Stereoselective esterase, coding gene, carrier, engineering bacteria and application thereof
A stereoselective, gene-encoding technology, applied in the direction of genetic engineering, vectors, applications, etc., can solve the problems of reducing the yield of target reaction products, side reactions, etc.
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Embodiment 1
[0039] Use the nucleic acid extraction kit to extract the genomic DNA of Pseudomonas saccharolyticum WZZ003 (PseudochrobactrumasaccharolyticumWZZ003), using the genomic DNA as a template, primer 1 (5'-ATGCAAAAAACAGCATGTTGAAAC-3'), primer 2 (5'-TTAACCGCGCAGGAAACG-3' ) for PCR amplification.
[0040] The amount of each component in the PCR reaction system (total volume 50 μL):
[0041] 10×TransStart rTaq Buffer (with Mg 2+ ) 5 μL, 10 mM dNTP mixture (2.5 mM each of dATP, dCTP, dGTP, and dTTP) 12.5 μL, 1 μL each of primer 1 and primer 2 at a concentration of 50 pM, 1 μL of genomic DNA, 1 μL of TransTaq DNA polymerase, and add water to make up to 50 μL.
[0042] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min, followed by a temperature cycle at 94°C for 30 s; 60°C for 30 s; 72°C for 1 min; a total of 30 cycles, and a final extension at 72°C for 10 min with a termination temperature of 4°C.
[0043] Take 6 μL of the PCR reaction solution and use 0....
Embodiment 2
[0045] The recombinant plasmid obtained according to Example 1 was chemically transformed into Escherichia coli Trans B (DE3) (the competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and the recombinant plasmid pEASY-E2-Est containing intracellular expression was obtained. Recombinant Escherichia coli Trans B(DE3) / pEASY-E2-YsEst. The recombinant Escherichia coli was cultured at 37°C for 16 hours with LB liquid medium containing ampicillin (50 μg / ml) and kanamycin (50 μg / ml), and then inoculated into fresh cells containing In LB liquid medium of ampicillin (50 μg / ml), cultivate at 37°C until the cell concentration OD600 is about 0.6, then add IPTG with a final concentration of 0.2mM to the LB liquid medium, and induce culture at 22°C for 6-8 hours , and then centrifuged at 10,000 rpm for 10 min at 4°C to collect the bacterial cells containing the recombinant esterase. The resulting recombinant genetically engineered bacterium cells were resuspended...
Embodiment 3
[0049] Obtain recombinant Escherichia coli Trans B(DE3) / pEASY-E2-Est wet thallus in embodiment 2, resuspend thallus with the Tris-HCl buffer solution of 50mM pH8.0, carry out sonication then (ultrasound 2s, interval 6s , the effective ultrasonic time is 5 min), the broken suspension is centrifuged at 10000 rpm for 10 min at 4°C, and the cell debris is discarded as much as possible to obtain the protein crude enzyme solution. According to the instructions of Ni-NTA metal chelate affinity chromatography, take the protein crude enzyme solution and load it on the pre-equilibrated Ni 2+ In the column, then use 10mM imidazole, 40mM imidazole, 100mM imidazole, 250mM imidazole aqueous solution to elute the impurity protein and the target protein successively. The effluent after being eluted with 100 mM imidazole aqueous solution is collected, desalted and concentrated by ultrafiltration membrane, and then stored at -20°C to obtain recombinant esterase.
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