WO2021057014A1 - Kluyveromyces marxianus aldo-keto reductase kmakr mutant and application thereof - Google Patents

Kluyveromyces marxianus aldo-keto reductase kmakr mutant and application thereof Download PDF

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WO2021057014A1
WO2021057014A1 PCT/CN2020/085538 CN2020085538W WO2021057014A1 WO 2021057014 A1 WO2021057014 A1 WO 2021057014A1 CN 2020085538 W CN2020085538 W CN 2020085538W WO 2021057014 A1 WO2021057014 A1 WO 2021057014A1
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bacteria
tert
mutant
kmakr
wet
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王亚军
邱帅
李树芳
程峰
翁春跃
郑裕国
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浙江工业大学
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group

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  • the present invention relates to the construction of a mutant of the aldehyde ketone reductase KmAKR derived from Kluyveromyces marxianus, and the development of aldehyde ketone reductase recombinant bacteria and enzymes in atorvastatin, rosuvastatin, pitavastatin and other "super statins" Application of side-chain chiral diol 6-substituted-(3R,5R/S)-dihydroxyhexanoate in chiral biocatalytic synthesis.
  • Atorvastatin, rosuvastatin, pitavastatin and other "super statins” are major lipid-lowering drugs for the treatment of cardiovascular and cerebrovascular diseases. They have high-efficiency lipid-lowering effects, long-term safety and clinical benefits, and significantly reduce cardiovascular and cerebrovascular diseases. Morbidity and mortality of diseases. So far, the cumulative sales of atorvastatin calcium has exceeded 100 billion US dollars, which is the most successful single drug variety in the history of the human pharmaceutical industry.
  • statins contain 6-substituted-(3R,5R/S)-dihydroxyhexanoic acid tert-butyl structure, which is not only an important pharmacodynamic group but also a key synthetic precursor.
  • 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl ester is the key chiral diol intermediate of atorvastatin calcium.
  • 6-Chloro-(3R,5S)-dihydroxyhexanoic acid tert-butyl ester is the synthetic precursor of "super statin" drugs such as rosuvastatin and pitavastatin.
  • the synthetic methods of 6-substituted-(3R,5R/S)-dihydroxyhexanoic acid tert-butyl ester that have been reported in the literature mainly include chemical catalysis 6-substituted-(5R/S)-hydroxy-3-carbonyl hexanoic acid tert-butyl ester
  • Asymmetric reduction and oxidoreductase differentially selectively reduce 6-substituted-(5R/S)-hydroxy-3-carbonylhexanoate tert-butyl ester.
  • Catalytic reduction processes with chemical catalysts such as borane have the disadvantages of high energy consumption, low conversion rate, low differential selectivity and high production cost.
  • enzymes as green natural biocatalysts, have the advantages of superior chemical selectivity, stereoselectivity and regioselectivity in catalyzing chemical reactions, with mild reaction conditions, few by-products, and environmental friendliness.
  • enzyme molecules when many enzyme molecules catalyze unnatural substrates, they often have problems such as low activity, poor stability, and inhibition of substrate products, so molecular modification of enzyme molecules is urgently needed.
  • the present invention establishes a high-throughput screening model, constructs a large-capacity mutant library, and screens and obtains robust super mutants, among which the "best"
  • the mutant KmAKR-W297H/Y296W/K29H/Y28A/T63M has the strongest catalytic performance.
  • the molecular mechanism of the improvement of the mutant's catalytic performance was further analyzed, the reaction process parameters were optimized, and the KmAKR-W297H/Y296W/K29H/Y28A/T63M catalytic synthesis was constructed. -Substituted-(3R,5R/S)-dihydroxyhexanoic acid tert-butyl ester process.
  • the purpose of the present invention is to solve the problems of low asymmetric reduction activity of 6-substituted-(5R/S)-hydroxy-3-carbonylhexanoate tert-butyl ester and low substrate feeding amount of the existing aldehyde and ketone reductase, and provide a solution Stereoselective aldehyde-ketone reductase series mutants and recombinant bacteria using the aldehyde-ketone reductase mutant or its crude enzyme solution as a catalyst for asymmetric reduction to synthesize 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert Butyl ester, 6-chloro-(3R,5S)-dihydroxyhexanoic acid tert-butyl ester and other chiral alcohol compounds, the catalyst activity is 4.1 times higher than that of KmAKR-W297H/Y296W/K29H/Y28A, and the substrate dosage is increased to
  • the present invention provides a Kluyveromyces marxianus aldehyde ketone reductase KmAKR mutant, which is obtained by site-directed saturation mutation at position 63 of the amino acid sequence shown in SEQ ID NO. 2, preferably the mutation
  • the body is one of the following: (1) Threonine at position 63 is mutated to alanine; (2) Threonine at position 63 is mutated to leucine; (3) Threonine at position 63 is mutated to methionine.
  • the nucleotide sequence of the coding gene corresponding to the amino acid sequence shown in SEQ ID NO. 2 is shown in SEQ ID NO. 1.
  • the present invention also provides an application of the aldehyde and ketone reductase KmAKR mutant in the preparation of chiral alcohols by asymmetric reduction of carbonyl compounds.
  • the specific application method is: the engineered bacteria containing the aldehyde and ketone reductase KmAKR mutant gene
  • the bacterial cells obtained by induction culture and the bacterial cells obtained by induction culture of engineered bacteria containing the glucose dehydrogenase gene are mixed, using the mixed bacteria or the crude enzyme solution extracted from the mixed bacteria as the catalyst, and the carbonyl compound as the substrate.
  • Glucose is the auxiliary substrate, and the conversion system is formed with pH 7.0, 100 mM PBS buffer as the reaction medium, and the reaction is carried out at 30-35° C. and 400-600 rpm. After the reaction is completed, the reaction solution is separated and purified to obtain the chiral alcohol compound.
  • the final concentration of substrate is 30-450g/L (preferably 200-400g/L)
  • the final concentration of glucose is 30-450g/L (preferably 200-400g/L)
  • the amount of catalyst is based on the total amount of mixed bacteria.
  • the dry weight is 0.1-20g DCW/L (the dry weight of DCW cells, preferably 20g DCW/L)
  • the wet bacterial cells obtained by induction culture of the engineered bacteria containing the glucose dehydrogenase gene are mixed at a dry weight ratio of 1.0-5.0:1 (w/w), preferably 3.5:1.
  • the glucose dehydrogenase gene (GenBank NO. KM817194.1) is from Exiguobacterium sibirium DSM 17290.
  • the carbonyl compound is one of the following: ethyl 4-chloro-3-carbonylbutyrate, tert-butyl 6-chloro-(5S)-hydroxy-3-carbonylhexanoate, ethyl 3-carbonylbutyrate, 4- Propyl bromo-3-carbonylbutyrate, 4,4,4,-trifluoro-3-carbonylbutyric acid ethyl ester, 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester, 3- Tert-Butyl carbonylbutyrate, acetophenone, preferably tert-butyl 6-chloro-(5S)-hydroxy-3-carbonylhexanoate, ethyl 3-carbonylbutyrate, 6-cyano-(5R)-hydroxy- Tert-Butyl 3-carbonylhexanoate.
  • the aldehyde and ketone reductase KmAKR mutant asymmetrically reduces 6-cyano-(5R)-
  • the method for preparing tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate from tert-butyl hydroxy-3-carbonylhexanoate is as follows: the engineered bacteria containing the aldehyde ketone reductase mutant gene are obtained by inducing cultivation The bacteria are mixed with the bacteria obtained by induction culture of the engineered bacteria containing the glucose dehydrogenase gene, using the mixed bacteria as the catalyst and 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester as the substrate The conversion system is constructed with glucose as the auxiliary substrate, and the pH 7.0, 100mM PBS buffer is used
  • the aldehyde and ketone reductase KmAKR mutant asymmetrically reduces 6-chloro-(5S)-hydroxy-
  • the method for preparing tert-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate from 3-carbonyl hexanoate tert-butyl ester is as follows: the bacteria containing the aldehyde ketone reductase KmAKR mutant gene are induced and cultured.
  • the bacteria and the bacteria obtained by the induction culture of the engineered bacteria containing the glucose dehydrogenase gene are mixed, using the mixed bacteria as the catalyst and 6-chloro-(5S)-hydroxy-3-carbonylhexanoate tert-butyl as the substrate,
  • the conversion system was constructed with glucose as the auxiliary substrate and pH 7.0, 100mM PBS buffer as the reaction medium. The reaction was carried out at 30°C and 400-600rpm. After the reaction was completed, the reaction solution was separated and purified to obtain 6-chloro-(3R). ,5S)-tert-butyl dihydroxyhexanoate.
  • the wet bacteria are prepared as follows: inoculate the engineered Escherichia coli bacteria carrying the aldehyde ketone reductase mutant gene into LB liquid medium containing a final concentration of 50 ⁇ g/mL kanamycin, and cultivate for 10 hours at 37°C, Inoculate 1.5% of the volume concentration into a fresh LB liquid medium containing a final concentration of 50 ⁇ g/mL kanamycin, incubate at 37°C and 180rpm for 2h, and then add isopropyl to the culture solution to a final concentration of 0.15mM Isopropyl ⁇ -D-thiogalactoside (IPTG), cultured at 28°C for 12 hours, and centrifuged at 4°C and 8000 rpm for 10 minutes to obtain wet cells containing the aldehyde ketone reductase mutant; the glucose dehydrogenase gene
  • IPTG Isopropyl ⁇ -D-thiogalactoside
  • the crude enzyme solution of the present invention is prepared as follows: the total amount of wet bacteria is 100g/L, resuspended in a pH 7.0, 100mM PBS buffer, and ultrasonically broken on an ice-water mixture for 6 minutes.
  • the ultrasonic breaking conditions are as follows: 400W, break for 1s, pause for 1s, take the broken mixture to obtain the crude enzyme solution.
  • the base sequence of the aldehyde-ketone reductase KmAKR and the aldehyde-ketone reductase KmAKR mutant of the present invention are both 933 bp in length, starting from the first base to the 933th base, the start codon is ATG, and the stop codon is TGA.
  • the KmAKR mutant of the aldehyde and ketone reductase of the present invention is obtained by using the site-specific saturation mutagenesis technology.
  • the KmAKR-W297H/Y296W/K29H/Y28A aldehyde ketone reductase gene (SEQ ID NO.1) is mutated using this technology to obtain
  • the mutant plasmid was introduced into E. coli BL21(DE3) competent cells by heat shock, and the obtained strains were inoculated, transferred, cultivated, induced culture, and bacteria were recovered.
  • the resuspended bacteria liquid was used to catalyze 6-cyano-(5R).
  • the specific method is as follows: The first step is to activate the control bacteria. Obtained the control bacteria E. coli BL21(DE3)/pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A, extracted the plasmid pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A, and stored it at -20°C .
  • the second step is to compare SWISS-MODEL with KmAKR-W297H/Y296W/K29H/Y28A to obtain the template protein crystal structure of homology modeling, use Modeller 9.19 homology modeling, perform molecular docking, and select the appropriate mutation site.
  • the selected sites are mainly the amino acid residues located near the active center and the amino acid residues on the loop loop of the active center.
  • the mutant primers are designed, using pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A as the template plasmid.
  • the mutant plasmid is obtained, and transformed, and the dominant mutant strain is screened to obtain the aldehyde ketone reductase mutant KmAKR-Y296W/W297H/K29H/Y28A/T63A (denoted as M5-A, KmAKR-Y296W/W297H/K29H) /Y28A/T63L (denoted as M5-L), KmAKR-Y296W/W297H/K29H/Y28A/T63M (denoted as M5-M), sample the superior mutants for sequencing and save.
  • the culture medium can be any medium in the art that can grow bacteria and produce the culture medium of the present invention, preferably LB Medium: tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, dissolved in distilled water, adjust pH 7.0.
  • LB Medium tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, dissolved in distilled water, adjust pH 7.0.
  • the culture method and conditions can be appropriately selected according to the host type and culture method and other factors according to common knowledge in the field.
  • the main beneficial effects of the present invention are mainly reflected in: the specific enzyme activities of the aldone reductase mutants M5-A, M5-L and M5-M constructed in the present invention are higher than those of the control group Increased by 1.1 times, 3.2 times, 4.1 times.
  • the mutant KmAKR-Y296W/W297H/K29H/Y28A/T63M, the largest substrate 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester can be charged up to 450g/L, and the product concentration changes with time.
  • Figure 1 shows the coupling of aldehyde ketone reductase and glucose dehydrogenase EsGDH to catalyze the asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester to prepare 6-cyano-(3R,5R)- Schematic diagram of the reaction of tert-butyl dihydroxyhexanoate.
  • Figure 2 shows the coupling of aldehyde ketone reductase and glucose dehydrogenase EsGDH to catalyze the asymmetric reduction of 6-chloro-(5S)-hydroxy-3-carbonylhexanoate tert-butyl ester to prepare 6-chloro-(3R,5S)-dihydroxyl
  • Figure 2 shows the coupling of aldehyde ketone reductase and glucose dehydrogenase EsGDH to catalyze the asymmetric reduction of 6-chloro-(5S)-hydroxy-3-carbonylhexanoate tert-butyl ester to prepare 6-chloro-(3R,5S)-dihydroxyl
  • FIG. 3 SDS-PAGE electrophoresis diagram of the supernatant of the aldehyde ketone reductase mutant and the pure enzyme.
  • Lane 1 Control KmAKR-W297H/Y296W/K29H/Y28A supernatant;
  • Lane 2 Control KmAKR-W297H/Y296W/K29H/Y28A pure enzyme;
  • Lane 3 KmAKR-W297H/Y296W/K29H/Y28A/T63A supernatant
  • Lane 4 KmAKR-W297H/Y296W/K29H/Y28A/T63A pure enzyme;
  • Lane 5 KmAKR-W297H/Y296W/K29H/Y28A/T63L supernatant;
  • Lane 6 KmAKR-W297H/Y296W/K29H/Y28A/T63L Pure enzyme;
  • Lane 7 KmAKR-W297H/Y2
  • Figure 4 is the use of aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A coupled with EsGDH asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester to prepare 6-cyano- Time course of (3R,5R)-dihydroxyhexanoate tert-butyl ester.
  • Figure 5 is the use of aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63A coupled with EsGDH asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonyl hexanoate tert-butyl ester to prepare 6-cyano Time course graph of tert-butyl dihydroxy-(3R,5R)-dihydroxyhexanoate.
