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A method for producing 5-aminovaleric acid by whole cell biocatalysis
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A kind of aminovaleric acid and biocatalysis technology, applied in the biological field, can solve the problems of low yield and long time consumption of 5-aminovaleric acid, and achieve the effect of solving the inhibition of enzyme activity, increasing the accumulation rate, and improving the activity
Active Publication Date: 2019-11-19
NANJING TECH UNIV
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Problems solved by technology
This method adopts the catalytic system of fermentation method, the output of 5-aminovaleric acid is low, and it takes a long time
[0007] After searching, a method for efficiently utilizing whole-cell biocatalysis to produce 5-aminovaleric acid has not been reported yet
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Embodiment 1
[0020] Embodiment 1: the liquid phase detection method of 5-aminovaleric acid
[0022] Embodiment 2: the construction process of overexpressing DavBA bacterial strain
[0023] E. coli Construction process of BL-22A-RB strain:
[0024] (1) davA was synthesized by Jinweizhi, and the enzyme cutting sites were NdeI and HindIII. After the davA fragment was treated and recovered by restriction endonucleases NdeI and HindIII, it was connected to the vector pET-22b treated with the same restriction endonuclease, Use T4 DNA ligase for 30 min at 25°C.
[0025] (2) Transform the connection solution in (1) into competent cells of Escherichia coli Trans1-T1 (provided by Quanshijin Biotechnology Co., Ltd.), spread on LB plates with ampicillin resistance, and overnight at 37°C nourish.
[0026](3) Pick a single colony grown on the plate in (2), transfer it to LB medium containing ampicillin resistance, then extract the plasmid, and then use restriction endonucleases NdeI and HindIII to carry out enzyme digestion verification, and finally get The vector pET-22b-davA ...
Embodiment 3
[0059] Example 3: Whole Cell Catalysis
[0060] (1) The bacterial strains preserved in the cryopreservation tube in Example 2 E. coli BL-22A-RB-YB was inoculated into shake tubes containing ampicillin resistance, kanamycin resistance, chloramphenicol resistance and LB, cultured at 37°C and 200rpm for 8 hours, and then transferred to a shake tube containing ampicillin resistance , kanamycin-resistant and chloramphenicol-resistant 500ML shake flasks, 37°C 200rpm shaking culture to OD 600nm is 0.3;
[0061] (2) Add IPTG with a final concentration of 0.5mM to the above shake flask for induction, and culture at 20°C and 200rpm for 12h;
[0062] (3) After 12 hours, centrifuge the above bacterial solution at 6000g for 5 minutes, discard the supernatant, and wash the collected cells twice with PBS of pH 7.0 to obtain the cells used for the catalytic reaction.
[0063] (4) Resuspend the collected cells with PBS of pH 7.0 and concentrate them into the catalytic system to make the OD ...
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Abstract
The invention discloses a method for biocatalytically producing 5-aminovaleric acid by using Escherichia coli cells with expressed L-lysine-2-monooxygenase DavB and delta-valeramide hydrolase DavA as a catalyst. According to method, DavBA-expressed E.coli BL21 (DE3) is subjected to low-temperature induction expression, bacteria are accumulated, and whole-cell catalysis is carried out in a buffer system by using the DavBA-expressed E.coli BL21 (DE3) as the catalyst under the auxiliary actions of metal ions and a surfactant to produce the 5-aminovaleric acid, wherein the cell OD600nm in the system is 5-90, and the lysine concentration is 0-400 g / L. The method solves the problems of long production cycle, complex metabolites, low substrate conversion rate, difficulty in product separation and extraction and high energy consumption in the fermentation method, overcomes the defect of difficulty in implementing the cascade catalytic process in the enzyme catalytic method, enhances the catalytic efficiency, and omits the complex enzyme purification process, so the production cost is lower.
Description
technical field [0001] The invention relates to a method for producing 5-aminovaleric acid by high-efficiency whole-cell biocatalysis, in particular to an Escherichia coli expressing L-lysine 2-monooxygenase DavB and δ-pentanamide hydrolase DavA The invention relates to a method for biocatalytically producing 5-aminovaleric acid with cells as catalysts, belonging to the field of biotechnology. Background technique [0002] 5-aminovaleric acid is an important C5 platform compound, which has a wide range of applications in medicine, such as the synthesis of 5-hydroxyvaleric acid, glutaric acid, 1,5-pentanediol and its derivatives, pentadiol An important precursor of acid anhydride, at the same time, it can also be used to synthesize nylon-5,5 (copolymer of cadaverine and pentamethylenediamine) and nylon-5 (5-aminovaleric acid homopolymer). Therefore, 5-aminopentane Acids are widely used in textile industry and pharmaceutical synthesis. [0003] At present, in the preparation...
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