Alcohol dehydrogenase mutant and use thereof

Abstract

The invention discloses an alcohol dehydrogenase mutant and use thereof. The alcohol dehydrogenase mutant of the present invention has high thermal stability and enables high catalytic efficiency and high conversion rate (i.e. space time yield) in the asymmetric reduction of prochiral diaryl ketones to produce chiral diaryl alcohols. Therefore, the alcohol dehydrogenase mutant of the present invention has extremely high prospect of application in the production of chiral diaryl alcohols, such as (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol, (R)-(4-chlorophenyl)-(pyridin-2-yl)-methanol.

Claims

1. An alcohol dehydrogenase mutant, wherein the mutant is obtained by mutating lysine at position 36, threonine at position 132, asparagine at position 159, lysine at position 210, threonine at position 248 and/or glutamine at position 272 in the starting amino acid sequence as shown in SEQ ID NO: 1 of alcohol dehydrogenase; or mutating lysine at position 36, threonine at position 132, asparagine at position 159, lysine at position 210, threonine at position 248, glutamine at position 272, glutamine at position 136, phenylalanine at position 161, serine at position 196, glutamate at position 214 and serine at position 237 in the starting amino acid sequence as shown in SEQ ID NO: 1 of alcohol dehydrogenase.

2. The alcohol dehydrogenase mutant according to claim 1, wherein the mutant is obtained by mutating lysine at position 36 to isoleucine, threonine at position 132 to aspartate, asparagine at position 159 to aspartate, lysine at position 210 to glutamate, threonine at position 248 to alanine and/or glutamine at position 272 to leucine; or mutating lysine at position 36 to isoleucine, threonine at position 132 to aspartate, asparagine at position 159 to aspartate, lysine at position 210 to glutamate, threonine at position 248 to alanine, glutamine at position 272 to leucine, glutamine at position 136 to asparagine, phenylalanine at position 161 to valine, serine at position 196 to glycine, glutamate at position 214 to glycine, and serine at position 237 to cysteine.

3. A gene encoding the alcohol dehydrogenase mutant according to claim 1.

4. A recombinant plasmid comprising the gene according to claim 3.

5. The recombinant plasmid according to claim 4, wherein the vector of the recombinant plasmid is pET-28a(+) plasmid, pET-28b(+) plasmid or pET-20b(+) plasmid.

6. A host cell comprising the gene according to claim 3.

7. A method for producing the alcohol dehydrogenase mutant according to claim 1, comprising steps of: inoculating a host cell comprising a gene encoding the alcohol dehydrogenase mutant into a fermentation medium for fermentation to obtain a fermentation broth; centrifuging the fermentation broth to collect the bacterial cells; homogenizing the bacterial cells and centrifuging to obtain a cell homogenate supernatant; and purifying the alcohol dehydrogenases from the cell homogenate supernatant.

8. A method for producing a chiral diaryl alcohol, comprising adding the alcohol dehydrogenase mutant according to claim 1 to a reaction system comprising a prochiral diaryl ketone for reaction; and extracting the resulting reaction solution to obtain a chiral diaryl alcohol.

9. The method for producing a chiral diaryl alcohol according to claim 8, wherein the reaction system further comprises a coenzyme, and a coenzyme regeneration system comprising D-glucose and a glucose dehydrogenase, or a phosphite and a phosphite dehydrogenase, or a formate and a formate dehydrogenase, or a lactate and a lactate dehydrogenase, or glycerol and a glycerol dehydrogenase.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1, 2, 3, and 4 are electrophoretograms of recombinant plasmids obtained by PCR amplification, where M: Marker; and Lanes 1-64: enzymatically cleaved products of recombinant plasmid pET28a-KpADH-1 to recombinant plasmid pET28a-KpADH-64.

(2) FIG. 5 shows results by SDS-PAGE electrophoresis analysis of expression products obtained from induced fermentation of recombinant E. coli in a shake flask, where M: Maker; and Lanes 1-64: pure enzymes, that are, mutants M1-M64 obtained from induced fermentation of recombinant E. coli BL21/pET28a-KpADH-1 to recombinant E. Coli BL21/pET28a-KpADH-64 in a shake flask.