  • Figure 6 is the use of aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63L coupled with EsGDH asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester to prepare 6-cyano Time course graph of tert-butyl dihydroxy-(3R,5R)-dihydroxyhexanoate.
  • Figure 7 is the use of aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63M coupled with EsGDH asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester to prepare 6-cyano Time course graph of tert-butyl dihydroxy-(3R,5R)-dihydroxyhexanoate.
  • Figure 8 is the use of aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63A coupled with EsGDH asymmetric reduction of 6-chloro-(5S)-hydroxy-3-carbonylhexanoate tert-butyl ester to prepare 6-chloro- Time course of (3R,5S)-dihydroxyhexanoate tert-butyl ester.
  • Figure 9 is the use of aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63L coupled with EsGDH asymmetric reduction of 6-chloro-(5S)-hydroxy-3-carbonyl hexanoic acid tert-butyl ester to prepare 6-chloro- Time course of (3R,5S)-dihydroxyhexanoate tert-butyl ester.
  • Figure 10 is the use of aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63M coupled with EsGDH asymmetric reduction of 6-chloro-(5S)-hydroxy-3-carbonylhexanoate tert-butyl ester to prepare 6-chloro- Time course of (3R,5S)-dihydroxyhexanoate tert-butyl ester.
  • Example 1 Construction and screening of aldehyde ketone reductase mutant library
  • KmAKR-W297H/Y296W/K29H/Y28A/T63M Marked as M5-M, that is, threonine at position 63 of the amino acid shown in SEQ ID NO. 2 is mutated to methionine.
  • site-specific saturation mutations were performed at positions 22 and 95, but none of the mutant strains with increased viability were obtained.
  • PCR reaction system 50 ⁇ L: 1 ⁇ L forward primer (100 ⁇ M), 1 ⁇ L reverse primer (100 ⁇ M), 25 ⁇ L 2 ⁇ Phanta buffer, 1 ⁇ L dNTP mixture (each 10mM), 1 ⁇ L plasmid template, 1 ⁇ L DNA polymerase and 21 ⁇ L ultrapure water.
  • the PCR program set up according to the Phanta Super-Fidelity DNA Polymerase Manual is as follows: 95°C pre-denaturation for 5 minutes, then 29 cycles (95°C denaturation for 15s, 55°C annealing for 15s, 72°C extension for 7s), 72°C final extension for 10 minutes, 16°C Keep warm. The obtained recombinant plasmid was transferred to E.
  • the obtained mutants were screened for dominant mutants, and the screening conditions were as follows: dry weight 25g/L (dry weight ratio of aldehyde ketone reductase mutant and glucose dehydrogenase bacteria to 3.5:1 (w/w) Resuspend the cells in PBS (100mM) at pH 7.0, and then add a final concentration of 50g/L 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester, 50g/L glucose to form a transformation system 10mL, at 35 The reaction is carried out under the conditions of °C and 600 rpm.
  • Example 2 Induced expression of aldehyde ketone reductase, mutants and glucose dehydrogenase in the control group
  • Glucose dehydrogenase genetically engineered bacteria Insert the 17290 glucose dehydrogenase gene from E.sibirium DSM (GenBank NO.KM817194.1) into pET28b(+) to construct a recombinant expression vector, and transfer this expression vector into E.coli BL21 (DE3) E. coli BL21(DE3)/pET28b(+)-esgdh was prepared.
  • coli BL21(DE3)/pET28b (+)-esgdh were respectively inoculated into LB liquid medium containing a final concentration of 50 ⁇ g/mL kanamycin, cultured at 37°C for 10 hours, and inoculated with a volume concentration of 1.5% (v/v) to freshly contain the final concentration In 50 ⁇ g/mL kanamycin LB liquid medium, incubate at 37°C and 180rpm for 2h, then add IPTG to the culture broth with a final concentration of 0.15mM IPTG. After incubation at 28°C for 12h, centrifuge at 4°C and 8000rpm for 10min to obtain the corresponding Wet bacterial cells.
  • the cells obtained above produce the corresponding protein, which can be used for the preparation of pure protein enzyme solution, and can also be used for the preparation of crude enzyme solution to catalyze asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl 6 -Cyano-(3R,5R)-dihydroxyhexanoate tert-butyl ester and catalytic asymmetric reduction of 6-chloro-(5S)-hydroxy-3-carbonylhexanoate tert-butyl ester to prepare 6-chloro-(3R,5S) -Tert-Butyl dihydroxyhexanoate.
  • the wet cells of the mutant strain and the wet cells of glucose dehydrogenase induced in Example 2 were mixed with a dry weight ratio of 3.5:1 (w/w) to form a mixed cell, and resuspended in a pH 7.0, 100mM PBS buffer. Obtain a mixed bacterial solution of the mutant strain.
  • the control strain E. coli BL21(DE3)/pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A was used to replace the wet bacteria of the mutant strain to prepare a mixed bacterial solution of the control strain.
  • the mixed bacterial liquid of the mutant strain and the mixed bacterial liquid of the control strain were used as catalysts, 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester was used as the substrate, glucose was used as the auxiliary substrate, and no external substances were added.
  • Source NADPH or NADP + use the endogenous NADPH of bacterial cells to establish a coenzyme circulation system.
  • the reaction system is 10mL, the amount of catalyst is 20g/L based on the total dry weight of the mixed bacteria, the final concentration of substrate is 30g/L, the final concentration of glucose is 30g/L, pH 7.0, 100mM PBS buffer is used as the reaction medium to construct the conversion system, 30 °C, 600rpm for 20 minutes to take samples, take 100 ⁇ L of the reaction solution and add 900 ⁇ L of absolute ethanol to precipitate the protein, that is, the reaction solution is diluted 10 times, -20°C overnight, centrifuged at 12000rpm for 3min, take the supernatant, pass through a 0.22 ⁇ m microfiltration membrane, as a liquid phase sample , HPLC detects 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl, 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl, 6-cyano-(3S, 5R) The amount and de p value of tert-but
  • Liquid phase detection conditions chromatographic column C18 (4.6 ⁇ 250mm, Acchrom, China) column, mobile phase acetonitrile: water volume ratio 1:3, flow rate 1.0mL/min, detection wavelength 210nm, injection volume 10 ⁇ L, column temperature 40°C.
  • the retention times of 6-cyano-(5R)-hydroxy-3-carbonylhexanoic acid tert-butyl ester and 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester were 13.9min and 9.8min, respectively.
  • Example 2 The superior mutants obtained in Example 3 (KmAKR-W297H/Y296W/K29H/Y28A/T63A, KmAKR-W297H/Y296W/K29H/Y28A/T63L, KmAKR-W297H/Y296W/K29H/Y28A/T63M in Table 2), According to the method described in Example 2, the wet cells of the aldehyde ketone reductase mutant were obtained, and the cells were collected by centrifugation at 8000 rpm and 4° C. for 10 minutes, and washed twice with 0.9% (w/v) saline.
  • the protein size was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
  • the pure aldehyde ketone reductase enzyme of the control strain E. coli BL21(DE3)/pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A was collected under the same conditions.
  • the electrophoresis results are shown in Figure 3.
  • the target enzyme expression level of the mutant strain Compared with E. coli BL21(DE3)/pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A strain, the enzyme expression level has no significant change. Therefore, the increase in the enzyme activity of the mutant is not caused by the increase in the expression level of the enzyme.
  • the enzyme itself is related to the increase in specific activity.
  • Example 5 Determination of the specific enzyme activity of the parent aldehyde ketone reductase and its mutants
  • Enzyme activity unit (U) is defined as: the amount of enzyme required to generate 1 micromole of 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl ester per minute at 35°C and pH 7.0 is defined as A unit of enzyme activity, U. Specific enzyme activity is defined as the number of units of activity per milligram of enzyme protein, U/mg.
  • Standard conditions for enzyme activity detection 5mM 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester, 0.25mM NADPH, appropriate amount of enzyme solution, 35°C, pH 7.0, 500rpm conditions for 3 minutes, sample processing and Perform HPLC detection and analysis.
  • the protein concentration was determined with a quinolinic acid protein determination kit (Nanjing KGI Biotechnology Development Co., Ltd., Nanjing).
  • Example 6 Aldone reductase control group KmAKR-W297H/Y296W/K29H/Y28A asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester
  • the aldehyde ketone reductase control group KmAKR-W297H/Y296W/K29H/Y28A wet bacteria and glucose dehydrogenase EsGDH wet bacteria were obtained through fermentation.
  • the wet bacteria KmAKR-W297H/Y296W/K29H/Y28A and the glucose dehydrogenase EsGDH catalyzed the 6-cyano-(5R)-hydroxy-3-carbonylhexanoic acid tert Butyl ester produces 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl ester.
  • the mixed bacteria with a dry weight ratio of 3.5:1 (w/w).
  • a 50mL reaction system first resuspend the mixed bacteria with pH 7.0, 100mM PBS buffer, and add dry weight to the mixed bacteria in the transformation system. 20g DCW/L, when the substrate 6-cyano-(5R)-hydroxy-3-carbonylhexanoic acid tert-butyl ester is 170g/L, the glucose concentration is 255g/L, and the pH is 7.0, 100mM PBS
  • the buffer is the reaction medium to form the conversion system.
  • Example 7 Asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester by aldehyde-ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63A
  • Example 2 the aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63A wet bacteria and the glucose dehydrogenase EsGDH wet bacteria were obtained through fermentation.
  • the wet bacteria KmAKR-W297H/Y296W/K29H/T63A and the glucose dehydrogenase EsGDH wet bacteria were mixed into a mixed bacteria with a dry weight ratio of 3.5:1 (w/w) , First resuspend the mixed bacteria in pH 7.0, 100mM PBS buffer, add 20g DCW/L to the mixed bacteria in the transformation system, the substrate 6-cyano-(5R)-hydroxy-3-carbonylhexyl
  • the dosage of tert-butyl ester is 200g/L
  • the glucose concentration is 200g/L
  • the pH 7.0 100mM PBS buffer
  • 100mM PBS buffer is used as the reaction medium to construct a 50mL conversion system, and the reaction is performed at 30°C and 400rpm.
  • the reaction process curve is shown in Figure 5.
  • the product can be completely converted into the product 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl within 2.5h, and the cumulative amount of the product reaches 549.3mM, the product de p value>99.5%, and the space-time yield reaches 1207.7 g/L d.
  • the tert-butyl 6-cyano-(5R)-hydroxy-3-carbonylhexanoate can be completely converted into tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate Butyl ester, and the reaction time is 6.0h, the reaction reaches the end, the conversion rate of the substrate is greater than 99%, and the de p value of the product is greater than 99.5%.
  • Example 8 Asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester by aldehyde-ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63L
  • the aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63L wet cell and the glucose dehydrogenase EsGDH wet cell were obtained by fermentation with the same method as in Example 7 with a dry weight ratio of 3.5:1 (w/w) Mix into a mixed bacteria.
  • the mixed bacteria are first resuspended in pH 7.0, 100mM PBS buffer.
  • the total dry weight of the mixed bacteria in the reaction system is 20g DCW/L, and the substrate 6-
  • the initial dosage of cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl is set to 300g/L, the concentration of glucose is 300g/L, pH 7.0, 100mM PBS buffer is used as the reaction medium to construct the conversion system, Reaction at 30°C and 600 rpm.
  • the reaction process curve is shown in Figure 6.
  • the product concentration gradually increases with the passage of time.
  • the reaction is completed in 5.0 hours and can be completely converted into 740.3mM product.
  • the product de p value>99.5%, and the space-time yield can reach 1152.2g/ L d.
  • tert-butyl 6-cyano-(5R)-hydroxy-3-carbonylhexanoate can be completely converted into tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate Ester, and the reaction time is 6.5h, the reaction reaches the end point, can be completely converted into 799.2mM product, the product de p value>99.5%.
  • Example 9 Asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester by aldehyde-ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63M
  • the aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63M wet cell and the glucose dehydrogenase EsGDH wet cell were obtained by fermentation with the same method as in Example 7 with a dry weight ratio of 3.5:1 (w/w) Mix into a mixed bacteria.
  • the mixed bacteria are first resuspended in pH 7.0, 100mM PBS buffer.
  • the total dry weight of the mixed bacteria in the reaction system is 20g DCW/L, and the substrate 6-
  • the initial dosage of cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl is set to 400g/L, the concentration of glucose is 400g/L, pH 7.0, 100mM PBS buffer is used as the reaction medium to construct the conversion system, Reaction at 30°C and 600 rpm.
  • the reaction process curve is shown in Figure 7.
  • the product concentration gradually increases with the passage of time.
  • the reaction is completed in 5.5 hours and can be completely converted into 829.6mM product.
  • the product de p value is >99.5%, and the space-time yield can reach 1427.2g/ L d.
  • Example 10 Asymmetric reduction of 6-chloro-(5S)-hydroxy-3-carbonylhexanoate tert-butyl ester by aldehyde and ketone reductase KmAKR-W297H/Y296W/K29H/Y28A/T63A
  • Example 2 the aldehyde ketone reductase control group KmAKR-W297H/Y296W/K29H/Y28A/T63A wet bacteria and glucose dehydrogenase EsGDH wet bacteria were obtained through fermentation.
  • the wet bacteria KmAKR-W297H/Y296W/K29H/Y28A/T63A and the glucose dehydrogenase EsGDH wet bacteria are mixed into a mixture with a dry weight ratio of 3.5:1 (w/w)
  • For the bacteria first resuspend the mixed bacteria with pH 7.0, 100mM PBS buffer, and add the dry weight of 20g DCW/L to the mixed bacteria in the transformation system.
  • the substrate When the substrate 6-chloro-(5S)-hydroxy-3-
  • the dosage of tert-butyl carbonylhexanoate is 300g/L and the glucose concentration is 300g/L
  • the substrate can be completely converted into the product 6-chloro-(3R,5S)-dihydroxyhexanoic acid after reacting at 30°C and 600rpm for 5 hours.
  • the substrate conversion rate is greater than 99%, and the de p value of the product always remains above 99.5%.
  • the concentration of the product was 632.9mM, and the space-time yield reached 720.2g/L d.
  • Example 11 Asymmetric reduction of 6-chloro-(5S)-hydroxy-3-carbonylhexanoate tert-butyl ester by aldehyde and ketone reductase KmAKR-W297H/Y296W/K29H/Y28A/T63L
  • Example 2 the aldehyde ketone reductase control group KmAKR-W297H/Y296W/K29H/Y28A/T63L wet bacteria and glucose dehydrogenase EsGDH wet bacteria were obtained through fermentation.
