A kind of aldehyde and ketone reductase mutant, gene, engineering bacteria and application thereof
A reductase and mutant technology, applied in genetic engineering, oxidoreductase, applications, etc., can solve the problem of low asymmetric reduction activity, and achieve the effect of good industrial application prospects
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Embodiment 1
[0023] Embodiment 1: Preparation of aldehyde and ketone reductase mutants
[0024] The aldehyde and ketone reductase K1AKR gene (GenBank accession number: KU145407) cloned from Kluyveromyces lactis CCTCC M 2014380 (the nucleotide sequence is shown in SEQ ID NO.4, and the encoded protein amino acid sequence is shown in SEQ ID NO.1), The expression vector pET28b-klakr has been successfully constructed.
[0025] Aldoketone reductase mutants were prepared by two rounds of site-directed saturation mutagenesis. In the first round, the tyrosine at position 295 of the amino acid sequence of aldehyde and ketone reductase KlAKR shown in SEQ ID NO.1 was mutated into tryptophan, and the recombinant plasmid pET28b-klakr was used as a template for full plasmid amplification, and the primer pair was designed as Y295- F and Y295-R (as shown in Table 1) to obtain the aldehyde and ketone reductase mutant Y295W (the amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence is sho...
Embodiment 2
[0029] Example 2: Induced expression of aldehyde and ketone reductase parents and mutants and glucose dehydrogenase
[0030] The aldehyde and ketone reductase mutant strain E.coli BL21 (DE3) / pET28b-klakr-Y295W (Y295W) and E.coli BL21 in the starting strain E.coli BL21(DE3) / pET28b-klakr and embodiment 1 (DE3) / pET28b-klakr-Y295W / W295L(Y295W / W295L) and recombinant glucose dehydrogenase strain E.coli BL21(DE3) / pET28b-esgdh were inoculated into LB liquid containing a final concentration of 50mg / L kanamycin culture medium at 37°C for 10 hours, then inoculate it at a volume concentration of 4% into fresh LB liquid medium containing a final concentration of 50 mg / L kanamycin, and cultivate at 37°C until the cell concentration is OD 600 0.6 to 0.8, add lactose with a final concentration of 9g / L to the culture medium, culture at 28°C for 12h, centrifuge at 8000g for 10min at 4°C, and collect the bacterial cells. It can be used for enzyme purification and biocatalysis to prepare 6-cyano...
Embodiment 3
[0031] Example 3: Purification of aldehyde and ketone reductase parents and mutants thereof
[0032] Suspend the aldehyde and ketone reductase bacterial cells described in Example 2 with buffer A (20 mM, pH 8.0 sodium phosphate buffer containing 500 mM NaCl and 20 mM imidazole), and ultrasonically break for 20 min (ice bath, power 400W, break for 1 s and stop for 1 s) ), centrifuged at 4°C and 12000rpm for 20min, and the supernatant was taken. Use a Ni affinity column (1.6cm × 10cm, Bio-Rad Company, USA) to purify the aldehyde and ketone reductase parent and its mutant protein, and the specific operations are as follows:
[0033] (1) Equilibrate the Ni column with the above buffer A;
[0034] (2) Pass the supernatant obtained by centrifugation after the above-mentioned ultrasonication through the Ni column at a flow rate of 1 mL / min, so that the target enzyme is adsorbed on the Ni column filler;
[0035] (3) Wash the Ni column with buffer A of 5 times the column volume above...
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