(3) FIG. 6 shows a chiral chromatogram of prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone asymmetrically reduced by the mutants M1-M64.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(4) The E. coli BL21(DE3) involved in the following examples is purchased from Beina Biotechnology; the pET-28a(+) plasmid and NADPH involved in the following examples are purchased from Novagen; the glucose dehydrogenase (GDH) and lactate dehydrogenase (LDH) involved in the following examples are purchased from Vazyme Biotech Co., Ltd; D-glucose involved in the following examples is purchased from Generay Biotech. Co., Ltd.; and the prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone involved in the following examples is purchased from Sangon Biotech. (Shanghhai) Co., Ltd. (the E. coli BL21(DE3) can be purchased and does not need to be preserved according to the patent procedures).

(5) The media involved in the examples are:

(6) LB liquid medium: yeast powder 5.0 g.Math.L.sup.−1, tryptone 10.0 g.Math.L.sup.−1, NaCl 10.0 g.Math.L.sup.−1, and kanamycin 100 mg.Math.L.sup.−1.

(7) LB solid medium: yeast powder 5.0 g.Math.L.sup.−1, tryptone 10.0 g.Math.L.sup.−1, NaCl 10.0 g.Math.L.sup.−1, agar powder 15 g/L, and kanamycin 50 mg.Math.L.sup.−1.

(8) Detection methods involved in examples:

(9) The detection method of enzyme activity of the alcohol dehydrogenase is as follows:

(10) A sodium phosphate buffer (PBS, 100 mM, pH 7.0) containing 1 mM NADPH and 1.0 mM substrate prochiral (4-chlorophenyl)-(pyridin-2-yl)-methanone is stood at 30° C. for 2 min, and then 10 μL of a pure enzyme solution is added to the sodium phosphate buffer, and reacted at 30° C. During the reaction, the change in absorbance of the reaction solution at 340 nm is measured on a microplate reader, and used to calculate the enzyme activity.

(11) The calculation formula of enzyme activity is as follows:
Enzyme activity(U/mL)=EW×V×10.sup.3/(6220×1)

(12) where EW is the change in absorbance at 340 nm in 1 min; V is the volume of the reaction solution, in mL; 6220 is the molar extinction coefficient of NADPH, in L/(mol.Math.cm); and 1 is the optical distance, in cm.

(13) Definition of enzyme activity: The amount of enzyme required for catalytic oxidation of 1 μmol NADPH per minute under these conditions is one enzyme activity unit (1 U).

(14) The detection methods of the conversion rate and stereoselectivity in the production of chiral diaryl alcohol (R)-(4-chlorophenyl)-(pyridin-2-yl)-methanol by the asymmetric reduction of prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone by alcohol dehydrogenase are as follows:

(15) A sodium phosphate buffer (PBS, 100 mM, pH 7.0) containing 1 mM NADPH and 1.0 mM substrate prochiral (4-chlorophenyl)-(pyridin-2-yl)-methanone is stood at 30° C. for 2 min, and then 10 μL of a pure enzyme solution is added to the sodium phosphate buffer and reacted at 30° C. for 60 min. Then, the reaction is terminated with boiling water. 100 μL of the reaction solution is added to 500 μL of ethyl acetate, shaken for 1-2 min, and centrifuged at 12000 rpm for 2-5 min. The supernatant is added to a centrifuge tube, the organic phase is allowed to evaporate completely, then 500 μL of chromatographic pure ethanol is added, and the conversion rate and e.e. value are analyzed by chiral liquid chromatography.

(16) Chromatographic conditions: Daicel Chiralcel OD-H (5 μm, 250 mm×4.6 mm) liquid chromatographic column, mobile phase n-hexane: ethanol: ethanolamine (90:10:0.01, v/v/v), flow rate 0.8 mL/min, column temperature 30° C., UV detection wavelength 254 nm, injection volume 10 μL, and retention times of (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol and (R)-(4-chlorophenyl)-(pyridin-2-yl)-methanol 11.12 min and 12.71 min.

(17) The calculation method of conversion rate is as follows:

(18) $\mathrm{Conversion}=\frac{A_s+A_R}{A_s+A_R+A_{\mathrm{sub}}}\times 100\%;$

(19) The calculation method of ee value is as follows:

(20) $\mathrm{ee}=\frac{A_s-A_R}{A_s+A_R}\times 100\%\left(S\right);$$\mathrm{ee}=\frac{A_R-A_S}{A_s+A_R}\times 100\%\left(R\right);$

(21) where As: molar concentration of (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol in the reaction solution; A.sub.R: molar concentration of (R)-(4-chlorophenyl)-(pyridin-2-yl)-methanol in the reaction solution; and A.sub.sub: molar concentration of unreacted (4-chlorophenyl)-(pyridin-2-yl)-methanone in the reaction solution.