  • the wet bacteria KmAKR-W297H/Y296W/K29H/Y28A/T63L and the glucose dehydrogenase EsGDH wet bacteria are mixed into a mixture with a dry weight ratio of 3.5:1 (w/w)
  • For the bacteria first resuspend the mixed bacteria with pH 7.0, 100mM PBS buffer, and add the dry weight of 20g DCW/L to the mixed bacteria in the transformation system.
  • the substrate When the substrate 6-chloro-(5S)-hydroxy-3-
  • the dosage of tert-butyl carbonylhexanoate is 350g/L and the glucose concentration is 350g/L, the substrate can be completely converted into the product 6-chloro-(3R,5S)-dihydroxyhexanoic acid after 4 hours at 30°C and 600rpm.
  • the substrate conversion rate is greater than 99%, and the de p value of the product always remains above 99.5%.
  • the concentration of the product was 738.4 mM, and the space-time yield reached 1050.4 g/L d.
  • Example 12 Asymmetric reduction of 6-chloro-(5S)-hydroxy-3-carbonylhexanoate tert-butyl ester by aldehyde and ketone reductase KmAKR-W297H/Y296W/K29H/Y28A/T63M
  • Example 2 the aldehyde ketone reductase control group KmAKR-W297H/Y296W/K29H/Y28A/T63M wet bacteria and glucose dehydrogenase EsGDH wet bacteria were obtained through fermentation.
  • the wet bacteria KmAKR-W297H/Y296W/K29H/Y28A/T63M and the glucose dehydrogenase EsGDH wet bacteria are mixed into a mixture with a dry weight ratio of 3.5:1 (w/w)
  • For the bacteria first resuspend the mixed bacteria with pH 7.0, 100mM PBS buffer, and add the dry weight of 20g DCW/L to the mixed bacteria in the transformation system.
  • the substrate 6-chloro-(5S)-hydroxy-3-
  • the dosage of tert-butyl carbonylhexanoate is 400g/L and the glucose concentration is 400g/L
  • the substrate can be completely converted into the product 6-chloro-(3R,5S)-dihydroxyhexanoic acid after 4 hours at 30°C and 600rpm.
  • the substrate conversion rate is greater than 99%, and the de p value of the product always remains above 99.5%.
  • the concentration of the product was 843.8mM, and the space-time yield reached 1188.2g/L d.
  • Example 13 Ald and Ketone Reductase KmAKR-W297H/Y296W/K29H/Y28A/T63A, KmAKR-W297H/Y296W/K29H/Y28A/T63L, KmAKR-W297H/Y296W/K29H/Y28A/T63M asymmetric reduction series of carbonyl compounds
  • the aldehyde ketone reductase mutants KmAKR-W297H/Y296W/K29H/Y28A/T63A, KmAKR-W297H/Y296W/K29H/Y28A/T63L, KmAKR-W297H/Y296W/K29H/Y28A/ T63M wet cells and glucose dehydrogenase EsGDH wet cells.
  • the aldehyde ketone reductase mutant wet cell and the glucose dehydrogenase EsGDH wet cell were mixed into a mixed cell at a dry weight ratio of 3.5:1 (w/w).
  • the conversion rate detection method refers to the article (Enzym.Microb.Technol.,2017,107:32-40) method. The results are shown in Table 4.

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Abstract

Disclosed are a Kluyveromyces marxianus aldo-keto reductase KmAKR mutant and application thereof, said aldo-keto reductase mutant being obtained by site-directed saturation mutagenesis at position 63 of the amino acid sequence shown in SEQ. ID NO. 2. The specific enzymatic activity of the aldo-keto reductase mutants M5-A, M5-L, and M5-M constructed by the present invention is increased by 1.1 times, 3.2 times, and 4.1 times, respectively, compared with a control group. The mutants KmAKR-Y296W/W297H/K29H/Y28A/T63M have significantly improved catalytic activity on 6-cyano-(5R)-hydroxy-3-carbonylhexanoate and 6-chloro-(5S)-hydroxy-3-carbonylhexanoate. The maximum substrate 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl dosage can reach 450 g/L, the substrate conversion rate is higher than 99%, the product dep value always remains above 99.5%, and the space–time yield of the biocatalytic process is as high as 1224.3 g/L d.

Description

马克斯克鲁维酵母醛酮还原酶KmAKR突变体及其应用Kluyveromyces marxianus aldone reductase KmAKR mutant and its application (一)技术领域(1) Technical field
本发明涉及一个源自马克斯克鲁维酵母的醛酮还原酶KmAKR的突变体构建，开发醛酮还原酶重组菌及酶在阿托伐他汀和瑞舒伐他汀、匹伐他汀等“超级他汀”侧链双手性二醇6-取代-(3R,5R/S)-二羟基己酸叔丁酯手性生物催化合成方面的应用。The present invention relates to the construction of a mutant of the aldehyde ketone reductase KmAKR derived from Kluyveromyces marxianus, and the development of aldehyde ketone reductase recombinant bacteria and enzymes in atorvastatin, rosuvastatin, pitavastatin and other "super statins" Application of side-chain chiral diol 6-substituted-(3R,5R/S)-dihydroxyhexanoate in chiral biocatalytic synthesis.
(二)背景技术(2) Background technology
阿托伐他汀和瑞舒伐他汀、匹伐他汀等“超级他汀”是治疗心脑血管疾病的重大降脂药品种，具有高效的降脂疗效、长期安全性和临床益处，显著降低心脑血管疾病的发病率和死亡率。迄今为止，阿托伐他汀钙的累计销售额已突破1000亿美元，是人类制药工业史上最成功的单一药物品种。Atorvastatin, rosuvastatin, pitavastatin and other "super statins" are major lipid-lowering drugs for the treatment of cardiovascular and cerebrovascular diseases. They have high-efficiency lipid-lowering effects, long-term safety and clinical benefits, and significantly reduce cardiovascular and cerebrovascular diseases. Morbidity and mortality of diseases. So far, the cumulative sales of atorvastatin calcium has exceeded 100 billion US dollars, which is the most successful single drug variety in the history of the human pharmaceutical industry.
他汀药物大多含有6-取代-(3R,5R/S)-二羟基己酸叔丁酯结构，既是重要的药效基团又是关键合成前体。6-氰基-(3R,5R)-二羟基己酸叔丁酯是阿托伐他汀钙的关键手性二醇中间体。6-氯-(3R,5S)-二羟基己酸叔丁酯是瑞舒伐他汀、匹伐他汀等“超级他汀”药物的合成前体。由于6-取代-(3R,5R/S)-二羟基己酸叔丁酯具有两个手性中心，因此，研究光学纯6-取代-(3R,5R/S)-二羟基己酸叔丁酯的手性合成方法学和合成技术具有重要意义。已有文献报道的6-取代-(3R,5R/S)-二羟基己酸叔丁酯合成方法主要包括化学催化6-取代-(5R/S)-羟基-3-羰基己酸叔丁酯不对称还原和氧化还原酶差向选择性还原6-取代-(5R/S)-羟基-3-羰基己酸叔丁酯。硼烷等化学催化剂催化还原工艺存在能耗高、转化率低、差向选择性低和生产成本高等缺陷。与化学催化剂相比，酶作为绿色天然生物催化剂，在催化化学反应中具有优越的化学选择性、立体选择性和区域选择性等优点，且反应条件温和、副产物少、环境友好。但许多酶分子在催化非天然底物时，往往存在活力较低、稳定性差、底物产物抑制等问题，亟需对酶分子实施分子改造。Most statins contain 6-substituted-(3R,5R/S)-dihydroxyhexanoic acid tert-butyl structure, which is not only an important pharmacodynamic group but also a key synthetic precursor. 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl ester is the key chiral diol intermediate of atorvastatin calcium. 6-Chloro-(3R,5S)-dihydroxyhexanoic acid tert-butyl ester is the synthetic precursor of "super statin" drugs such as rosuvastatin and pitavastatin. Since 6-substituted-(3R,5R/S)-dihydroxyhexanoic acid tert-butyl ester has two chiral centers, the optically pure 6-substituted-(3R,5R/S)-dihydroxyhexanoic acid tert-butyl ester has been studied The chiral synthesis methodology and synthesis technology of esters are of great significance. The synthetic methods of 6-substituted-(3R,5R/S)-dihydroxyhexanoic acid tert-butyl ester that have been reported in the literature mainly include chemical catalysis 6-substituted-(5R/S)-hydroxy-3-carbonyl hexanoic acid tert-butyl ester Asymmetric reduction and oxidoreductase differentially selectively reduce 6-substituted-(5R/S)-hydroxy-3-carbonylhexanoate tert-butyl ester. Catalytic reduction processes with chemical catalysts such as borane have the disadvantages of high energy consumption, low conversion rate, low differential selectivity and high production cost. Compared with chemical catalysts, enzymes, as green natural biocatalysts, have the advantages of superior chemical selectivity, stereoselectivity and regioselectivity in catalyzing chemical reactions, with mild reaction conditions, few by-products, and environmental friendliness. However, when many enzyme molecules catalyze unnatural substrates, they often have problems such as low activity, poor stability, and inhibition of substrate products, so molecular modification of enzyme molecules is urgently needed.
得益于蛋白质工程的科技进步，生物催化已广泛应用于工业化生产。在我们前期发明(CN 201710282633.X，CN 201910155559.4)的基础上，本发明通过建立高通量筛选模型，构建大容量突变体文库，筛选获得具有鲁棒性的超级突变体，其中“最佳”突变体KmAKR-W297H/Y296W/K29H/Y28A/T63M催化性能最强，进一步分析了突变体催化性能提升的分子机理，优化反应工艺参数，构建KmAKR-W297H/Y296W/K29H/Y28A/T63M催化合成6-取代-(3R,5R/S)-二羟基己酸叔丁酯工艺。Thanks to the technological advancement of protein engineering, biocatalysis has been widely used in industrial production. On the basis of our previous inventions (CN 201710282633.X, CN 201910155559.4), the present invention establishes a high-throughput screening model, constructs a large-capacity mutant library, and screens and obtains robust super mutants, among which the "best" The mutant KmAKR-W297H/Y296W/K29H/Y28A/T63M has the strongest catalytic performance. The molecular mechanism of the improvement of the mutant's catalytic performance was further analyzed, the reaction process parameters were optimized, and the KmAKR-W297H/Y296W/K29H/Y28A/T63M catalytic synthesis was constructed. -Substituted-(3R,5R/S)-dihydroxyhexanoic acid tert-butyl ester process.
(三)发明内容(3) Contents of the invention
本发明目的是针对现有醛酮还原酶对6-取代-(5R/S)-羟基-3-羰基己酸叔丁酯不对称还原活性不高及底物投料量低的问题，提供一种立体选择性醛酮还原酶系列突变体及利用该醛酮还原酶突变体的重组菌或其粗酶液作为催化剂，不对称还原合成6-氰基-(3R,5R)-二羟基己酸叔丁酯、6-氯-(3R,5S)-二羟基己酸叔丁酯等手性醇化合物，催化剂活性较KmAKR-W297H/Y296W/K29H/Y28A提高了4.1倍，底物投料量提高至450g/L，这是所有文献报道中的最高水平。The purpose of the present invention is to solve the problems of low asymmetric reduction activity of 6-substituted-(5R/S)-hydroxy-3-carbonylhexanoate tert-butyl ester and low substrate feeding amount of the existing aldehyde and ketone reductase, and provide a solution Stereoselective aldehyde-ketone reductase series mutants and recombinant bacteria using the aldehyde-ketone reductase mutant or its crude enzyme solution as a catalyst for asymmetric reduction to synthesize 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert Butyl ester, 6-chloro-(3R,5S)-dihydroxyhexanoic acid tert-butyl ester and other chiral alcohol compounds, the catalyst activity is 4.1 times higher than that of KmAKR-W297H/Y296W/K29H/Y28A, and the substrate dosage is increased to 450g /L, this is the highest level in all literature reports.
本发明采用的技术方案是：The technical scheme adopted by the present invention is:
本发明提供一种马克斯克鲁维酵母醛酮还原酶KmAKR突变体，所述醛酮还原酶突变体是将SEQ ID NO.2所示氨基酸序列第63位点进行定点饱和突变获得的，优选突变体为下列之一：(1)第63位苏氨酸突变为丙氨酸；(2)第63位苏氨酸突变为亮氨酸；(3)第63位苏氨酸突变为蛋氨酸。SEQ ID NO.2所示氨基酸序列对应的编码基因核苷酸序列为SEQ ID NO.1所示。The present invention provides a Kluyveromyces marxianus aldehyde ketone reductase KmAKR mutant, which is obtained by site-directed saturation mutation at position 63 of the amino acid sequence shown in SEQ ID NO. 2, preferably the mutation The body is one of the following: (1) Threonine at position 63 is mutated to alanine; (2) Threonine at position 63 is mutated to leucine; (3) Threonine at position 63 is mutated to methionine. The nucleotide sequence of the coding gene corresponding to the amino acid sequence shown in SEQ ID NO. 2 is shown in SEQ ID NO. 1.
本发明还提供一种所述醛酮还原酶KmAKR突变体在不对称还原羰基化合物制备手性醇中的应用，具体所述的应用方法为：将含醛酮还原酶KmAKR突变体基因的工程菌经诱导培养获得的菌体和含葡萄糖脱氢酶基因的工程菌经诱导培养获得的菌体混合，以混合菌体或混合菌体提取的粗酶液为催化剂，以羰基化合物为底物，以葡萄糖为辅底物，以pH 7.0、100mM的PBS缓冲液为反应介质构成转化体系，在30-35℃、400～600rpm条件下进行反应，反应结束，反应液分离纯化，获得手性醇化合物。The present invention also provides an application of the aldehyde and ketone reductase KmAKR mutant in the preparation of chiral alcohols by asymmetric reduction of carbonyl compounds. The specific application method is: the engineered bacteria containing the aldehyde and ketone reductase KmAKR mutant gene The bacterial cells obtained by induction culture and the bacterial cells obtained by induction culture of engineered bacteria containing the glucose dehydrogenase gene are mixed, using the mixed bacteria or the crude enzyme solution extracted from the mixed bacteria as the catalyst, and the carbonyl compound as the substrate. Glucose is the auxiliary substrate, and the conversion system is formed with pH 7.0, 100 mM PBS buffer as the reaction medium, and the reaction is carried out at 30-35° C. and 400-600 rpm. After the reaction is completed, the reaction solution is separated and purified to obtain the chiral alcohol compound.
