Restructuring carbonyl reductase mutant ReCR-Mut, encoding gene, engineering bacteria and application
A technology for encoding genes and mutants, which is applied in the field of biocatalysis and can solve problems such as reducing catalytic efficiency
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Embodiment 1
[0022] Example 1: Construction of recombinant expression plasmid pEASY-E2-recr and genetically engineered bacteria E.coliBL21(DE3) / pEASY-E2-recr
[0023] Using the genomic DNA of Rhodococcus erythropolis WZ010 strain as a template, the designed primers F1 and R1 were used for PCR amplification. The amplification system is shown in Table 1.
[0024] Table 1 PCR amplification reaction system
[0025]
[0026]The primers are as follows: F1, 5'-ATGAAGGCAATCCAGTACAC-3'; R1, 5'-CTACAGACCAGGGACCACA-3'. The PCR reaction process is as follows: pre-denaturation at 94°C for 5 minutes; after that, denaturation at 94°C for 30 s, renaturation at 55°C for 30 s, and 72°C for 1 min as a cycle, repeating this cycle 30 times; finally, 72°C for 10 min. PCR products were detected by 0.8% agarose gel electrophoresis. figure 2 A bright band can be seen at about 1000bp, which is consistent with the theoretical value of 1047bp.
[0027] The PCR product fragment (the nucleotide sequence is shown...
Embodiment 2
[0029] Example 2: Construction of recombinant expression plasmid pEASY-E2-recr-mut and genetically engineered bacteria E.coliBL21(DE3) / pEASY-E2-recr-mut
[0030] Using the plasmid pEASY-E2-recr as a template, primers F2 and R2 were designed, and the mutant plasmid was amplified by inverse PCR. The amplification system is shown in Table 2.
[0031] Table 2 PCR amplification reaction system
[0032]
[0033] The primers are as follows: F2, 5'-TACACCTTCGGCCTTCCTCTCACGC-3'; R2, 5'-AAGGCCGAAGGTGTACTGCTCCTCG-3'. The PCR reaction process is as follows: pre-denaturation at 95°C for 2 min; then, denaturation at 95°C for 20 s, renaturation at 68°C for 20 s, and 3 min at 72°C as a cycle, repeating this cycle 30 times; finally, maintaining 10 min at 72°C. PCR products were detected by 0.8% agarose gel electrophoresis. image 3 A bright band at about 6000bp can be seen in , which is consistent with the theoretical value of the plasmid.
[0034] The PCR product was digested at 37°C fo...
Embodiment 3
[0038] Example 3: Induced expression and isolation and purification of recombinant carbonyl reductase ReCR and its mutant ReCR-Mut
[0039] The constructed genetic engineering bacteria E.coliBL21(DE3) / pEASY-E2-recr and E.coliBL21(DE3) / pEASY-E2-recr-mut were respectively inoculated into LB liquid medium containing 100 μg / mL ampicillin, 37 Cultivate at ℃ for 12 hours to obtain seed liquid, inoculate the seed liquid with an inoculum volume concentration of 2% into fresh LB liquid medium containing 100 μg / mL ampicillin, and cultivate to OD at 37°C 600 Then add IPTG with a final concentration of 0.3mM, induce at 20°C for 12h, obtain the induction culture medium, then centrifuge the culture medium at 4°C and 10000rpm for 10min, discard the supernatant to collect the wet cells, and use it as a whole Catalyst for cellular catalysis.
[0040] Add an appropriate amount of Tris-HCl (pH8.0) buffer solution to the above-mentioned wet bacteria according to the ratio of 1g wet bacteria plus...
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