Example 1: Production, Expression and Purification of Alcohol Dehydrogenase Mutants

(22) A gene encoding the alcohol dehydrogenase having an amino acid sequence as shown in SEQ ID NO: 1 was chemically synthesized (the nucleotide sequence of the gene is as shown in SEQ ID NO: 2, see Table 1 for details). The obtained gene and the pET-28a(+) plasmid were respectively cleaved by Nde I and Xho I and then ligated. The ligation product was transformed into E. coli BL21(DE3), and then the transformed cells were coated on LB solid medium, and incubated at 37° C. for 8-10 h. 5 transformants were picked up from the LB solid medium, inoculated into LB liquid medium, and incubated at 37° C. for 10 hrs. Then the plasmid was extracted, and the extracted plasmid was enzymatically cleaved and sequenced for verification. The plasmid was verified to be correct. That is, a recombinant plasmid pET28a-KpADH comprising the gene encoding wild-type alcohol dehydrogenase and recombinant E. coli BL21/pET28a-KpADH comprising the gene encoding wild-type alcohol dehydrogenase were obtained.

(23) Site-directed mutation was performed by whole plasmid PCR using the obtained recombinant plasmid pET28a-KpADH as a template, to obtain a recombinant plasmid comprising a gene encoding the alcohol dehydrogenase mutant K36I (mutation of lysine at position 36 to isoleucine), T132D (mutation of threonine at position 132 to aspartate), N159D (mutation of asparagine at position 159 to aspartate), K210E (mutation of lysine at position 210 to glutamate), T248A (mutation of threonine at position 248 to alanine), Q272L (mutation of glutamine at position 272 to leucine), K36I/T132D, K36I/N159D, K26I/K210E, K26I/T248A, K26I/Q272L, T132D/N159D, T132D/K210E, T132D/T248A, T132D/Q272L, N159D/K210E, N159D/T248A, N159D/Q272L, K210E/T248A, K210E/Q272L, T248A/Q272L, K36I/T132D/N159D, K36I/T132D/K210E, K36I/T132D/T248A, K36I/T132D/Q272L, K36I/N159D/K210E, K36I/N159D/T248A, K36I/N159D/Q272L, K36I/K219E/T248A, K36I/K210E/Q272L, K36I/T248A/Q272L, T132D/N159D/K210E, T132D/N159D/T248A, T132D/N159D/Q272L, T132D/K210E/T248A, T132D/K210E/Q272L, T132D/T248A/Q272L, N159D/K210E/T248A, N159D/K210E/Q272L, N159D/K248A/Q272L, K210E/T248A/Q272L, K36I/T132D/N159D/K210E, K36I/T132D/N159D/T248A, K36I/T132D/N159D/Q272L, K36I/T132D/K210E/T248A, K36I/T132D/K210E/Q272L, K36I/T132D/T248A/Q272L, K36I/N159D/K210E/T248A, K36I/N159D/K210E/Q272L, K36I/N159D/T248A/Q272L, K36I/K210E/T248A/Q272L, T132D/N159D/K210E/Q272L, T132D/N159D/T248A/Q272L, T132D/K210E/T248A/Q272L, T132D/N159D/K210E/T248A, N159D/K210E/T248A/Q272L, K36I/T132D/N159D/K210E/T248A, K36I/T132D/N159D/K210E/Q272L, K36I/T132D/N159D/T248A/Q272L, K36I/T132D/K210E/T248A/Q272L, K36I/N159D/K210E/T248A/Q272L, T132D/N159D/K210E/T248A/Q272L, K36I/T132D/N159D/K210E/T248A/Q272L, K36I/T132D/N159D/K210E/T248A/Q272L/Q136N/F161V/S196G/E214G/S237C, or Q136N/F161V/S196G/E214G/S237C (mutation of glutamine at position 136 to asparagine, mutation of phenylalanine at position 161 to valine, mutation of serine at position 196 to glycine, mutation of glutamate at position 214 to glycine, and mutation of serine at position 237 to cysteine) respectively. The alcohol dehydrogenase mutants were respectively designated as M1-M64 and S5.