进一步，所述转化体系中，底物终浓度30～450g/L(优选200～400g/L)，葡萄糖终浓度30～450g/L(优选200～400g/L)，催化剂用量以混合菌体总量干重计为0.1～20g DCW/L(DCW细胞干重，优选20g DCW/L)，所述混合菌体中含醛酮还原酶突变体基因的工程菌经诱导培养获得的湿菌体与含葡萄糖脱氢酶基因的工程菌经诱导培养获得的湿菌体以干重比1.0～5.0:1(w/w)，优选3.5:1混合。所述葡萄糖脱氢酶基因(GenBank NO.KM817194.1)来自Exiguobacterium sibirium DSM 17290。Furthermore, in the conversion system, the final concentration of substrate is 30-450g/L (preferably 200-400g/L), the final concentration of glucose is 30-450g/L (preferably 200-400g/L), and the amount of catalyst is based on the total amount of mixed bacteria. The dry weight is 0.1-20g DCW/L (the dry weight of DCW cells, preferably 20g DCW/L), and the wet bacterial cells obtained by induction culture of the engineered bacteria containing the aldehyde ketone reductase mutant gene in the mixed bacterial cells and The wet bacterial cells obtained by induction culture of the engineered bacteria containing the glucose dehydrogenase gene are mixed at a dry weight ratio of 1.0-5.0:1 (w/w), preferably 3.5:1. The glucose dehydrogenase gene (GenBank NO. KM817194.1) is from Exiguobacterium sibirium DSM 17290.
进一步，羰基化合物为下列之一：4-氯-3-羰基丁酸乙酯、6-氯-(5S)-羟基-3-羰基己酸叔丁酯、3-羰基丁酸乙酯、4-溴-3-羰基丁酸丙酯、4,4,4,-三氟-3-羰基丁酸乙酯、6-氰基-(5R)-羟基-3-羰基己酸叔丁酯、3-羰基丁酸叔丁酯、苯乙酮，优选6-氯-(5S)-羟基-3-羰基己酸叔丁酯、3-羰基丁酸乙酯、6-氰基-(5R)-羟基-3-羰基己酸叔丁酯。Further, the carbonyl compound is one of the following: ethyl 4-chloro-3-carbonylbutyrate, tert-butyl 6-chloro-(5S)-hydroxy-3-carbonylhexanoate, ethyl 3-carbonylbutyrate, 4- Propyl bromo-3-carbonylbutyrate, 4,4,4,-trifluoro-3-carbonylbutyric acid ethyl ester, 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester, 3- Tert-Butyl carbonylbutyrate, acetophenone, preferably tert-butyl 6-chloro-(5S)-hydroxy-3-carbonylhexanoate, ethyl 3-carbonylbutyrate, 6-cyano-(5R)-hydroxy- Tert-Butyl 3-carbonylhexanoate.
本发明所述羰基化合物为6-氰基-(5R)-羟基-3-羰基己酸叔丁酯时，所述醛酮还原酶KmAKR突变体在不对称还原6-氰基-(5R)-羟基-3-羰基己酸叔丁酯制备6-氰基-(3R,5R)-二羟基己酸叔丁酯的方法为：将含醛酮还原酶突变体基因的工程菌经诱导培养获得的菌体和含葡萄糖脱氢酶基因的工程菌经诱导培养获得的菌体混合，以混合菌体为催化剂，以6-氰基-(5R)-羟基-3-羰基己酸叔丁酯为底物，以葡萄糖为辅底物，以pH 7.0、100mM的PBS缓冲液为反应介质构成转化体系，在30℃、400～600rpm条件下进行反应，反应结束，反应液分离纯化，获得6-氰基-(3R,5R)-二羟基己酸叔丁酯。When the carbonyl compound of the present invention is tert-butyl 6-cyano-(5R)-hydroxy-3-carbonylhexanoate, the aldehyde and ketone reductase KmAKR mutant asymmetrically reduces 6-cyano-(5R)- The method for preparing tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate from tert-butyl hydroxy-3-carbonylhexanoate is as follows: the engineered bacteria containing the aldehyde ketone reductase mutant gene are obtained by inducing cultivation The bacteria are mixed with the bacteria obtained by induction culture of the engineered bacteria containing the glucose dehydrogenase gene, using the mixed bacteria as the catalyst and 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester as the substrate The conversion system is constructed with glucose as the auxiliary substrate, and the pH 7.0, 100mM PBS buffer is used as the reaction medium to form the conversion system. The reaction is carried out at 30°C and 400-600rpm. After the reaction is completed, the reaction solution is separated and purified to obtain 6-cyano -(3R,5R)-tert-butyl dihydroxyhexanoate.
本发明所述底物为6-氯-(5S)-羟基-3-羰基己酸叔丁酯时，所述醛酮还原酶KmAKR突变体在不对称还原6-氯-(5S)-羟基-3-羰基己酸叔丁酯制备6-氯-(3R,5S)-二羟基己酸叔丁酯中的方法为：将含醛酮还原酶KmAKR突变体基因的工程菌经诱导培养获得的菌体和含葡萄糖脱氢酶基因的工程菌经诱导培养获得的菌体混合，以混合菌体为催化剂，以6-氯-(5S)-羟基-3-羰基己酸叔丁酯为底物，以葡萄糖为辅底物，以pH 7.0、100mM的PBS缓冲液为反应介质构成转化体系，在30℃、400～600rpm条件下进行反应，反应结束，反应液分离纯化，获得6-氯-(3R,5S)-二羟基己酸叔丁酯。When the substrate of the present invention is 6-chloro-(5S)-hydroxy-3-carbonylhexanoate tert-butyl ester, the aldehyde and ketone reductase KmAKR mutant asymmetrically reduces 6-chloro-(5S)-hydroxy- The method for preparing tert-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate from 3-carbonyl hexanoate tert-butyl ester is as follows: the bacteria containing the aldehyde ketone reductase KmAKR mutant gene are induced and cultured. The bacteria and the bacteria obtained by the induction culture of the engineered bacteria containing the glucose dehydrogenase gene are mixed, using the mixed bacteria as the catalyst and 6-chloro-(5S)-hydroxy-3-carbonylhexanoate tert-butyl as the substrate, The conversion system was constructed with glucose as the auxiliary substrate and pH 7.0, 100mM PBS buffer as the reaction medium. The reaction was carried out at 30°C and 400-600rpm. After the reaction was completed, the reaction solution was separated and purified to obtain 6-chloro-(3R). ,5S)-tert-butyl dihydroxyhexanoate.
进一步，所述湿菌体按如下方法制备：将携带醛酮还原酶突变体基因的大肠杆菌工程菌接种到含终浓度50μg/mL卡那霉素的LB液体培养基中，37℃培养10h，以体积浓度1.5％的接种量接种到新鲜的含终浓度50μg/mL卡那霉素的LB液体培养基中，37℃、180rpm培养2h，再向培养液中加入终浓度为0.15mM异丙基硫代半乳糖苷(Isopropyl β-D-thiogalactoside，IPTG)，28℃培养12h后，4℃、8000rpm离心10min，获得含醛酮还原酶突变体的湿菌体；所述含葡萄糖脱氢酶基因的工程菌经诱导培养获得的湿菌体制备方法同含醛酮还原酶基因的湿菌体。Furthermore, the wet bacteria are prepared as follows: inoculate the engineered Escherichia coli bacteria carrying the aldehyde ketone reductase mutant gene into LB liquid medium containing a final concentration of 50 μg/mL kanamycin, and cultivate for 10 hours at 37°C, Inoculate 1.5% of the volume concentration into a fresh LB liquid medium containing a final concentration of 50μg/mL kanamycin, incubate at 37°C and 180rpm for 2h, and then add isopropyl to the culture solution to a final concentration of 0.15mM Isopropyl β-D-thiogalactoside (IPTG), cultured at 28°C for 12 hours, and centrifuged at 4°C and 8000 rpm for 10 minutes to obtain wet cells containing the aldehyde ketone reductase mutant; the glucose dehydrogenase gene The preparation method of the wet bacterial cells obtained by induction culture of the engineered bacteria is the same as that of the wet bacterial cells containing the aldehyde ketone reductase gene.
本发明所述粗酶液按如下方法制备：按照湿菌体总量100g/L的用量重悬于pH 7.0、100mM PBS缓冲液中，在冰水混合物上超声破碎6min，超声破碎条件：功率为400W，破碎1s、暂停1s，取破碎混合液，获得粗酶液。The crude enzyme solution of the present invention is prepared as follows: the total amount of wet bacteria is 100g/L, resuspended in a pH 7.0, 100mM PBS buffer, and ultrasonically broken on an ice-water mixture for 6 minutes. The ultrasonic breaking conditions are as follows: 400W, break for 1s, pause for 1s, take the broken mixture to obtain the crude enzyme solution.
本发明醛酮还原酶KmAKR及醛酮还原酶KmAKR突变体碱基序列全长均为933bp，从第一个碱基起至第933个碱基止，起始密码子为ATG，终止密码子为TGA。The base sequence of the aldehyde-ketone reductase KmAKR and the aldehyde-ketone reductase KmAKR mutant of the present invention are both 933 bp in length, starting from the first base to the 933th base, the start codon is ATG, and the stop codon is TGA.
本发明所述醛酮还原酶KmAKR突变体的获取是采用定点饱和突变技术，使用该技术对KmAKR-W297H/Y296W/K29H/Y28A醛酮还原酶基因(SEQ ID NO.1)进行突变，将获得的突变质粒以热击方式导入E.coli BL21(DE3)感受态细胞，对获得菌株进 行接种、转接、培养、诱导培养、菌体回收，利用重悬菌液催化6-氰基-(5R)-羟基-3-羰基己酸叔丁酯不对称还原，制备光学纯6-氰基-(3R,5R)-二羟基己酸叔丁酯，具体方法如下：第一步将对照菌活化，获得了对照菌E.coli BL21(DE3)/pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A，提取质粒pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A，并保存于-20℃。第二步通过SWISS-MODEL与KmAKR-W297H/Y296W/K29H/Y28A比较，获得同源建模的模板蛋白晶体结构，利用Modeller 9.19同源建模，并进行分子对接，选择合适的突变位点，选择的位点主要是位于活性中心附近的氨基酸残基以及活性中心loop环上的氨基酸残基，设计突变的引物，以pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A为模板质粒，进行定点饱和突变，获得突变质粒，并转化，进行优势突变菌的筛选，获得醛酮还原酶突变体KmAKR-Y296W/W297H/K29H/Y28A/T63A(记为M5-A、KmAKR-Y296W/W297H/K29H/Y28A/T63L(记为M5-L)、KmAKR-Y296W/W297H/K29H/Y28A/T63M(记为M5-M)，将优势突变体送样测序并保存。The KmAKR mutant of the aldehyde and ketone reductase of the present invention is obtained by using the site-specific saturation mutagenesis technology. The KmAKR-W297H/Y296W/K29H/Y28A aldehyde ketone reductase gene (SEQ ID NO.1) is mutated using this technology to obtain The mutant plasmid was introduced into E. coli BL21(DE3) competent cells by heat shock, and the obtained strains were inoculated, transferred, cultivated, induced culture, and bacteria were recovered. The resuspended bacteria liquid was used to catalyze 6-cyano-(5R). )-Hydroxy-3-carbonylhexanoate tert-butyl ester is asymmetrically reduced to prepare optically pure 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl ester. The specific method is as follows: The first step is to activate the control bacteria. Obtained the control bacteria E. coli BL21(DE3)/pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A, extracted the plasmid pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A, and stored it at -20℃ . The second step is to compare SWISS-MODEL with KmAKR-W297H/Y296W/K29H/Y28A to obtain the template protein crystal structure of homology modeling, use Modeller 9.19 homology modeling, perform molecular docking, and select the appropriate mutation site. The selected sites are mainly the amino acid residues located near the active center and the amino acid residues on the loop loop of the active center. The mutant primers are designed, using pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A as the template plasmid. Site-directed saturation mutagenesis, the mutant plasmid is obtained, and transformed, and the dominant mutant strain is screened to obtain the aldehyde ketone reductase mutant KmAKR-Y296W/W297H/K29H/Y28A/T63A (denoted as M5-A, KmAKR-Y296W/W297H/K29H) /Y28A/T63L (denoted as M5-L), KmAKR-Y296W/W297H/K29H/Y28A/T63M (denoted as M5-M), sample the superior mutants for sequencing and save.
本发明醛酮还原酶突变体和葡萄糖脱氢酶基因工程菌的接种、转接、诱导、菌体回收，培养基可为本领域任何可使菌体生长并产生本发明的培养基，优选LB培养基：胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，蒸馏水溶解，调节pH 7.0。培养方法和培养条件没有特殊的限制，培养方法和条件可以根据宿主类型和培养方法等因素的不同按本领域普通知识进行适当的选择。For the inoculation, transfer, induction, and cell recovery of the aldehyde ketone reductase mutant and glucose dehydrogenase genetically engineered bacteria of the present invention, the culture medium can be any medium in the art that can grow bacteria and produce the culture medium of the present invention, preferably LB Medium: tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, dissolved in distilled water, adjust pH 7.0. There are no special restrictions on the culture method and culture conditions, and the culture method and conditions can be appropriately selected according to the host type and culture method and other factors according to common knowledge in the field.
与现有技术相比，本发明主要的有益效果主要体现在：本发明构建的醛酮还原酶突变体M5-A、M5-L、M5-M的比酶活较对照组醛酮还原酶分别增加了1.1倍、3.2倍、4.1倍。其中突变体KmAKR-Y296W/W297H/K29H/Y28A/T63M最大底物6-氰基-(5R)-羟基-3-羰基己酸叔丁酯投料量可达到450g/L，产物浓度随时间的推移而逐渐升高，7.0h内可反应完成，底物转化率大于99％，产物de p值始终保持在99.5％以上。时空产率达到1224.3g/L d。而利用对照组KmAKR-W297H/Y296W/K29H/Y28A催化6-氰基-(5R)-羟基-3-羰基己酸叔丁酯时最大底物投料量可达到200g/L，因此醛酮还原酶突变体KmAKR-Y296W/W297H/K29H/Y28A/T63A、KmAKR-Y296W/W297H/K29H/Y28A/T63L、KmAKR-Y296W/W297H/K29H/Y28A/T63M更具工业应用前景。 Compared with the prior art, the main beneficial effects of the present invention are mainly reflected in: the specific enzyme activities of the aldone reductase mutants M5-A, M5-L and M5-M constructed in the present invention are higher than those of the control group Increased by 1.1 times, 3.2 times, 4.1 times. Among them, the mutant KmAKR-Y296W/W297H/K29H/Y28A/T63M, the largest substrate 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester, can be charged up to 450g/L, and the product concentration changes with time. But gradually increase, the reaction can be completed within 7.0h, the substrate conversion rate is greater than 99%, and the product de p value is always maintained above 99.5%. The space-time yield reached 1224.3g/L d. When using the control group KmAKR-W297H/Y296W/K29H/Y28A to catalyze 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester, the maximum substrate feeding amount can reach 200g/L, so the aldehyde ketone reductase Mutants KmAKR-Y296W/W297H/K29H/Y28A/T63A, KmAKR-Y296W/W297H/K29H/Y28A/T63L, KmAKR-Y296W/W297H/K29H/Y28A/T63M have more industrial application prospects.
