Alcohol dehydrogenase mutant and its preparation method and application
An alcohol dehydrogenase and mutant technology, which is applied in the field of enzymes and enzyme engineering, can solve the problems of low stereoselectivity of diaryl alcohols, and achieve the effect of good industrial application prospects.
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Embodiment 1
[0051] Construction of embodiment 1 mutant
[0052] According to the crystal structure of the wild-type protein (PDB ID 4RF2; SEQ ID NO: 1, the wild-type protein is derived from Lactobacillus kefir (Lactobacillus Kefir)), the reaction mechanism of alcohol dehydrogenase, and the steric position of the double aryl substrate According to the results of the resistance and hydrophobic properties, the size of the enzyme active pocket, molecular docking and molecular dynamics experiments, P190, I144, E145, L199, and M206, which constitute the active pocket, were selected as the sites for modification. Specifically, using pET21b containing the nucleic acid sequence of SEQ ID NO: 1 as a PCR template, using the mutation primer design website https: / / www.agilent.com / store / primerDesignProgram.jsp to design complementary primers, can be used to construct various Primers for amino acid mutations. The PCR reaction system is shown in Table 1, and the PCR reaction cycle temperature is shown i...
Embodiment 2
[0058] Screening of embodiment 2 mutants
[0059] Inoculate the mutant single clone of E.coli BL21(DE3) obtained in Example 1 into LB liquid medium containing 3 ml of ampicillin resistance, grow to about OD0.6, add IPTG to make the final concentration 0.1mM, Mutant protein expression was induced at 20 °C. After 20 hours, the bacterial solution was collected by centrifugation, washed with physiological saline and resuspended with 400 μl of 100 mM Tris-HCL (pH 8.0). Reaction system: more than 400 μl of suspended bacterial liquid, 50 μl of 200 mM 4-chlorophenyl-phenyl ketone (dissolved in isopropanol) and 50 μl of 5 mM NADP were added to form a reaction system with a total volume of 500 μl.
[0060] After reacting at 30° C. for 0.5-2 h, 1 ml of isopropanol was added to terminate the reaction, and the sample solution obtained after centrifugation was analyzed by chiral HPLC. Conversion determination and chiral analysis methods are as follows: Daicel liquid phase chiral analysis ...
Embodiment 3
[0065] Example 3 Alcohol dehydrogenase mutant catalyzes the asymmetric reduction of 4-chlorophenyl-phenyl ketone to prepare 500mM (R)-4-chlorophenylbenzyl alcohol
[0066] This embodiment includes the following steps:
[0067] 1. Preparation of mutant wet cells: pick mutant single clones (containing the alcohol dehydrogenase mutant whose sequence is shown in SEQ ID NO: 2) on a petri dish and inoculate them in 3 ml of LB medium containing Amp resistance Cultivate at 37°C for 16 hours, inoculate fresh medium with 1% inoculum, continue to cultivate for about 2.5 hours, and the OD reaches 0.6-0.8. Add IPTG at a final concentration of 0.1 mM, and continue to culture at 20° C. for 20 h to obtain mutant resting cells (wet cells). Resting cells (wet cells) containing glucose dehydrogenase were prepared in the same manner.
[0068] The 50ml reaction system includes: 2.4g mutant resting cells (wet cells), 0.8g glucose dehydrogenase wet cells, 32.5ml pH8.0 Tris-HCl, 2.5ml NADP (final c...
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