(24) The primers for mutations K36I, T132D, N159D, K210E, T248A, Q272L, Q136N, F161V, S196G, E214G, and S237C are:

(25) TABLE-US-00001 K36I-F: (SEQ ID NO: 3) AGAAGTCAAGACattGCTGAT; K36I-R: (SEQ ID NO: 4) TAACTTATCAGCaatGTCTTG; T132D-F: (SEQ ID NO: 5) GCTTCAATTATGgatCCACATAGA; T132D-R: (SEQ ID NO: 6) TTGTCTATGTGGatcCATAATTGA; N159D-F: (SEQ ID NO: 7) AATGCTTATGAAgatGTCGTT; N159D-R: (SEQ ID NO: 8) AGCAGTAACGAcatCTTCATA; K210E-F: (SEQ ID NO: 9) GAAGACGTCACTgaaAAACTAAAT; K210E-R: (SEQ ID NO: 10) TTCATTTAGTTTttcAGTGACGTC; T248A-F: (SEQ ID NO: 11) GATGTCGCCAAAgcaCACGTTTTG; T248A-R: (SEQ ID NO: 12) ACCCAAAACGTGtgcTTTGGCGAC; Q272L-F: (SEQ ID NO: 13) GGCGCCTTCTCTctgCAAGATATT; Q272L-R: (SEQ ID NO: 14) AACAATATCTTGcagAGAGAAGGC; Q136N-F: (SEQ ID NO: 15) CCACATAGAaatAATGATCCA; Q136N-R: (SEQ ID NO: 16) TGGATCATTATTTCTATGTGG; F161V-F: (SEQ ID NO: 17) TATGAAAATGTCgttACTGCT; F161V-R: (SEQ ID NO: 18) ACAATAAGCAGTAACGACATT; S196G-F: (SEQ ID NO: 19) ACTATCCACCCAggtTTCGTT; S196G-R: (SEQ ID NO: 20) TCCGAAAACGAAACCTGGGTG; E214G-F: (SEQ ID NO: 21) CTAAATggtACTTGTGAAATT; E214G-R: (SEQ ID NO: 22) AATTTCACAAGTACCATTTAG; S237C-F: (SEQ ID NO: 23) AAGACTCACTTCtgtCAATTC; S237C-R: (SEQ ID NO: 24) ATCAATGAATTGACAGAAGTG,

(26) PCR reaction system (50 μL): KOD enzyme (2.5 U/mL) 1.0 μL, template (5-50 ng) 1.0 μL, dNTP 4.0 μL, 10× reaction buffer 5.0 μL, upstream and downstream primers each 1.0 μL, and ddH.sub.2O q.s. to 50 μL.

(27) Conditions for PCR amplification of products: (1) denaturation at 94° C. for 3 min, 10-15 cycles of (2) denaturation at 94° C. for 30 sec, (3) annealing at 54° C. for 30 sec, and (4) extension at 72° C. for 150 sec, and final extension at 72° C. for 10 min. The PCR product was stored at 4° C.

(28) The product after PCR amplification was detected by 1% agarose gel electrophoresis. After the detection, 0.5 μL of a methylation template digestive enzyme (Dpn I) was added to 10 μL of the amplified product, and mixed uniformly by blowing and aspirating with a pipette tip. After reaction for 1 hr at 37° C., the amplified product after treatment with Dpn I was transformed into E. coli BL21(DE3), and then the transformed cells were coated on LB solid medium, and incubated at 37° C. for 8-10 h. 5 transformants were picked up from the LB solid medium, inoculated into LB liquid medium, and incubated at 37° C. for 10 h. Then the plasmid was extracted, and the extracted plasmid was enzymatically cleaved (the verification results are shown in FIGS. 1-4) and sequenced for verification. The plasmid was verified to be correct. That is, recombinant plasmid pET28a-KpADH-1 to recombinant plasmid pET28a-KpADH-65 comprising respectively the genes encoding alcohol dehydrogenase mutants M1-M64 and S5 and recombinant E. coli BL21/pET28a-KpADH-1 to recombinant E. coli BL21/pET28a-KpADH-65 comprising respectively the genes encoding alcohol dehydrogenase mutants M1-M64 and S5 were obtained.