(四)附图说明(4) Description of the drawings
图1是醛酮还原酶与葡萄糖脱氢酶EsGDH偶联催化6-氰基-(5R)-羟基-3-羰基己酸叔丁酯不对称还原制备6-氰基-(3R,5R)-二羟基己酸叔丁酯的反应示意图。Figure 1 shows the coupling of aldehyde ketone reductase and glucose dehydrogenase EsGDH to catalyze the asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester to prepare 6-cyano-(3R,5R)- Schematic diagram of the reaction of tert-butyl dihydroxyhexanoate.
图2是醛酮还原酶与葡萄糖脱氢酶EsGDH偶联催化6-氯-(5S)-羟基-3-羰基己酸叔丁酯不对称还原制备6-氯-(3R,5S)-二羟基己酸叔丁酯的反应示意图Figure 2 shows the coupling of aldehyde ketone reductase and glucose dehydrogenase EsGDH to catalyze the asymmetric reduction of 6-chloro-(5S)-hydroxy-3-carbonylhexanoate tert-butyl ester to prepare 6-chloro-(3R,5S)-dihydroxyl Schematic diagram of the reaction of tert-butyl caproate
图3醛酮还原酶突变体上清液和纯酶的SDS-PAGE电泳图。泳道1：对照KmAKR-W297H/Y296W/K29H/Y28A上清液；泳道2：对照KmAKR-W297H/Y296W/K29H/Y28A纯酶；泳道3：KmAKR-W297H/Y296W/K29H/Y28A/T63A上清液；泳道4：KmAKR-W297H/Y296W/K29H/Y28A/T63A纯酶；泳道5：KmAKR-W297H/Y296W/K29H/Y28A/T63L上清液；泳道6：KmAKR-W297H/Y296W/K29H/Y28A/T63L纯酶；泳道7：KmAKR-W297H/Y296W/K29H/Y28A/T63M上清液；泳道8：KmAKR-W297H/Y296W/K29H/Y28A/T63M纯酶；M：标准蛋白分子。Figure 3 SDS-PAGE electrophoresis diagram of the supernatant of the aldehyde ketone reductase mutant and the pure enzyme. Lane 1: Control KmAKR-W297H/Y296W/K29H/Y28A supernatant; Lane 2: Control KmAKR-W297H/Y296W/K29H/Y28A pure enzyme; Lane 3: KmAKR-W297H/Y296W/K29H/Y28A/T63A supernatant Lane 4: KmAKR-W297H/Y296W/K29H/Y28A/T63A pure enzyme; Lane 5: KmAKR-W297H/Y296W/K29H/Y28A/T63L supernatant; Lane 6: KmAKR-W297H/Y296W/K29H/Y28A/T63L Pure enzyme; Lane 7: KmAKR-W297H/Y296W/K29H/Y28A/T63M supernatant; Lane 8: KmAKR-W297H/Y296W/K29H/Y28A/T63M pure enzyme; M: Standard protein molecule.
图4是利用醛酮还原酶突变体KmAKR-W297H/Y296W/K29H/Y28A偶联EsGDH不对称还原6-氰基-(5R)-羟基-3-羰基己酸叔丁酯制备6-氰基-(3R,5R)-二羟基己酸叔丁酯时间进程图。Figure 4 is the use of aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A coupled with EsGDH asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester to prepare 6-cyano- Time course of (3R,5R)-dihydroxyhexanoate tert-butyl ester.
图5是利用醛酮还原酶突变体KmAKR-W297H/Y296W/K29H/Y28A/T63A偶联EsGDH不对称还原6-氰基-(5R)-羟基-3-羰基己酸叔丁酯制备6-氰基-(3R,5R)-二羟基己酸叔丁酯时间进程图。Figure 5 is the use of aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63A coupled with EsGDH asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonyl hexanoate tert-butyl ester to prepare 6-cyano Time course graph of tert-butyl dihydroxy-(3R,5R)-dihydroxyhexanoate.
图6是利用醛酮还原酶突变体KmAKR-W297H/Y296W/K29H/Y28A/T63L偶联EsGDH不对称还原6-氰基-(5R)-羟基-3-羰基己酸叔丁酯制备6-氰基-(3R,5R)-二羟基己酸叔丁酯时间进程图。Figure 6 is the use of aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63L coupled with EsGDH asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester to prepare 6-cyano Time course graph of tert-butyl dihydroxy-(3R,5R)-dihydroxyhexanoate.
图7是利用醛酮还原酶突变体KmAKR-W297H/Y296W/K29H/Y28A/T63M偶联EsGDH不对称还原6-氰基-(5R)-羟基-3-羰基己酸叔丁酯制备6-氰基-(3R,5R)-二羟基己酸叔丁酯时间进程图。Figure 7 is the use of aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63M coupled with EsGDH asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester to prepare 6-cyano Time course graph of tert-butyl dihydroxy-(3R,5R)-dihydroxyhexanoate.
图8是利用醛酮还原酶突变体KmAKR-W297H/Y296W/K29H/Y28A/T63A偶联EsGDH不对称还原6-氯-(5S)-羟基-3-羰基己酸叔丁酯制备6-氯-(3R,5S)-二羟基己酸叔丁酯时间进程图。Figure 8 is the use of aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63A coupled with EsGDH asymmetric reduction of 6-chloro-(5S)-hydroxy-3-carbonylhexanoate tert-butyl ester to prepare 6-chloro- Time course of (3R,5S)-dihydroxyhexanoate tert-butyl ester.
图9是利用醛酮还原酶突变体KmAKR-W297H/Y296W/K29H/Y28A/T63L偶联EsGDH不对称还原6-氯-(5S)-羟基-3-羰基己酸叔丁酯制备6-氯-(3R,5S)-二羟基己酸叔 丁酯时间进程图。Figure 9 is the use of aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63L coupled with EsGDH asymmetric reduction of 6-chloro-(5S)-hydroxy-3-carbonyl hexanoic acid tert-butyl ester to prepare 6-chloro- Time course of (3R,5S)-dihydroxyhexanoate tert-butyl ester.
图10是利用醛酮还原酶突变体KmAKR-W297H/Y296W/K29H/Y28A/T63M偶联EsGDH不对称还原6-氯-(5S)-羟基-3-羰基己酸叔丁酯制备6-氯-(3R,5S)-二羟基己酸叔丁酯时间进程图。Figure 10 is the use of aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63M coupled with EsGDH asymmetric reduction of 6-chloro-(5S)-hydroxy-3-carbonylhexanoate tert-butyl ester to prepare 6-chloro- Time course of (3R,5S)-dihydroxyhexanoate tert-butyl ester.
(五)具体实施方式(5) Specific implementation methods
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited to this:
实施例1：醛酮还原酶突变体文库的构建及筛选Example 1: Construction and screening of aldehyde ketone reductase mutant library
马克斯克鲁维酵母醛酮还原酶突变体文库的制备通过1轮定点饱和突变来实现，引物设计如表1，以E.coli BL21(DE3)/pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A(构建参见专利申请CN201910072740.9)中载体pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A为模板，以表1中Thr63-F和Thr63-R为引物，经饱和突变PCR，将SEQ ID NO.2所示醛酮还原酶KmAKR-W297H/Y296W/K29H/Y28A氨基酸序列的第63位苏氨酸突变为其余19种氨基酸，并转化，涂平板，通过优势菌株筛选(表2)，获得醛酮还原酶突变体KmAKR-W297H/Y296W/K29H/Y28A/T63A(记为M5-A，即SEQ ID NO.2所示氨基酸第63位苏氨酸突变为丙氨酸)、KmAKR-W297H/Y296W/K29H/Y28A/T63L(记为M5-L，即SEQ ID NO.2所示氨基酸第63位苏氨酸突变为亮氨酸)和KmAKR-W297H/Y296W/K29H/Y28A/T63M(记为M5-M，即SEQ ID NO.2所示氨基酸第63位苏氨酸突变为蛋氨酸)。同时，以pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A为模板，对22位点和95位点进行定点饱和突变，但均未获得活力提高的突变菌株。The preparation of the Kluyveromyces marxianus aldehyde ketone reductase mutant library was achieved by a round of site-directed saturation mutagenesis. The primer design is shown in Table 1. E. coli BL21(DE3)/pET28a(+)-kmakr-W297H/Y296W/K29H /Y28A (see patent application CN201910072740.9 for construction) in the vector pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A as the template, using Thr63-F and Thr63-R in Table 1 as primers, and by saturation mutation PCR, The amino acid sequence of the aldehyde and ketone reductase KmAKR-W297H/Y296W/K29H/Y28A shown in SEQ ID NO. 2 has a mutation of threonine at position 63 to the remaining 19 amino acids, and transforms, spreads, and screens by dominant strains (Table 2) , Obtain the aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63A (denoted as M5-A, that is, the amino acid shown in SEQ ID NO. 2 threonine mutated to alanine), KmAKR- W297H/Y296W/K29H/Y28A/T63L (denoted as M5-L, that is, threonine at position 63 of the amino acid shown in SEQ ID NO. 2 is mutated to leucine) and KmAKR-W297H/Y296W/K29H/Y28A/T63M( Marked as M5-M, that is, threonine at position 63 of the amino acid shown in SEQ ID NO. 2 is mutated to methionine). At the same time, using pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A as a template, site-specific saturation mutations were performed at positions 22 and 95, but none of the mutant strains with increased viability were obtained.
PCR反应体系(50μL)：1μL正向引物(100μM)，1μL反向引物(100μM)，25μL 2×Phanta缓冲液，1μL dNTP混合物(各10mM)，1μL质粒模板，1μL DNA聚合酶和21μL超纯水。根据Phanta Super-Fidelity DNA聚合酶手册设置的PCR程序如下：95℃预变性5min，然后29个循环(95℃变性15s，55℃退火15s，72℃延伸7s)，72℃终延伸10min，16℃保温。将得到的重组质粒转移到大肠杆菌BL21(DE3)感受态细胞中，并将克隆子在37℃培养12h。然后挑出克隆并转接到10mL含50μg/mL卡那霉素的LB液体培养基中，并在37℃，180rpm培养10h。对获得的突变体进行优势突变体的筛选，筛选条件如下：以干重25g/L(醛酮还原酶突变体和葡萄糖脱氢酶 菌体干重比3.5:1(w/w)的量加入pH 7.0的PBS(100mM)重悬细胞，再加入终浓度50g/L 6-氰基-(5R)-羟基-3-羰基己酸叔丁酯，50g/L的葡萄糖构成转化体系10mL，在35℃、600rpm条件下进行反应，反应结束，取样检测6-氰基-(3R,5R)-二羟基己酸叔丁酯浓度，筛选获得优势菌株。将获得优势菌株送杭州擎科生物技术有限公司测序，并保存在-80℃冰箱。PCR reaction system (50μL): 1μL forward primer (100μM), 1μL reverse primer (100μM), 25μL 2×Phanta buffer, 1μL dNTP mixture (each 10mM), 1μL plasmid template, 1μL DNA polymerase and 21μL ultrapure water. The PCR program set up according to the Phanta Super-Fidelity DNA Polymerase Manual is as follows: 95°C pre-denaturation for 5 minutes, then 29 cycles (95°C denaturation for 15s, 55°C annealing for 15s, 72°C extension for 7s), 72°C final extension for 10 minutes, 16°C Keep warm. The obtained recombinant plasmid was transferred to E. coli BL21(DE3) competent cells, and the clones were cultured at 37°C for 12h. Then the clones were picked and transferred to 10 mL of LB liquid medium containing 50 μg/mL kanamycin, and cultured at 37° C. and 180 rpm for 10 h. The obtained mutants were screened for dominant mutants, and the screening conditions were as follows: dry weight 25g/L (dry weight ratio of aldehyde ketone reductase mutant and glucose dehydrogenase bacteria to 3.5:1 (w/w) Resuspend the cells in PBS (100mM) at pH 7.0, and then add a final concentration of 50g/L 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester, 50g/L glucose to form a transformation system 10mL, at 35 The reaction is carried out under the conditions of ℃ and 600 rpm. After the reaction is completed, samples are taken to detect the concentration of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate, and the dominant strains will be screened. The dominant strains will be sent to Hangzhou Qingke Biotechnology Co., Ltd. Sequencing and storing in a refrigerator at -80°C.
表1醛酮还原酶定点饱和突变引物设计Table 1 Design of primers for site-directed saturation mutagenesis of aldehyde and ketone reductase
Figure PCTCN2020085538-appb-000001
Figure PCTCN2020085538-appb-000001
实施例2：对照组醛酮还原酶、突变体和葡萄糖脱氢酶的诱导表达Example 2: Induced expression of aldehyde ketone reductase, mutants and glucose dehydrogenase in the control group
葡萄糖脱氢酶基因工程菌：将来自E.sibirium DSM 17290葡萄糖脱氢酶基因(GenBank NO.KM817194.1)***到pET28b(+)构建重组表达载体，并将此表达载体转入E.coli BL21(DE3)制得E.coli BL21(DE3)/pET28b(+)-esgdh。Glucose dehydrogenase genetically engineered bacteria: Insert the 17290 glucose dehydrogenase gene from E.sibirium DSM (GenBank NO.KM817194.1) into pET28b(+) to construct a recombinant expression vector, and transfer this expression vector into E.coli BL21 (DE3) E. coli BL21(DE3)/pET28b(+)-esgdh was prepared.
