Method for preparing 1-cyancyclohexylacetic acid by using nitrilase engineering bacterium
A technology of nitrilase and engineering bacteria, which is applied in the field of preparing 1-cyanocyclohexylacetic acid by using nitrilase engineering bacteria, can solve the problems of high cost, low yield, environmental pollution, etc., and achieve good regioselectivity, high The effect of catalytic activity
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Embodiment 1
[0026] Example 1: Synthesis of Nitrilase Gene
[0027] A nitrilase gene (GeneBank accession no. D12583) was obtained by screening the nitrilase gene sequence using the protein PDB and NCBI databases. The nitrilase was derived from Rhodococcus rhodochrous K22 (Rhodococcus rhodochrous K22). According to the sequence of Rhodococcus rhodochrous K22 nitrilase (GeneBank accession no.D12583), the codon was optimized according to the preferred codon of Escherichia coli. According to the expression vector pET28b ( +) The enzyme cutting sites Nco Ⅰ and Xho Ⅰ were designed, and the nitrilase gene RkN (shown in SEQ ID NO.2) was synthesized by the method of total synthesis through the conventional operation of genetic engineering, encoding the amino acid of the enzyme The sequence is shown in SEQ ID NO.1.
Embodiment 2
[0028] Embodiment 2: the construction of nitrilase gene expression vector and its recombinant transformant
[0029] The synthesized nitrilase genes RkN and pET28b(+) were double-digested with Nco I and Xho I respectively, and after about 6 hours of digestion, the digested products were recovered and ligated with T4 ligase at 16°C for 16 hours to obtain the recombinant Expression plasmid pET28b(+)-RkN. Transform the expression vector pET28b(+)-RkN into E.coli BL21(DE3) recipient bacteria, smear it on the LB agar plate containing kanamycin (final concentration 50mg / L), cultivate overnight at 37°C, and plate Colonies (yellow-white round colonies) grew on the A single clone was randomly picked, and after culture, the plasmid was extracted for sequencing. The sequencing results showed that a positive clone E.coli BL21(DE3) / pET28b(+)-RkN was obtained.
Embodiment 3
[0030] Example 3: Expression of Nitrilase
[0031] The recombinant transformant E.coli BL21(DE3) / pET28b(+)-RkN obtained in Example 2 was inoculated into LB liquid medium, cultivated at 37°C and 150rpm for 10-12 hours, and then the inoculation amount was 2% by volume Inoculate into LB liquid culture medium containing kanamycin (final concentration 50mg / L) and carry out expansion culture, 37 ℃, 150rpm cultivate to the OD of culture medium 600 Between 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM, induce culture at 28° C., 150 rpm for 10 hours. The bacterial cells were collected by centrifugation of the culture solution, and washed twice with normal saline to obtain wet bacterial cells.
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