(29) The obtained recombinant E. coli BL21/pET28a-KpADH and recombinant E. coli BL21/pET28a-KpADH-1 to recombinant E. coli BL21/pET28a-KpADH-65 were respectively coated on LB solid medium, and incubated at 37° C. for 8-10 h, to obtain single colonies. A single colony was picked up and inoculated into LB liquid medium, and incubated at 37° C. for 12-14 h to obtain a seed suspension. The seed suspension was inoculated into LB liquid medium in an amount of 2% (v/v), and incubated at 37° C. and 200 rpm until OD.sub.600 reached 0.8. IPTG was added to at a final concentration of 0.2 mM, and the induction culture was continued for 8 h at 25° C. to obtain a fermentation broth. The fermentation broth was centrifuged at 4° C. and 8000 rpm for 10 min, and the cells were collected. The collected cells were suspended in potassium phosphate buffer (100 mmol.Math.L.sup.−1, pH 6.0) and ultrasonically homogenized. A cell homogenate supernatant containing respectively wild-type alcohol dehydrogenase, alcohol dehydrogenase mutants M1-M64 or alcohol dehydrogenase mutant S5 was collected.

(30) The obtained cell homogenate supernatant was purified by running through an affinity column HisTrap FF crude (nickel column). The purification process was as follows. The nickel column was equilibrated with buffer A (20 mmol.Math.L.sup.−1 sodium phosphate, 500 mmol.Math.L.sup.−1 NaCl, 20 mmol.Math.L.sup.−1 imidazole, pH 7.4), and the cell homogenate supernatant obtained in Example 1 was allowed to run through the nickel column. The proteins that were not bound to the nickel column were eluted off using buffer A. After the flow-through peak ran out, elution with a gradient from buffer A to buffer B (20 mmol.Math.L.sup.−1 sodium phosphate, 500 mmol.Math.L.sup.−1 NaCl, 500 mmol.Math.L.sup.−1 imidazole, pH 7.4) was carried out, to elute off the recombinant protein binding to the nickel column. In this way, a pure enzyme solution of wild-type alcohol dehydrogenase, alcohol dehydrogenase mutants M1-M64, or alcohol dehydrogenase mutant S5 was obtained.

(31) The obtained pure enzyme solution of alcohol dehydrogenase mutants M1-M64 or alcohol dehydrogenase mutant S5 was analyzed by SDS-PAGE. The analysis results are shown in FIG. 5.

(32) It can be seen from FIG. 5 that the pure enzyme solution of alcohol dehydrogenase mutants M1-M64 or alcohol dehydrogenase mutant S5 shows a single band at about 45 kDa, and contains less protein impurities, indicating that the purification effect of the nickel column is good.

Example 2: Thermal Stability of Alcohol Dehydrogenase Mutants

(33) The pure enzyme solution of wild-type alcohol dehydrogenase, alcohol dehydrogenase mutants M1-M64 or alcohol dehydrogenase mutant S5 obtained in Example 1 was allowed to stand for 15 min in a water bath at a temperature of 40-60° C. respectively. After 15 min, the enzyme activity of the wild-type alcohol dehydrogenase, alcohol dehydrogenase mutants M1-M64 and alcohol dehydrogenase mutant S5 was determined. The relative activity was calculated by the percentages of the remaining activity after standing in the water bath relative to the activity before standing in the water bath at different temperatures taken as 100%, to determine the T.sub.50.sup.15 values of the wild-type alcohol dehydrogenase, alcohol dehydrogenase mutants M1-M64 and alcohol dehydrogenase mutant S5 (where T.sub.50.sup.15 is the temperature at which the activity of the alcohol dehydrogenase after standing 15 min in the water bath is half of the activity before standing in the water bath. The test results are shown in Table 2).

(34) The pure enzyme solution of wild-type alcohol dehydrogenase, alcohol dehydrogenase mutants M1-M64 or alcohol dehydrogenase mutant S5 obtained in Example 1 was allowed to stand in a water bath at a temperature of 30-45° C. respectively. A sample was taken periodically to determine the activity of wide-type alcohol dehydrogenase, alcohol dehydrogenase mutants M1-M64 and alcohol dehydrogenase mutant S5. The relative activity was calculated by the percentages of the remaining activity after standing in the water bath relative to the activity before standing in the water bath at different temperatures taken as 100%, to determine the half-life t.sub.1/2 of wide-type alcohol dehydrogenase, alcohol dehydrogenase mutants M1-M64 and alcohol dehydrogenase mutant S5 (where the half-life t.sub.1/2 is the time over which the activity is halved upon standing at a certain temperature). A fitted inactivation curve was plotted from the half-life of wide-type alcohol dehydrogenase, alcohol dehydrogenase mutants M1-M64 and alcohol dehydrogenase mutant S5 at different temperatures according to the Arrhennius equation, and the deactivation activation energy E.sub.d of wide-type alcohol dehydrogenase, alcohol dehydrogenase mutants M1-M64 and alcohol dehydrogenase mutant S5 was calculated (the calculation results as shown in Table 2).