将实施例1出发菌株E.coli BL21(DE3)/pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A和实施例1筛选的醛酮还原酶突变菌株以及E.coli BL21(DE3)/pET28b(+)-esgdh分别接种到含有终浓度50μg/mL卡那霉素的LB液体培养基中，37℃培养10h，以体积浓度1.5％(v/v)的接种量接种到新鲜的含有终浓度50μg/mL卡那霉素的LB液体培养基中，37℃、180rpm培养2h，再向培养液中加入终浓度为0.15mM IPTG，28℃培养12h后，4℃、8000rpm离心10min，获得相应的湿菌体细胞。以上获得的细胞产有相应的蛋白，可用于蛋白纯酶液的制备，也可用于粗酶液催化不对称还原6-氰基-(5R)-羟基-3-羰基己酸叔丁酯制备6-氰基-(3R,5R)-二羟基己酸叔丁酯以及催化不对称还原6-氯-(5S)-羟基-3-羰基己酸叔丁酯制备6-氯-(3R,5S)-二羟基己酸叔丁酯。The starting strain E.coli BL21(DE3)/pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A and the aldehyde ketone reductase mutant strain screened in Example 1 and E. coli BL21(DE3)/pET28b (+)-esgdh were respectively inoculated into LB liquid medium containing a final concentration of 50μg/mL kanamycin, cultured at 37°C for 10 hours, and inoculated with a volume concentration of 1.5% (v/v) to freshly contain the final concentration In 50μg/mL kanamycin LB liquid medium, incubate at 37°C and 180rpm for 2h, then add IPTG to the culture broth with a final concentration of 0.15mM IPTG. After incubation at 28°C for 12h, centrifuge at 4°C and 8000rpm for 10min to obtain the corresponding Wet bacterial cells. The cells obtained above produce the corresponding protein, which can be used for the preparation of pure protein enzyme solution, and can also be used for the preparation of crude enzyme solution to catalyze asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl 6 -Cyano-(3R,5R)-dihydroxyhexanoate tert-butyl ester and catalytic asymmetric reduction of 6-chloro-(5S)-hydroxy-3-carbonylhexanoate tert-butyl ester to prepare 6-chloro-(3R,5S) -Tert-Butyl dihydroxyhexanoate.
实施例3：突变文库筛选Example 3: Screening of mutation libraries
将实施例2诱导表达的突变株湿菌体及葡萄糖脱氢酶湿菌体以干重比3.5:1(w/w)混合成混合菌体，加入pH 7.0、100mM PBS缓冲液中重悬，获得突变株混合菌液。同样条件下，用对照菌株E.coli BL21(DE3)/pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A替换突变体菌株湿菌体制备对照株混合菌液。The wet cells of the mutant strain and the wet cells of glucose dehydrogenase induced in Example 2 were mixed with a dry weight ratio of 3.5:1 (w/w) to form a mixed cell, and resuspended in a pH 7.0, 100mM PBS buffer. Obtain a mixed bacterial solution of the mutant strain. Under the same conditions, the control strain E. coli BL21(DE3)/pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A was used to replace the wet bacteria of the mutant strain to prepare a mixed bacterial solution of the control strain.
分别将突变株混合菌液和对照株混合菌液作为催化剂，以6-氰基-(5R)-羟基-3-羰 基己酸叔丁酯为底物，以葡萄糖为辅助底物，不添加外源性NADPH或NADP +，利用菌体细胞内源性NADPH，建立起辅酶循环***。反应体系选择为10mL，催化剂用量以混合菌体总干重计20g/L，底物终浓度30g/L，葡萄糖终浓度30g/L，pH 7.0、100mM PBS缓冲液为反应介质构建转化体系，30℃、600rpm反应20min取样，取反应液100μL加入900μL无水乙醇沉淀蛋白，即反应液稀释10倍，-20℃过夜，12000rpm离心3min，取上清，过0.22μm微滤膜，作为液相样品，HPLC检测6-氰基-(5R)-羟基-3-羰基己酸叔丁酯、6-氰基-(3R,5R)-二羟基己酸叔丁酯、6-氰基-(3S,5R)-二羟基己酸叔丁酯的生成量及de p值。以产物6-氰基-(3R,5R)-二羟基己酸叔丁酯和de p为指标，筛选优势突变体，实验结果示于表2。 The mixed bacterial liquid of the mutant strain and the mixed bacterial liquid of the control strain were used as catalysts, 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester was used as the substrate, glucose was used as the auxiliary substrate, and no external substances were added. Source NADPH or NADP + , use the endogenous NADPH of bacterial cells to establish a coenzyme circulation system. The reaction system is 10mL, the amount of catalyst is 20g/L based on the total dry weight of the mixed bacteria, the final concentration of substrate is 30g/L, the final concentration of glucose is 30g/L, pH 7.0, 100mM PBS buffer is used as the reaction medium to construct the conversion system, 30 ℃, 600rpm for 20 minutes to take samples, take 100μL of the reaction solution and add 900μL of absolute ethanol to precipitate the protein, that is, the reaction solution is diluted 10 times, -20℃ overnight, centrifuged at 12000rpm for 3min, take the supernatant, pass through a 0.22μm microfiltration membrane, as a liquid phase sample , HPLC detects 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl, 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl, 6-cyano-(3S, 5R) The amount and de p value of tert-butyl dihydroxycaproate. Using the product 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester and de p as indicators, the dominant mutants were screened. The experimental results are shown in Table 2.
液相检测条件：色谱柱
Figure PCTCN2020085538-appb-000002
C18(4.6×250mm，Acchrom，China)柱，流动相乙腈:水体积比为1:3，流速1.0mL/min，检测波长210nm，进样量10μL，柱温40℃。6-氰基-(5R)-羟基-3-羰基己酸叔丁酯、6-氰基-(3R,5R)-二羟基己酸叔丁酯保留时间分别为13.9min和9.8min。 Liquid phase detection conditions: chromatographic column
Figure PCTCN2020085538-appb-000002
C18 (4.6×250mm, Acchrom, China) column, mobile phase acetonitrile: water volume ratio 1:3, flow rate 1.0mL/min, detection wavelength 210nm, injection volume 10μL, column temperature 40℃. The retention times of 6-cyano-(5R)-hydroxy-3-carbonylhexanoic acid tert-butyl ester and 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester were 13.9min and 9.8min, respectively.
表2 KmAKR-W297H/Y296W/K29H/Y28A及其突变体的催化性能和立体选择性Table 2 The catalytic performance and stereoselectivity of KmAKR-W297H/Y296W/K29H/Y28A and its mutants
Figure PCTCN2020085538-appb-000003
Figure PCTCN2020085538-appb-000003
实施例4：醛酮还原酶母本及其突变体的纯化Example 4: Purification of aldehyde ketone reductase parent and its mutant
将实施例3获得的优势突变体(表2中KmAKR-W297H/Y296W/K29H/Y28A/T63A，KmAKR-W297H/Y296W/K29H/Y28A/T63L，KmAKR-W297H/Y296W/K29H/Y28A/T63M)，根据实施例2所述方法获得醛酮还原酶突变体湿菌体，分别在8000rpm，4℃下离心10min收集菌体，并用0.9％(w/v)盐水洗涤两次。按照湿菌体总量100g/L的量加入pH 7.0、100mM PBS缓冲液中重悬，在冰水混合物上超声破碎6min，超声破碎条件：功率为400W，破碎1s、暂停1s，获得突变株粗酶液。通过在8000rpm，4℃下离心10min收集上清液(电泳图见图3所示)，将其通过0.45μm膜微过滤后，采用Ni亲和柱纯化突变体蛋白。The superior mutants obtained in Example 3 (KmAKR-W297H/Y296W/K29H/Y28A/T63A, KmAKR-W297H/Y296W/K29H/Y28A/T63L, KmAKR-W297H/Y296W/K29H/Y28A/T63M in Table 2), According to the method described in Example 2, the wet cells of the aldehyde ketone reductase mutant were obtained, and the cells were collected by centrifugation at 8000 rpm and 4° C. for 10 minutes, and washed twice with 0.9% (w/v) saline. Add 100g/L of the total amount of wet bacteria to the pH 7.0, 100mM PBS buffer and resuspend, sonicate for 6min in the ice-water mixture. The conditions of ultrasonication: power 400W, break for 1s, pause for 1s, and obtain the crude mutant strain. Enzyme solution. The supernatant was collected by centrifugation at 8000 rpm and 4°C for 10 min (the electrophoresis diagram is shown in Figure 3), and after microfiltration through a 0.45 μm membrane, the mutant protein was purified using a Ni affinity column.
使用镍亲和柱(1.6×10cm，Bio-Rad公司，美国)纯化突变体蛋白，具体操作如下：①用缓冲液A(含0.3M NaCl、20mM咪唑的pH 8.0、20mM PBS缓冲液)进行预平衡。②用缓冲液A以1.0mL/min的流速洗去未结合的杂质，直至电导率稳定。③然后用缓冲液B(含0.3M NaCl、500mM咪唑的pH 8.0、20mM PBS缓冲液)洗脱目的蛋白。将收集的洗脱液用20mM PBS缓冲液(pH 7.0)透析过夜。所有纯化步骤均在4℃下进行。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定蛋白质大小。对照菌株E.coli BL21(DE3)/pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A的醛酮还原酶纯酶采用相同条件收集，电泳结果示于图3，突变菌株的目标酶表达量较E.coli BL21(DE3)/pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A菌株的酶表达量没有显著变化，因此，突变体的酶活提高不是酶的表达量增加引起的，与酶自身比活力增加有关。Use a nickel affinity column (1.6×10cm, Bio-Rad, USA) to purify the mutant protein. The specific operation is as follows: ①Prepare with buffer A (containing 0.3M NaCl, 20mM imidazole, pH 8.0, 20mM PBS buffer) balance. ② Wash away unbound impurities with buffer A at a flow rate of 1.0 mL/min until the conductivity is stable. ③Then use buffer B (pH 8.0 containing 0.3M NaCl, 500mM imidazole, 20mM PBS buffer) to elute the target protein. The collected eluate was dialyzed overnight with 20mM PBS buffer (pH 7.0). All purification steps were performed at 4°C. The protein size was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The pure aldehyde ketone reductase enzyme of the control strain E. coli BL21(DE3)/pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A was collected under the same conditions. The electrophoresis results are shown in Figure 3. The target enzyme expression level of the mutant strain Compared with E. coli BL21(DE3)/pET28a(+)-kmakr-W297H/Y296W/K29H/Y28A strain, the enzyme expression level has no significant change. Therefore, the increase in the enzyme activity of the mutant is not caused by the increase in the expression level of the enzyme. The enzyme itself is related to the increase in specific activity.
实施例5：母本醛酮还原酶及其突变体酶比酶活的测定Example 5: Determination of the specific enzyme activity of the parent aldehyde ketone reductase and its mutants
酶活单位(U)定义为：在35℃、pH 7.0条件下，每分钟每生成1微摩尔6-氰基-(3R,5R)-二羟基己酸叔丁酯所需的酶量定义为一个酶活单位，U。比酶活定义为每毫克酶蛋白所具有的活力单位数，U/mg。Enzyme activity unit (U) is defined as: the amount of enzyme required to generate 1 micromole of 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl ester per minute at 35°C and pH 7.0 is defined as A unit of enzyme activity, U. Specific enzyme activity is defined as the number of units of activity per milligram of enzyme protein, U/mg.
酶活检测标准条件：5mM 6-氰基-(5R)-羟基-3-羰基己酸叔丁酯，0.25mM NADPH，适量酶液，35℃、pH 7.0，500rpm条件下反应3min，样品处理并进行HPLC检测分析。Standard conditions for enzyme activity detection: 5mM 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester, 0.25mM NADPH, appropriate amount of enzyme solution, 35℃, pH 7.0, 500rpm conditions for 3 minutes, sample processing and Perform HPLC detection and analysis.
蛋白浓度用二喹啉甲酸蛋白测定试剂盒(南京凯基生物科技发展有限公司，南京)测定。The protein concentration was determined with a quinolinic acid protein determination kit (Nanjing KGI Biotechnology Development Co., Ltd., Nanjing).
表3醛酮还原酶及其突变体的相对酶活和差向对应异构体选择性(de p)值 Table 3 Relative enzyme activity and epimeric selectivity (de p ) values of aldehyde ketone reductase and its mutants
Figure PCTCN2020085538-appb-000004
Figure PCTCN2020085538-appb-000004
实施例6：醛酮还原酶对照组KmAKR-W297H/Y296W/K29H/Y28A不对称还原6-氰基-(5R)-羟基-3-羰基己酸叔丁酯Example 6: Aldone reductase control group KmAKR-W297H/Y296W/K29H/Y28A asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester
根据实施例2的描述，通过发酵获得醛酮还原酶对照组KmAKR-W297H/Y296W/K29H/Y28A湿菌体和葡萄糖脱氢酶EsGDH湿菌体。在建立的双酶偶联体系中，将湿菌体KmAKR-W297H/Y296W/K29H/Y28A和葡萄糖脱氢酶EsGDH湿菌体催化6-氰基-(5R)-羟基-3-羰基己酸叔丁酯生成6-氰基-(3R,5R)-二羟基己酸叔丁酯。According to the description of Example 2, the aldehyde ketone reductase control group KmAKR-W297H/Y296W/K29H/Y28A wet bacteria and glucose dehydrogenase EsGDH wet bacteria were obtained through fermentation. In the established two-enzyme coupling system, the wet bacteria KmAKR-W297H/Y296W/K29H/Y28A and the glucose dehydrogenase EsGDH catalyzed the 6-cyano-(5R)-hydroxy-3-carbonylhexanoic acid tert Butyl ester produces 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl ester.
以干重比3.5:1(w/w)混合成混合菌体，在50mL反应体系中，先将混合菌体用pH 7.0、100mM的PBS缓冲液重悬，转化体系中混合菌体加入干重量为20g DCW/L，当底物6-氰基-(5R)-羟基-3-羰基己酸叔丁酯的投料量为170g/L，葡萄糖浓度为255g/L，以pH 7.0、100mM的PBS缓冲液为反应介质构成转化体系，在30℃、600rpm条件下反应，4.5h能完全转化成产物6-氰基-(3R,5R)-二羟基己酸叔丁酯，底物转化率大于99％，产物de p值始终保持在99.5％以上。产物的浓度为549mM，时空产率达到670.5g/L d。 Mix the mixed bacteria with a dry weight ratio of 3.5:1 (w/w). In a 50mL reaction system, first resuspend the mixed bacteria with pH 7.0, 100mM PBS buffer, and add dry weight to the mixed bacteria in the transformation system. 20g DCW/L, when the substrate 6-cyano-(5R)-hydroxy-3-carbonylhexanoic acid tert-butyl ester is 170g/L, the glucose concentration is 255g/L, and the pH is 7.0, 100mM PBS The buffer is the reaction medium to form the conversion system. It can be completely converted into the product 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester in 4.5h under the conditions of 30℃ and 600rpm. %, the product de p value always remains above 99.5%. The concentration of the product was 549mM, and the space-time yield reached 670.5g/L d.
