A novel nitrilase and its application
A technology of nitrile hydrolysis and phenylacetonitrile, applied in the field of genetic engineering, to achieve the effects of short fermentation cycle, high nitrilase activity and high catalytic efficiency
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Embodiment 1
[0036] The acquisition of embodiment 1 nitrilase
[0037] 1. Experimental method
[0038] The genome data (NCBI Reference Sequence: NZ_KQ033901.1) of Nitriliruptor alkaliphilus ANL-iso2 (preservation number DSM 45188) was retrieved by NCBI database data, and the genome was screened based on the prediction and annotation of functional genes to obtain a Nitrilase gene (Nit2). According to the amino acid sequence of the nitrilase, the codons were optimized according to the codons preferred by Escherichia coli, and the restriction sites Nde I and Xba I were designed according to the characteristics of the expression vector pColdI DNA.
[0039] 2. Experimental results
[0040] The nitrilase gene Nit2 (shown in SEQ ID: 2) was synthesized by a total synthesis method through the conventional operation of genetic engineering, and the amino acid sequence of the encoded enzyme is shown in SEQ ID: 1.
Embodiment 2
[0041] The construction of embodiment 2 recombinant expression vector pCold I DNA-Nit2 and the construction of recombinant engineering bacteria
[0042] 1. Experimental method
[0043]Use Nde I and Xba I restriction endonucleases to carry out double digestion and recovery treatment of the Nit2 gene fragment, and use T4 DNA ligase to combine the fragment with the commercial vector pCold I DNA treated with the same restriction endonuclease at 16 The ligation was carried out at ℃ for 6 hours to construct the intracellular recombinant expression vector pCold I DNA-Nit2. Transform the constructed intracellular expression vector pCold I DNA-Nit2 into E.coli BL21(DE3) recipient bacteria, spread on the LB agar plate containing ampicillin (final concentration: 50 μg / mL), at 37°C After culturing overnight, randomly pick clones from the colonies grown on the plate the next day and extract the plasmids for identification by agarose gel electrophoresis.
[0044] 2. Experimental results ...
Embodiment 3
[0046] Embodiment 3 contains the preparation of nitrilase BrNit bacterial cell
[0047] 1. Experimental method
[0048] The genetically engineered bacteria E.coli BL21(DE3) / pCold I DNA-Nit2 constructed in Example 2 was inoculated into the LB liquid medium containing 50 μg / mL ampicillin, cultivated at 37° C. for 12 h, and then inoculated with 2% inoculum ( v / v) Inoculate into fresh LB liquid medium containing 50 μg / mL ampicillin, and cultivate at 37°C until the cell concentration is OD 600 About 0.5, then add IPTG with a final concentration of 0.5mM to the LB liquid medium, after induction culture at 15°C for 20h, centrifuge the culture solution at 4°C, 8000rpm for 5min, discard the supernatant, and collect the recombinant nitrilase-containing The wet cells of Escherichia coli BL21(DE3) / pCold I DNA-Nit2 containing intracellular expression of recombinant nitrilase. Then SDS-PAGE electrophoresis was performed to verify the size and expression of the target protein.
[0049] 2....
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