(35) The calculation formula of deactivation activation energy is as follows:

(36) $E_d=\text{-}\mathrm{RT}\left(\mathrm{lnk}-\ln A\right);$$k=\frac{\ln 2}{t_{1,2}};$

(37) where R: molar gas constant; T: corresponding temperature; k: deactivation rate, t.sub.1/2: half-life at corresponding temperature, and A: pre-exponential factor.

(38) It can be seen from Table 2 that the thermal stability of alcohol dehydrogenase mutants M1-M64 is significantly improved compared with the wild-type alcohol dehydrogenase. Among them, the alcohol dehydrogenase mutant M63 has the most excellent thermal stability, has a T.sub.50.sup.15 and an E.sub.d reaching 53.1° C. and 989.13 kJ/mol respectively, which are 11.1° C. and 32.06 kJ/mol higher than the wild type; and has a half-life t.sub.1/2 at 45° C. that is 3000 times that of the wild-type alcohol dehydrogenase. The thermal stability of alcohol dehydrogenase mutant S5 decreases obviously compared with the wild-type alcohol dehydrogenase.

(39) TABLE-US-00002 TABLE 2 T.sub.50.sup.15, E.sub.d, and t.sub.1/2 at 45° C. of wild-type alcohol dehydrogenase, alcohol dehydrogenase mutants M1-M64 and alcohol dehydrogenase mutant S5 T.sub.50.sup.15 Ed t.sub.1/2 No. ° C. kJ/mol min Wild type .sup. 42 ± 0.1 957.07 1.1 M1 43.5 ± 0.2 959.95 nd M2 44.7 ± 0.2 965.39 nd M3 43.9 ± 0.1 960.71 nd M4 43.8 ± 0.2 960.56 nd M5 45.8 ± 0.3 968.26 nd M6 43.6 ± 0.2 960.10 nd M7 45.6 ± 0.1 966.78 nd M8 .sup. 44 ± 0.3 962.23 nd M9 45.2 ± 0.2 965.57 nd M10 46.8 ± 0.3 970.42 nd M11 44.5 ± 0.2 963.45 nd M12 44.6 ± 0.2 963.90 nd M13 46.6 ± 0.3 970.06 nd M14 47.5 ± 0.1 972.79 nd M15 45.8 ± 0.2 967.99 nd M16 44.4 ± 0.1 963.29 nd M17 45.8 ± 0.2 967.84 nd M18 .sup. 44 ± 0.3 962.23 nd M19 .sup. 47 ± 0.2 971.27 nd M20 45.5 ± 0.2 966.78 nd M21 .sup. 47 ± 0.3 971.27 nd M22 45.8 ± 0.2 967.39 nd M23 47.6 ± 0.4 972.54 nd M24 49.1 ± 0.3 977.65 nd M25 46.9 ± 0.1 970.72 nd M26 45.7 ± 0.1 967.23 nd M27 46.9 ± 0.3 970.72 nd M28 45.2 ± 0.3 965.87 nd M29 48.5 ± 0.2 975.83 nd M30 46.6 ± 0.2 969.66 nd M31 48.5 ± 0.3 975.83 nd M32 46.4 ± 0.4 969.36 nd M33 .sup. 48 ± 0.3 973.45 nd M34 46.4 ± 0.2 968.60 nd M35 .sup. 49 ± 0.1 977.34 nd M36 47.8 ± 0.1 973.30 nd M37 .sup. 49 ± 0.3 977.34 nd M38 47.9 ± 0.3 973.15 nd M39 46.7 ± 0.2 966.48 nd M40 47.5 ± 0.3 971.93 nd M41 49.1 ± 0.1 976.63 nd M42 48.2 ± 0.2 974.05 nd M43 49.1 ± 0.3 976.78 nd M44 47.4 ± 0.4 971.63 nd M45 51.1 ± 0.2 983.7 nd M46 48.9 ± 0.2 976.48 nd M47 50.6 ± 0.1 981.33 nd M48 49.2 ± 0.2 977.39 nd M49 46.4 ± 0.1 968.90 nd M50 48.8 ± 0.1 976.02 nd M51 50.3 ± 0.3 980.87 nd M52 49.7 ± 0.3 979.21 nd M53 47.8 ± 0.1 976.32 nd M54 49.3 ± 0.4 978.26 nd M55 50.9 ± 0.2 983.11 nd M56 48.9 ± 0.2 976.48 nd M57 51.3 ± 0.3 984.33 nd M58 48.7 ± 0.1 976.43 nd M59 50.7 ± 0.3 982.50 nd M60 52.4 ± 0.4 986.93 nd M61 50.5 ± 0.2 981.63 nd M62 51.3 ± 0.1 984.21 nd M63 53.1 ± 0.4 989.13 3000 M64 51.5 ± 0.1 984.14 2400 S5 .sup. 41 ± 0.4 953.10 0.9 nd: not detected.