实施例7：醛酮还原酶突变体KmAKR-W297H/Y296W/K29H/Y28A/T63A不对称还原6-氰基-(5R)-羟基-3-羰基己酸叔丁酯Example 7: Asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester by aldehyde-ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63A
根据实施例2的描述，通过发酵获得醛酮还原酶突变体KmAKR-W297H/Y296W/K29H/Y28A/T63A湿菌体和葡萄糖脱氢酶EsGDH湿菌体。在建立的的双酶偶联体系中，将湿菌体KmAKR-W297H/Y296W/K29H/T63A和葡萄糖脱氢酶EsGDH湿菌体以干重比3.5:1(w/w)混合成混合菌体，先将混合菌体用pH 7.0、100mM的PBS缓冲液重悬，转化体系中混合菌体加入干重量为20g DCW/L，底物6-氰基-(5R)-羟基-3-羰基己酸叔丁酯的投料量为200g/L，葡萄糖浓度为200g/L，pH 7.0、100mM PBS缓冲液为反应介质构建转化体系50mL，30℃、400rpm反应，反应进程曲线如图5所示，底物在2.5h能完全转化成产物6-氰基-(3R,5R)-二羟基己酸叔丁酯，且产物的累积量达到549.3mM，产物de p值>99.5％，时空产率达到1207.7 g/L d。继续增加底物浓度到300g/L时，6-氰基-(5R)-羟基-3-羰基己酸叔丁酯能完全转化成6-氰基-(3R,5R)-二羟基己酸叔丁酯，且在反应时间为6.0h，反应达到终点，底物的转化率大于99％，产物de p值>99.5％。 According to the description of Example 2, the aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63A wet bacteria and the glucose dehydrogenase EsGDH wet bacteria were obtained through fermentation. In the established two-enzyme coupling system, the wet bacteria KmAKR-W297H/Y296W/K29H/T63A and the glucose dehydrogenase EsGDH wet bacteria were mixed into a mixed bacteria with a dry weight ratio of 3.5:1 (w/w) , First resuspend the mixed bacteria in pH 7.0, 100mM PBS buffer, add 20g DCW/L to the mixed bacteria in the transformation system, the substrate 6-cyano-(5R)-hydroxy-3-carbonylhexyl The dosage of tert-butyl ester is 200g/L, the glucose concentration is 200g/L, the pH 7.0, 100mM PBS buffer is used as the reaction medium to construct a 50mL conversion system, and the reaction is performed at 30°C and 400rpm. The reaction process curve is shown in Figure 5. The product can be completely converted into the product 6-cyano-(3R,5R)-dihydroxyhexanoate tert-butyl within 2.5h, and the cumulative amount of the product reaches 549.3mM, the product de p value>99.5%, and the space-time yield reaches 1207.7 g/L d. When the substrate concentration is increased to 300g/L, the tert-butyl 6-cyano-(5R)-hydroxy-3-carbonylhexanoate can be completely converted into tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate Butyl ester, and the reaction time is 6.0h, the reaction reaches the end, the conversion rate of the substrate is greater than 99%, and the de p value of the product is greater than 99.5%.
实施例8：醛酮还原酶突变体KmAKR-W297H/Y296W/K29H/Y28A/T63L不对称还原6-氰基-(5R)-羟基-3-羰基己酸叔丁酯Example 8: Asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester by aldehyde-ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63L
同实施例7的方法通过发酵获得醛酮还原酶突变体KmAKR-W297H/Y296W/K29H/Y28A/T63L湿菌体和葡萄糖脱氢酶EsGDH湿菌体以干重比3.5:1(w/w)混合成混合菌体，在50mL反应体系中，混合菌体先用pH 7.0、100mM PBS缓冲液重悬，反应体系中混合菌体的投加总干重量为20g DCW/L，将底物6-氰基-(5R)-羟基-3-羰基己酸叔丁酯的初始投料量设定为300g/L，葡萄糖的浓度为300g/L，pH 7.0、100mM PBS缓冲液为反应介质构建转化体系，30℃、600rpm反应。反应进程曲线如图6所示，产物浓度随时间的推移而逐渐升高，5.0h反应完成，可完全转化成740.3mM的产物，产物de p值>99.5％，时空产率可达1152.2g/L d。将底物浓度提高为370g/L，6-氰基-(5R)-羟基-3-羰基己酸叔丁酯能完全转化成6-氰基-(3R,5R)-二羟基己酸叔丁酯，且在反应时间为6.5h，反应达到终点，可完全转化成799.2mM的产物，产物de p值>99.5％。 The aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63L wet cell and the glucose dehydrogenase EsGDH wet cell were obtained by fermentation with the same method as in Example 7 with a dry weight ratio of 3.5:1 (w/w) Mix into a mixed bacteria. In a 50mL reaction system, the mixed bacteria are first resuspended in pH 7.0, 100mM PBS buffer. The total dry weight of the mixed bacteria in the reaction system is 20g DCW/L, and the substrate 6- The initial dosage of cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl is set to 300g/L, the concentration of glucose is 300g/L, pH 7.0, 100mM PBS buffer is used as the reaction medium to construct the conversion system, Reaction at 30°C and 600 rpm. The reaction process curve is shown in Figure 6. The product concentration gradually increases with the passage of time. The reaction is completed in 5.0 hours and can be completely converted into 740.3mM product. The product de p value>99.5%, and the space-time yield can reach 1152.2g/ L d. Increasing the substrate concentration to 370g/L, tert-butyl 6-cyano-(5R)-hydroxy-3-carbonylhexanoate can be completely converted into tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate Ester, and the reaction time is 6.5h, the reaction reaches the end point, can be completely converted into 799.2mM product, the product de p value>99.5%.
实施例9：醛酮还原酶突变体KmAKR-W297H/Y296W/K29H/Y28A/T63M不对称还原6-氰基-(5R)-羟基-3-羰基己酸叔丁酯Example 9: Asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl ester by aldehyde-ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63M
同实施例7的方法通过发酵获得醛酮还原酶突变体KmAKR-W297H/Y296W/K29H/Y28A/T63M湿菌体和葡萄糖脱氢酶EsGDH湿菌体以干重比3.5:1(w/w)混合成混合菌体，在50mL反应体系中，混合菌体先用pH 7.0、100mM PBS缓冲液重悬，反应体系中混合菌体的投加总干重量为20g DCW/L，将底物6-氰基-(5R)-羟基-3-羰基己酸叔丁酯的初始投料量设定为400g/L，葡萄糖的浓度为400g/L，pH 7.0、100mM PBS缓冲液为反应介质构建转化体系，30℃、600rpm反应。反应进程曲线如图7所示，产物浓度随时间的推移而逐渐升高，5.5h反应完成，可完全转化成829.6mM的产物，产物de p值>99.5％，时空产率可达1427.2g/L d。继续增加底物的浓度，在同样的反应体系中加入终浓度450g/L的6-氰基-(5R)-羟基-3-羰基己酸叔丁酯，反应条件与上述方法相同，产物浓度随时间的推移而逐渐升高，7.0h内反应完成，底物转化率大于99％，产物de p值始终保持在99.5％以上。产物的浓度为881.1mM，产物de p值>99.5％，时空产率达到1224.3g/L d。 The aldehyde ketone reductase mutant KmAKR-W297H/Y296W/K29H/Y28A/T63M wet cell and the glucose dehydrogenase EsGDH wet cell were obtained by fermentation with the same method as in Example 7 with a dry weight ratio of 3.5:1 (w/w) Mix into a mixed bacteria. In a 50mL reaction system, the mixed bacteria are first resuspended in pH 7.0, 100mM PBS buffer. The total dry weight of the mixed bacteria in the reaction system is 20g DCW/L, and the substrate 6- The initial dosage of cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl is set to 400g/L, the concentration of glucose is 400g/L, pH 7.0, 100mM PBS buffer is used as the reaction medium to construct the conversion system, Reaction at 30°C and 600 rpm. The reaction process curve is shown in Figure 7. The product concentration gradually increases with the passage of time. The reaction is completed in 5.5 hours and can be completely converted into 829.6mM product. The product de p value is >99.5%, and the space-time yield can reach 1427.2g/ L d. Continue to increase the concentration of the substrate, add 6-cyano-(5R)-hydroxy-3-carbonylhexanoate tert-butyl at a final concentration of 450g/L to the same reaction system. The reaction conditions are the same as the above method, and the product concentration varies with It gradually increased with the passage of time, the reaction was completed within 7.0h, the substrate conversion rate was greater than 99%, and the product de p value remained above 99.5%. The product concentration is 881.1mM, the product de p value is >99.5%, and the space-time yield reaches 1224.3g/L d.
实施例10：醛酮还原酶KmAKR-W297H/Y296W/K29H/Y28A/T63A不对称还原6-氯-(5S)-羟基-3-羰基己酸叔丁酯Example 10: Asymmetric reduction of 6-chloro-(5S)-hydroxy-3-carbonylhexanoate tert-butyl ester by aldehyde and ketone reductase KmAKR-W297H/Y296W/K29H/Y28A/T63A
根据实施例2的描述，通过发酵获得醛酮还原酶对照组KmAKR-W297H/Y296W/K29H/Y28A/T63A湿菌体和葡萄糖脱氢酶EsGDH湿菌体。在建立的双酶偶联催化体系中，将湿菌体KmAKR-W297H/Y296W/K29H/Y28A/T63A和葡萄糖脱氢酶EsGDH湿菌体以干重比3.5:1(w/w)混合成混合菌体，先将混合菌体用pH 7.0、100mM的PBS缓冲液重悬，转化体系中混合菌体加入干重量为20g DCW/L，当底物6-氯-(5S)-羟基-3-羰基己酸叔丁酯的投料量为300g/L，葡萄糖浓度为300g/L时，底物在30℃、600rpm反应5h能完全转化成产物6-氯-(3R,5S)-二羟基己酸叔丁酯，底物转化率大于99％，产物de p值始终保持在99.5％以上。产物的浓度为632.9mM，时空产率达到720.2g/L d。 According to the description of Example 2, the aldehyde ketone reductase control group KmAKR-W297H/Y296W/K29H/Y28A/T63A wet bacteria and glucose dehydrogenase EsGDH wet bacteria were obtained through fermentation. In the established double enzyme coupling catalytic system, the wet bacteria KmAKR-W297H/Y296W/K29H/Y28A/T63A and the glucose dehydrogenase EsGDH wet bacteria are mixed into a mixture with a dry weight ratio of 3.5:1 (w/w) For the bacteria, first resuspend the mixed bacteria with pH 7.0, 100mM PBS buffer, and add the dry weight of 20g DCW/L to the mixed bacteria in the transformation system. When the substrate 6-chloro-(5S)-hydroxy-3- When the dosage of tert-butyl carbonylhexanoate is 300g/L and the glucose concentration is 300g/L, the substrate can be completely converted into the product 6-chloro-(3R,5S)-dihydroxyhexanoic acid after reacting at 30℃ and 600rpm for 5 hours. For tert-butyl ester, the substrate conversion rate is greater than 99%, and the de p value of the product always remains above 99.5%. The concentration of the product was 632.9mM, and the space-time yield reached 720.2g/L d.
实施例11：醛酮还原酶KmAKR-W297H/Y296W/K29H/Y28A/T63L不对称还原6-氯-(5S)-羟基-3-羰基己酸叔丁酯Example 11: Asymmetric reduction of 6-chloro-(5S)-hydroxy-3-carbonylhexanoate tert-butyl ester by aldehyde and ketone reductase KmAKR-W297H/Y296W/K29H/Y28A/T63L
根据实施例2的描述，通过发酵获得醛酮还原酶对照组KmAKR-W297H/Y296W/K29H/Y28A/T63L湿菌体和葡萄糖脱氢酶EsGDH湿菌体。在建立的双酶偶联催化体系中，将湿菌体KmAKR-W297H/Y296W/K29H/Y28A/T63L和葡萄糖脱氢酶EsGDH湿菌体以干重比3.5:1(w/w)混合成混合菌体，先将混合菌体用pH 7.0、100mM的PBS缓冲液重悬，转化体系中混合菌体加入干重量为20g DCW/L，当底物6-氯-(5S)-羟基-3-羰基己酸叔丁酯的投料量为350g/L，葡萄糖浓度为350g/L时，底物在30℃、600rpm反应4h能完全转化成产物6-氯-(3R,5S)-二羟基己酸叔丁酯，底物转化率大于99％，产物de p值始终保持在99.5％以上。产物的浓度为738.4mM，时空产率达到1050.4g/L d。 According to the description of Example 2, the aldehyde ketone reductase control group KmAKR-W297H/Y296W/K29H/Y28A/T63L wet bacteria and glucose dehydrogenase EsGDH wet bacteria were obtained through fermentation. In the established double enzyme coupling catalytic system, the wet bacteria KmAKR-W297H/Y296W/K29H/Y28A/T63L and the glucose dehydrogenase EsGDH wet bacteria are mixed into a mixture with a dry weight ratio of 3.5:1 (w/w) For the bacteria, first resuspend the mixed bacteria with pH 7.0, 100mM PBS buffer, and add the dry weight of 20g DCW/L to the mixed bacteria in the transformation system. When the substrate 6-chloro-(5S)-hydroxy-3- When the dosage of tert-butyl carbonylhexanoate is 350g/L and the glucose concentration is 350g/L, the substrate can be completely converted into the product 6-chloro-(3R,5S)-dihydroxyhexanoic acid after 4 hours at 30℃ and 600rpm. For tert-butyl ester, the substrate conversion rate is greater than 99%, and the de p value of the product always remains above 99.5%. The concentration of the product was 738.4 mM, and the space-time yield reached 1050.4 g/L d.
