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  • Method for producing D-amino acid oxidase through fermentation
  • Method for producing D-amino acid oxidase through fermentation
  • Method for producing D-amino acid oxidase through fermentation

Examples

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Effect test

Embodiment 1

[0046] The genetically engineered bacteria are activated on a slant, and the colonies are picked up and inoculated into a shaker flask equipped with a first-level seed medium for cultivation as a first-level seed solution. The cultivation conditions of the first-level seed solution are: pick a single colony into Streptomycin was used as an antibiotic in a 250mL shake flask with a liquid volume of 50mL, placed in a constant temperature shaking incubator at 37°C, and shaken at 180rpm for 12h.

[0047] Inoculate the first-level seed liquid into the shaking flask equipped with the second-level seed medium and cultivate it as the second-level seed liquid. g / L streptomycin sulfate was used as an antibiotic in a 500mL shake flask with a liquid volume of 100mL, placed in a constant temperature shaking incubator at 37°C, and shaken at 180rpm for 10-12h.

[0048] Take the inoculation amount as 10% to inoculate the secondary seed liquid into the 2L fermentation medium in the 5L fermenter...

Embodiment 2

[0056] Fermentation method, condition are all the same as embodiment 1 except following changes. Set the feeding mode to DO-STAT, where the DO setting value is 30%, and when DO exceeds 0.1%, automatic feeding. The fermentation time is 22-24h, and when the biomass growth of the bacteria slows down, the fermentation is stopped. After the fermentation is completed, the fermentation broth is centrifuged at 8000 rpm for 10 minutes with a freeze-drying centrifuge to collect the bacteria. It is determined by experiments that the biomass of the thalline obtained by fed-batch fermentation by the method is 23.4g DCW / L, and the specific enzyme activity of the thalline is 388.3U / gDCW. The parameters of the fermentation process are shown in figure 1 .

Embodiment 3

[0058] Fermentation method, condition are all the same as embodiment 1 except following changes. Set the feeding mode to DO-STAT, where the DO setting value is 50%, and when DO exceeds 0.1%, automatic feeding. The fermentation time is 22-24h, and when the biomass growth of the bacteria slows down, the fermentation is stopped. After the fermentation is completed, the fermentation broth is centrifuged at 8000 rpm for 10 minutes with a freeze-drying centrifuge to collect the bacteria. It is determined by experiments that the biomass of the thalli obtained by fed-batch fermentation by the method is 20.4g DCW / L, and the specific enzyme activity of the thalline is 377.3U / gDCW.

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Abstract

The invention discloses a method for producing D-amino acid oxidase through fermentation, and belongs to the technical field of biological enzyme preparation. The method comprises the following steps: taking recombinant escherichia coli for expressing D-amino acid oxidase as a production strain, inoculating the recombinant escherichia coli into a fermentation culture medium for fermentation culture, supplementing materials in batches in a DO-STAT mode after dissolved oxygen is reduced to 1%, and setting the DO value to be 10-50%; in the fermentation process, adding 10-50 g / L lactose serving as an inducer, meanwhile, adding D-amino acid into the fermentation liquor, continuing culture till fermentation is finished, and collecting thalli containing D-amino acid oxidase in cells. In the fed-batch fermentation process, high-concentration lactose is added to serve as an inducer to improve the enzyme activity of DAAO, meanwhile, a proper amount of D-amino acid is added to reduce inhibition of DAAO on growth of host cells, the production efficiency of DAAO is greatly improved, and conditions are provided for industrial production.

Description

technical field [0001] The invention relates to the technical field of biological enzyme preparation, in particular to a method for fermenting and producing D-amino acid oxidase by genetically engineered bacteria. Background technique [0002] D-amino acid oxidase (DAAO, EC 1.4.3.3) is a flavonoid enzyme with flavin adenine dinucleotide as the prosthetic group, which can catalyze the D-amino acid oxidase with absolute stereospecificity in the presence of oxygen molecules. Oxidative deamination of amino acids to produce α-keto acids, ammonia and hydrogen peroxide (H 2 o 2 ). Due to its strict stereoselectivity, DAAO has been used to produce unnatural L-amino acids. [0003] L-amino acid is an important intermediate compound in drug synthesis. At present, DAAO has been widely used in the industrial catalytic preparation of L-amino acids. For example, DAAO acts as a catalyst to convert cephalosporin C into 7-aminocephalosporanic acid (7-ACA), which is a key compound in the...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12N1/20C12R1/19
CPCC12N9/0024C12N1/20C12Y104/03003
Inventor 徐建妙郑裕国曹慧婷薛亚平程峰邹树平
Owner ZHEJIANG UNIV OF TECH
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