A kind of penicillinase mutant with improved enzyme activity and its construction method
A penicillinase and mutant technology, applied in the field of genetic engineering, can solve the problems of low penicillinase activity and low protein expression, and achieve the effects of improving enzyme activity, reducing production costs, and meeting the needs of industrialized production of penicillinase
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Embodiment 1
[0022] Example 1 Site-directed mutagenesis
[0023] Using the recombinant plasmid containing SEQ ID NO: 4 as a template plasmid, refer to TaKaRa biological products and operation manuals, use TaKaRa MuTanBEST mutation kit, and use the designed corresponding mutation primer pair to amplify the site-directed mutation sequence of BmPGA by PCR. The specific operation steps as follows:
[0024] (1) Mutation primers
[0025] Forward primer BLAA-150-F is 5'-GACCTGAACACCGCTATCRHTGGTGACG AACGTGACACC-3',
[0026] The reverse primer BLAA-150-R is 5'-GGTGTCACGTTCGTCACCADYGATAGCGG TGTTCAGGTC-3'.
[0027] (2) Reaction system (10ul)
[0028] 10ul of 10xPfu polymerase buffer, 2ul of 10mmol / L dNTP solution, 25pmoles of forward primer, 25pmoles of reverse primer, 5U of Pfu DNA polymerase, 100-200pg of plasmid DNA template, add water to 100ul.
[0029] (3) PCR conditions: 94°C for 3 minutes, 94°C for 30s, 55°C for 30s, 72°C for 300s, 30 cycles, 72°C for 10 minutes.
[0030] Refer to the ope...
Embodiment 2
[0032] The acquisition of embodiment 2 engineering bacteria
[0033] Referring to the method of "Molecular Cloning Experiment Guide", transform the mutated plasmid into Escherichia coli competent cells or yeast competent cells to obtain genetically engineered strains expressing mutant enzymes, and finally apply the engineered strains to kanamycin sulfate On the LB selective plate of antibiotics, culture upside down at 37°C for about 12-18 hours, and those that can grow on the LB plate with kanamycin sulfate antibiotics are transformants transformed with recombinant plasmids.
Embodiment 3
[0034] Embodiment 3 Engineering bacteria fermentation culture
[0035] Pick a single colony from the LB selective culture plate, inoculate it into 3 mL of LB liquid medium, add kanamycin to a final concentration of 100 μg / mL, and culture at 250 r / min at 37°C overnight; take 2 mL and culture overnight Inoculated into 200mL of LB liquid medium, cultured at 250r / min at 37°C for 4-6h, and the OD600 reached between 1.0-1.6, to obtain the seed bacterial liquid. Insert the inoculation amount of 1:15 into a 5-liter fermenter containing 3 liters of TB medium. Under the fermentation conditions of 37°C, 400rpm, and 1.3vvm, ferment until the OD600 reaches 25, and adjust the temperature to 30°C. Induce with IPTG with a final concentration of 1%, continue culturing for 2 hours, then add IPTG with a final concentration of 0.5% for induction, continue to cultivate for about 14-16 hours, and the fermentation ends.
[0036] The fermentation curve of engineering bacteria is as follows: figure 1 ...
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