实施例12：醛酮还原酶KmAKR-W297H/Y296W/K29H/Y28A/T63M不对称还原6-氯-(5S)-羟基-3-羰基己酸叔丁酯Example 12: Asymmetric reduction of 6-chloro-(5S)-hydroxy-3-carbonylhexanoate tert-butyl ester by aldehyde and ketone reductase KmAKR-W297H/Y296W/K29H/Y28A/T63M
根据实施例2的描述，通过发酵获得醛酮还原酶对照组KmAKR-W297H/Y296W/K29H/Y28A/T63M湿菌体和葡萄糖脱氢酶EsGDH湿菌体。在建立的双酶偶联催化体系中，将湿菌体KmAKR-W297H/Y296W/K29H/Y28A/T63M和葡萄糖脱氢酶EsGDH湿菌体以干重比3.5:1(w/w)混合成混合菌体，先将混合菌体用pH 7.0、100mM的PBS缓冲液重悬，转化体系中混合菌体加入干重量为20g DCW/L，当底物6-氯-(5S)-羟基-3-羰基己酸叔丁酯的投料量为400g/L，葡萄糖浓度为 400g/L时，底物在30℃、600rpm反应4h能完全转化成产物6-氯-(3R,5S)-二羟基己酸叔丁酯，底物转化率大于99％，产物de p值始终保持在99.5％以上。产物的浓度为843.8mM，时空产率达到1188.2g/L d。 According to the description of Example 2, the aldehyde ketone reductase control group KmAKR-W297H/Y296W/K29H/Y28A/T63M wet bacteria and glucose dehydrogenase EsGDH wet bacteria were obtained through fermentation. In the established double enzyme coupling catalytic system, the wet bacteria KmAKR-W297H/Y296W/K29H/Y28A/T63M and the glucose dehydrogenase EsGDH wet bacteria are mixed into a mixture with a dry weight ratio of 3.5:1 (w/w) For the bacteria, first resuspend the mixed bacteria with pH 7.0, 100mM PBS buffer, and add the dry weight of 20g DCW/L to the mixed bacteria in the transformation system. When the substrate 6-chloro-(5S)-hydroxy-3- When the dosage of tert-butyl carbonylhexanoate is 400g/L and the glucose concentration is 400g/L, the substrate can be completely converted into the product 6-chloro-(3R,5S)-dihydroxyhexanoic acid after 4 hours at 30℃ and 600rpm. For tert-butyl ester, the substrate conversion rate is greater than 99%, and the de p value of the product always remains above 99.5%. The concentration of the product was 843.8mM, and the space-time yield reached 1188.2g/L d.
实施例13：醛酮还原酶KmAKR-W297H/Y296W/K29H/Y28A/T63A、KmAKR-W297H/Y296W/K29H/Y28A/T63L、KmAKR-W297H/Y296W/K29H/Y28A/T63M不对称还原系列羰基化合物Example 13: Ald and Ketone Reductase KmAKR-W297H/Y296W/K29H/Y28A/T63A, KmAKR-W297H/Y296W/K29H/Y28A/T63L, KmAKR-W297H/Y296W/K29H/Y28A/T63M asymmetric reduction series of carbonyl compounds
根据实施例2的描述，通过发酵获得醛酮还原酶突变体KmAKR-W297H/Y296W/K29H/Y28A/T63A、KmAKR-W297H/Y296W/K29H/Y28A/T63L、KmAKR-W297H/Y296W/K29H/Y28A/T63M湿菌体和葡萄糖脱氢酶EsGDH湿菌体。在建立的双酶偶联催化体系中，分别将醛酮还原酶突变体湿菌体和葡萄糖脱氢酶EsGDH湿菌体以干重比3.5:1(w/w)混合成混合菌体，先将混合菌体用pH 7.0、100mM的PBS缓冲液重悬，转化体系中混合菌体加入干重量为4.0g DCW/L，总体系为10mL，系列羰基化合物底物投料量50g/L、葡萄糖浓度50g/L、35℃，600rpm反应12h，进行转化率的检测。转化率检测方法参照文章(Enzym.Microb.Technol.,2017,107:32–40)方法。结果示于表4。According to the description of Example 2, the aldehyde ketone reductase mutants KmAKR-W297H/Y296W/K29H/Y28A/T63A, KmAKR-W297H/Y296W/K29H/Y28A/T63L, KmAKR-W297H/Y296W/K29H/Y28A/ T63M wet cells and glucose dehydrogenase EsGDH wet cells. In the established two-enzyme coupling catalytic system, the aldehyde ketone reductase mutant wet cell and the glucose dehydrogenase EsGDH wet cell were mixed into a mixed cell at a dry weight ratio of 3.5:1 (w/w). Resuspend the mixed bacteria in a pH 7.0, 100mM PBS buffer, add 4.0g DCW/L to the mixed bacteria in the transformation system, the total system is 10mL, the serial carbonyl compound substrate dosage is 50g/L, and the glucose concentration The reaction was carried out at 50g/L, 35°C, 600rpm for 12h, and the conversion rate was detected. The conversion rate detection method refers to the article (Enzym.Microb.Technol.,2017,107:32-40) method. The results are shown in Table 4.
表4醛酮还原酶KmAKR突变体催化系列羰基化合物不对称还原反应的结果Table 4 Results of asymmetric reduction of carbonyl compounds catalyzed by the KmAKR mutant of aldehyde and ketone reductase
Figure PCTCN2020085538-appb-000005
Figure PCTCN2020085538-appb-000005
Figure PCTCN2020085538-appb-000006
Figure PCTCN2020085538-appb-000006

Claims (9)

  1. 一种马克斯克鲁维酵母醛酮还原酶KmAKR突变体，其特征在于所述醛酮还原酶KmAKR突变体是将SEQ ID NO.2所示氨基酸序列第63位点进行定点饱和突变获得的。A Kluyveromyces marxianus aldehyde ketone reductase KmAKR mutant is characterized in that the aldehyde ketone reductase KmAKR mutant is obtained by site-directed saturation mutation at position 63 of the amino acid sequence shown in SEQ ID NO.2.
  2. 如权利要求1所述醛酮还原酶KmAKR突变体，其特征在于所述突变体为下列之一：(1)第63位苏氨酸突变为丙氨酸；(2)第63位苏氨酸突变为亮氨酸；(3)第63位苏氨酸突变为蛋氨酸。The aldehyde-ketone reductase KmAKR mutant of claim 1, wherein the mutant is one of the following: (1) Threonine at position 63 is mutated to alanine; (2) Threonine at position 63 Mutation to leucine; (3) Threonine at position 63 was mutated to methionine.
  3. 一种权利要求1所述醛酮还原酶KmAKR突变体在不对称还原羰基化合物制备手性醇中的应用，其特征在于所述应用方法为：将含醛酮还原酶突变体基因的工程菌经诱导培养获得的湿菌体和含葡萄糖脱氢酶基因的工程菌经诱导培养获得的湿菌体混合，以混合菌体或混合菌体提取的粗酶液为催化剂，以羰基化合物为底物，以葡萄糖为辅底物，采用pH 7.0、100mM的PBS缓冲液为反应介质构成转化体系，在30-35℃、400～600rpm条件下进行反应，反应结束，反应液分离纯化，获得手性醇化合物。An application of the KmAKR mutant of aldehyde and ketone reductase in claim 1 in the preparation of chiral alcohols by asymmetric reduction of carbonyl compounds, characterized in that the application method is: the engineered bacteria containing the aldehyde and ketone reductase mutant gene are subjected to The wet bacteria obtained by the induction culture are mixed with the wet bacteria obtained by the induction culture of the engineered bacteria containing the glucose dehydrogenase gene. The mixed bacteria or the crude enzyme solution extracted from the mixed bacteria are used as the catalyst, and the carbonyl compound is used as the substrate. Glucose is used as the auxiliary substrate, and the pH 7.0, 100mM PBS buffer is used as the reaction medium to form the conversion system. The reaction is carried out at 30-35°C and 400-600rpm. After the reaction is completed, the reaction solution is separated and purified to obtain the chiral alcohol compound. .
  4. 如权利要求3所述的应用，其特征在于所述转化体系中，底物终浓度30～450g/L，葡萄糖终浓度30～450g/L，催化剂用量以混合菌体总量干重计为0.1～20g DCW/L，所述混合菌体中含醛酮还原酶突变体基因的工程菌经诱导培养获得的湿菌体与含葡萄糖脱氢酶基因的工程菌经诱导培养获得的湿菌体以干重比1.0～5.0:1。The application according to claim 3, characterized in that in the conversion system, the final concentration of substrate is 30-450g/L, the final concentration of glucose is 30-450g/L, and the amount of catalyst is 0.1 based on the total dry weight of the mixed bacteria. ~20g DCW/L, in the mixed bacteria, the wet bacteria obtained by induction culture of the engineered bacteria containing the aldone reductase mutant gene and the wet bacteria obtained by the induction culture of the engineered bacteria containing the glucose dehydrogenase gene are based on The dry weight ratio is 1.0～5.0:1.
  5. 如权利要求3所述的应用，其特征在于所述羰基化合物为下列之一：4-氯-3-羰基丁酸乙酯、6-氯-(5S)-羟基-3-羰基己酸叔丁酯、3-羰基丁酸乙酯、4-溴-3-羰基丁酸丙酯、4,4,4,-三氟-3-羰基丁酸乙酯、6-氰基-(5R)-羟基-3-羰基己酸叔丁酯、3-羰基丁酸叔丁酯、苯乙酮。The application according to claim 3, wherein the carbonyl compound is one of the following: ethyl 4-chloro-3-carbonylbutyrate, tert-butyl 6-chloro-(5S)-hydroxy-3-carbonylhexanoate Ester, ethyl 3-carbonylbutyrate, propyl 4-bromo-3-carbonylbutyrate, ethyl 4,4,4,-trifluoro-3-carbonylbutyrate, 6-cyano-(5R)-hydroxyl Tert-Butyl-3-carbonylhexanoate, tert-butyl 3-carbonylbutanoate, acetophenone.
  6. 如权利要求3所述的应用，其特征在于当底物为6-氰基-(5R)-羟基-3-羰基己酸叔丁酯时，所述应用方法为：将含醛酮还原酶突变体基因的工程菌经诱导培养获得的湿菌体和含葡萄糖脱氢酶基因的工程菌经诱导培养获得的湿菌体混合，以混合菌体为催化剂，以6-氰基-(5R)-羟基-3-羰基己酸叔丁酯为底物，以葡萄糖为辅底物，以pH 7.0、100mM的PBS缓冲液为反应介质构成转化体系，在30℃、400～600rpm条件下进行反应，反应结束，反应液分离纯化，获得6-氰基-(3R,5R)-二羟基己酸叔丁酯。The application according to claim 3, wherein when the substrate is tert-butyl 6-cyano-(5R)-hydroxy-3-carbonylhexanoate, the application method is: mutating the aldehyde-containing ketone reductase The wet bacterial cells obtained by the induction culture of the engineered bacteria containing the glucose dehydrogenase gene are mixed with the wet bacterial cells obtained by the induction culture of the engineered bacteria containing the glucose dehydrogenase gene. The mixed bacterial cells are used as a catalyst and 6-cyano-(5R)- Tert-butyl hydroxy-3-carbonylhexanoate is the substrate, glucose is the auxiliary substrate, and the pH 7.0, 100mM PBS buffer is used as the reaction medium to form the conversion system. The reaction is carried out at 30°C and 400-600rpm. At the end, the reaction solution was separated and purified to obtain 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester.
  7. 如权利要求3所述的应用，其特征在于当底物为6-氯-(5S)-羟基-3-羰基己酸叔丁酯时，所述应用方法为：将含醛酮还原酶突变体基因的工程菌经诱导培养获得的湿菌体和含葡萄糖脱氢酶基因的工程菌经诱导培养获得的湿菌体混合，以混合菌体为催化剂，以6-氯-(5S)-羟基-3-羰基己酸叔丁酯为底物，以葡萄糖为辅底物，以pH 7.0、100mM的PBS 缓冲液为反应介质构成转化体系，在30℃、400～600rpm条件下进行反应，反应结束，反应液分离纯化，获得6-氯-(3R,5S)-二羟基己酸叔丁酯。The application according to claim 3, wherein when the substrate is tert-butyl 6-chloro-(5S)-hydroxy-3-carbonylhexanoate, the application method is: adding a mutant containing aldehyde and ketone reductase The wet bacteria obtained by induction culture of genetically engineered bacteria and the wet bacteria obtained by induction culture of engineered bacteria containing glucose dehydrogenase gene are mixed, using the mixed bacteria as a catalyst, and 6-chloro-(5S)-hydroxy- Tert-butyl 3-carbonylhexanoate is used as the substrate, glucose is used as the auxiliary substrate, and the pH 7.0, 100mM PBS buffer is used as the reaction medium to form the conversion system. The reaction is carried out at 30°C and 400-600rpm, and the reaction is complete. The reaction liquid was separated and purified to obtain 6-chloro-(3R,5S)-dihydroxyhexanoic acid tert-butyl ester.
  8. 如权利要求3所述的应用，其特征在于所述湿菌体按如下方法制备：将携带醛酮还原酶突变体基因的大肠杆菌工程菌接种到含终浓度50μg/mL卡那霉素的LB液体培养基中，37℃培养10h，以体积浓度1.5％的接种量接种到新鲜的含终浓度50μg/mL卡那霉素的LB液体培养基中，37℃、180rpm培养2h，再向培养液中加入终浓度为0.15mM异丙基硫代半乳糖苷，28℃培养12h后，4℃、8000rpm离心10min，获得含醛酮还原酶突变体的湿菌体；所述含葡萄糖脱氢酶基因的工程菌经诱导培养获得的湿菌体制备方法同含醛酮还原酶基因的湿菌体。The application according to claim 3, characterized in that the wet bacteria are prepared as follows: the engineered Escherichia coli bacteria carrying the aldehyde ketone reductase mutant gene are inoculated into LB with a final concentration of 50 μg/mL kanamycin Incubate in liquid medium at 37°C for 10 hours, inoculate fresh LB liquid medium with a final concentration of 50μg/mL kanamycin at a volume concentration of 1.5% inoculum, incubate at 37°C, 180rpm for 2 hours, and then add to the culture medium After adding 0.15mM isopropylthiogalactoside at a final concentration of 0.15mM, cultured at 28°C for 12h, and then centrifuged at 4°C and 8000rpm for 10min to obtain a wet bacterial cell containing the aldone reductase mutant; the glucose dehydrogenase gene The preparation method of the wet bacterial cells obtained by induction culture of the engineered bacteria is the same as that of the wet bacterial cells containing the aldehyde ketone reductase gene.
  9. 如权利要求3所述的应用，其特征在于所述粗酶液按如下方法制备：按照湿菌体总量100g/L的量加入pH 7.0、100mM PBS缓冲液中，在冰水混合物上超声破碎6min，超声破碎条件：功率为400W，破碎1s、暂停1s，取破碎混合液，获得粗酶液。The application according to claim 3, characterized in that the crude enzyme solution is prepared by the following method: adding 100g/L of the total amount of wet bacteria to the pH 7.0, 100mM PBS buffer, and ultrasonically disintegrating on an ice-water mixture 6min, ultrasonic crushing conditions: power 400W, crush for 1s, pause for 1s, take the crushing mixture to obtain the crude enzyme solution.
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