A kind of method utilizing l-lysine 2-monooxygenase and delta-valeramide hydrolase to catalyze the preparation of 5-aminovaleric acid
A technology of monooxygenase and aminovaleric acid, applied in the field of bioengineering, can solve problems such as unfavorable separation and purification, low yield of 5-aminovaleric acid, complex reaction system, etc., and achieve easy extraction and separation, simple composition, and high product concentration Effect
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Embodiment 1
[0032] Embodiment 1: Acquisition of davA gene
[0033] (1) Genomic DNA of Pseudomonas putida KT2440 (Pseudomonas putida KT2440, ATCC No.47054) was prepared by conventional methods. For this process, please refer to the small section of the bacterial genome in the "Guide to Molecular Biology" published by Science Press. method of preparation. The gene encoding DavA was amplified from the genome of Pseudomonas putida KT2440 using a PCR reaction.
[0034] The primers used are:
[0035] Upstream primer davA.f: 5'- GGATCC GATGCGCATCGCTCTGTACCAG-3', containing BamHI restriction site
[0036] Downstream primer davA.r: 5'- GAATTC TCAGCCTTTACGCAGGTGCAGC-3', with EcoRI restriction site
[0037] The enzyme used in the PCR reaction system is TransStart FastPfu DNA Polymerase. The resulting PCR product with a single band and high brightness was ligated to the blunt-ended cloning vector pEASY-Blunt Simple. Transform the connecting liquid into E. coli Trans1-T1 competent cells, and ...
Embodiment 2
[0045] Embodiment 2: Expression and purification of DavA gene
[0046] (1) Pick a single colony from the resistant plate and inoculate it in a shake tube containing LB medium, and culture overnight at 37°C;
[0047] (2) Transfer the bacterial solution obtained in step (1) to a 300ml Erlenmeyer flask containing 50ml LB medium at an inoculum size of 1%, and culture it with shaking at 37°C until OD 600nm 0.4~0.6;
[0048] (3) Add IPTG at a final concentration of 1 mM to the above culture medium, and induce expression overnight on a shaker at 16° C. at 175 rpm.
[0049] (4) After the strain was induced and expressed according to the above method, the bacteria were collected by centrifugation, washed twice with PBS and then resuspended to OD with solution A 600nm =30, add 10% glycerol and 1‰ PMSF to the bacterial suspension, mix well, and crush the bacterial cells with an ultrasonic breaker. The bacterial cell disruption solution was centrifuged at 12,000 rpm at 4°C for 30 minut...
Embodiment 3
[0051] Embodiment 3: acquisition of davB gene
[0052] (1) Genomic DNA of Pseudomonas putida KT2440 (Pseudomonas putida KT2440, ATCC No.47054) was prepared by conventional methods. For this process, please refer to the small section of the bacterial genome in the "Guide to Molecular Biology" published by Science Press. method of preparation. The gene encoding DavB was amplified from the genome of Pseudomonas putida KT2440 using a PCR reaction.
[0053] The primers used are:
[0054] Upstream primer davB.f: 5'- GGATCC GATGAACAAGAAGAACCGCCAC-3', containing BamHI restriction site
[0055] Downstream primer davB.r: 5'- GAATTC TCAATCCGCCAGGGCGATCGGG-3', with EcoRI restriction site
[0056] The enzyme used in the PCR reaction system is TransStart FastPfu DNA Polymerase. The resulting PCR product with a single band and high brightness was ligated to the blunt-ended cloning vector pEASY-Blunt Simple. Transform the connecting liquid into E. coli Trans1-T1 competent cells, and ...
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