Example 3: Kinetic Parameters and Stereoselectivity in the Production of Chiral Diaryl Alcohol (4-Chlorophenyl)-(Pyridin-2-Yl)-Methanol by the Asymmetric Reduction of Prochiral Diaryl Ketone (4-Chlorophenyl)-(Pyridin-2-Yl)-Methanone by Alcohol Dehydrogenase Mutants

(40) The initial reduction activity of wild-type alcohol dehydrogenase and alcohol dehydrogenase mutants M63, M64 and S5 obtained in Example 1 were respectively determined with 0.1-5 mM prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone as a substrate. The data was fitted by the nonlinear regression method in Graph Pad Prism 7.0 software, to obtain the K.sub.m and V.sub.max in Michaelis-Menten equation. Then k.sub.cat and k.sub.cat/K.sub.m were calculated. The calculation results are shown in Table 3.

(41) k.sub.cat is calculated by the formula k.sub.cat=V.sub.max.Math.M/60, where M is the molecular weight of the enzyme, in kDa.

(42) It can be known from Table 3 that the catalytic efficiency of alcohol dehydrogenase mutant M63 in the asymmetric reduction of prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone to produce (R)-(4-chlorophenyl)-(pyridin-2-yl)-methanol is obviously improved compared with the wild-type alcohol dehydrogenase, and is 1.3 times that of the wild-type alcohol dehydrogenase. The catalytic efficiency of alcohol dehydrogenase mutant M64 in the asymmetric reduction of prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone to produce (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol is close to that of the wild type, but is 1.35 times that of the alcohol dehydrogenase mutant S5. The catalytic efficiency of alcohol dehydrogenase mutant S5 in the asymmetric reduction of prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone to produce (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol is decreased compared with the wild type.

(43) The stereoselectivity of wild-type alcohol dehydrogenase and alcohol dehydrogenase mutants M63, M64 and S5 obtained in Example 1 in the asymmetric reduction of prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone to produce chiral diaryl alcohol (4-chlorophenyl)-(pyridin-2-yl)-methanol was detected. The detection results are shown in Table 3.

(44) It can be known from Table 3 that the stereoselectivity of alcohol dehydrogenase mutants M63, M64 and S5 in the asymmetric reduction of prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone to produce chiral diaryl alcohol (4-chlorophenyl)-(pyridin-2-yl)-methanol is not decreased compared with the wild-type alcohol dehydrogenase. Moreover, it can be known from Table 3 that the wild-type alcohol dehydrogenase can asymmetrically reduce the prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone to produce (R)-(4-chlorophenyl)-(pyridin-2-yl)-methanol, with an e.e. of 87.1% (R). Compared with the wild-type alcohol dehydrogenase, the alcohol dehydrogenase mutant M64 has reversed stereoselectivity in the asymmetric reduction of prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone to produce chiral diaryl alcohol (4-chlorophenyl)-(pyridin-2-yl)-methanol, and can asymmetrically reduce the prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone to produce (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol, with an e.e. value of up to 97.3% (S).

(45) TABLE-US-00003 TABLE 3 Kinetic parameters and stereoselectivity in the production of chiral diaryl alcohol (4-chlorophenyl)-(pyridin-2-yl)-methanol by the asymmetric reduction of prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone by the wild-type alcohol dehydrogenase and alcohol dehydrogenase mutants M63, M64 and S5 K.sub.m V.sub.max k.sub.cat k.sub.cat/K.sub.m e.e. No. (mM) (μmol .Math. min.sup.−1 .Math. mg.sup.−1) (s.sup.−1) (s.sup.−1 .Math. mM.sup.−1) (%) Wild 0.95 ± 0.1 31.75 ± 1.4 20.09 ± 1.0 21.15 87.1 (R) type S5 1.09 ± 0.1 24.44 ± 2.6 16.09 ± 1.7 14.76 97.3 (S).sup.  M63 0.83 ± 0.2 32.83 ± 3    22.8 ± 2.14 27.47 87.2 (R) M64  0.86 ± 0.14 25.12 ± 1.6 17.44 ± 1.1 20.27 97.2 (S).sup. 

