EstWY enzyme mutant having improved activity
A technology of mutants and activity, applied in the field of enzyme engineering, can solve the problems of poor enzyme activity of EstWY, and achieve the effect of simple culture conditions, good activity and high-efficiency expression
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Embodiment 1
[0027] Embodiment 1: the construction of single point EstWY enzyme mutant
[0028] The wild-type EstWY enzyme plasmid pET-28a was preserved in the laboratory, and a single-point EstWY enzyme mutant was constructed by the whole plasmid PCR method. The schematic diagram of the crystal structure of the EstWY enzyme ENND-TOP protein and the schematic diagram of the mutation site are as follows figure 1 shown. The details are as follows: using pET-28a as a template, the upstream and downstream primers of each mutation site are listed in Table 1, and are named in the format of "mutation site substitution amino acid". Using the high-fidelity DNA polymerase PrimeSTAR HS DNA Polymerase Kit, a round of PCR amplification was performed in order to obtain a gene recombinant plasmid containing the mutant. The reaction system is shown in Table 2. PCR conditions: pre-denaturation: 95°C for 3min; denaturation: 98°C for 10s; annealing: 58°C for 10s; extension: 72°C for 5min; cycle 35 times; fu...
Embodiment 2
[0034] Embodiment 2: the construction of multi-point EstWY enzyme mutant
[0035] In order to further analyze the effect of different amino acid species at each site on the catalytic properties of the enzyme, referring to the method of site-directed mutagenesis, the whole plasmid PCR technology is still used to obtain the saturated mutation library gene. The details are as follows: Use the high-fidelity DNA polymerase PrimeSTAR HS DNA Polymerase Kit, carry out multiple rounds of PCR amplification in order to obtain gene recombinant plasmids containing mutants. The reaction system, PCR conditions and transformation conditions are the same as those for site-directed mutagenesis.
Embodiment 3
[0036] Embodiment 3: construction of mutant engineered bacteria
[0037] Refer to the instructions of the Super Competent Kit to construct engineering bacteria. The specific operation is as follows. Firstly, confirm that E.coliBL21(DE3) cannot grow under Kan resistance; secondly, streak and activate the E.coli BL21(DE3); thirdly, take a single colony and add it to LB culture without resistance culture medium until the OD600 is between 0.6 and 0.8, and prepare competent cells with the kit’s own solution; the fourth step is to transform and spread on the LB solid medium plate containing Kan resistance, and cultivate for 16 hours; finally, pick For 3 single colonies, the target gene was amplified by bacterial liquid PCR, and the target band was identified by agarose gel electrophoresis, and then sent to Suzhou Jinweizhi for sequencing to confirm the engineering bacteria.
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