Example 4. Conversion Efficiency in the Production of (S)-(4-Chlorophenyl)-(Pyridin-2-Yl)-Methanol by the Asymmetric Reduction of Prochiral Diaryl Ketone (4-Chlorophenyl)-(Pyridin-2-Yl)-Methanone by Alcohol Dehydrogenase Mutant

(46) The wild-type alcohol dehydrogenase and alcohol dehydrogenase mutant M64 obtained in Example 1 were added in an amount of 7 g/L respectively to 100 mM potassium phosphate buffer (pH 7.0) containing 100 mM, 200 mM, or 500 mM prochiral diaryl ketone(4-chlorophenyl)-(pyridin-2-yl)-methanone, reacted at 30° C., pH 7.0, and 200 rpm for 12 h to obtain a reaction solution. In addition to the prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone, the potassium phosphate buffer further contained 0.02 mM coenzyme NADP.sup.+, 1.5 mM glucose, 1.5 mM glucose dehydrogenase GDH and 5% (v/v) ethanol.

(47) The conversion rates in the asymmetric reduction of prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone to produce (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol by the wild-type alcohol dehydrogenase and alcohol dehydrogenase mutant M64 at different reaction times were detected, and the conversion efficiency (that is, space time yield) in the asymmetric reduction of prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone to produce (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol by the wild-type alcohol dehydrogenase and alcohol dehydrogenase mutant M64 was calculated. The detection results were shown in Tables 4-5.

(48) It can be known from Tables 4-5 that in case of 100 mM substrate, the wild-type alcohol dehydrogenase and alcohol dehydrogenase mutant M64 achieve a conversion rate of >99.9% respectively at 4 h and 2 h of reaction. Therefore, compared with the wild-type alcohol dehydrogenase, the alcohol dehydrogenase mutant M64 enables an obviously improved conversion efficiency in the asymmetric reduction of prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone to produce (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol. Also, it can be known from Tables 4-5 that when 500 mM prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone is added, the wild-type alcohol dehydrogenase fails to convert it completely, but the conversion efficiency enabled by the alcohol dehydrogenase mutant M64 is not affected by high concentration of prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone. It can be seen that the alcohol dehydrogenase mutant M64 is adapted to an environment with a high concentration of prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone, and can asymmetrically reduce the high concentration of prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone to produce (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol with a high conversion efficiency (that is, space time yield) that is up to 651 g/(L.Math.d).

(49) TABLE-US-00004 TABLE 4 Conversion rate in the production of (S)-(4-chlorophenyl)- (pyridin-2-yl)-methanol by the asymmetric reduction of prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2- yl)-methanone by the wide-type alcohol dehydrogenase Reaction time Conversion rate (%) (h) 100 mM 200 mM 500 mM 0.5 60.5 35.6 19.7 1 95.5 56.9 24.1 2 97.5 82.0 26.2 3 99.8 95.5 27.6 4 >99.9 98.6 28 6 >99.9 >99.9 28.1 8 >99.9 >99.9 28.1 12 >99.9 >99.9 28.2

(50) TABLE-US-00005 TABLE 5 Conversion rate in the production of (S)-(4-chlorophenyl)- (pyridin-2-yl)-methanol by the asymmetric reduction of prochiral diaryl ketone (4-chlorophenyl)-(pyridin-2-yl)- methanone by the alcohol dehydrogenase mutant M64 Reaction time Conversion rate (%) (h) 100 mM 200 mM 500 mM 0.5 95 81.3 43 1 98.3 95.2 58.6 2 >99.9 98.9 86.2 3 >99.9 >99.9 98.3 4 >99.9 >99.9 99.3 6 >99.9 >99.9 >99.9 8 >99.9 >99.9 >99.9 12 >99.9 >99.9 >99.9

(51) While the present invention has been described above by way of preferred examples, the present invention is not limited thereto. Various modifications and changes can be made by those skilled in the art without departing from the spirit and scope of the present invention as defined by the appended claims.