WO2020087626A1 - 一种醇脱氢酶突变体及其在辅酶再生中的应用 - Google Patents

一种醇脱氢酶突变体及其在辅酶再生中的应用 Download PDF

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WO2020087626A1
WO2020087626A1 PCT/CN2018/118368 CN2018118368W WO2020087626A1 WO 2020087626 A1 WO2020087626 A1 WO 2020087626A1 CN 2018118368 W CN2018118368 W CN 2018118368W WO 2020087626 A1 WO2020087626 A1 WO 2020087626A1
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alcohol dehydrogenase
mutant
seq
sodium
buffer
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倪晔
朱诚
许国超
周婕妤
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江南大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C12P13/222—Phenylalanine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)

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  • the invention provides an alcohol dehydrogenase mutant and its application in coenzyme regeneration, which belongs to the technical field of enzyme engineering and biological engineering.
  • the coenzyme NAD (P) H-dependent biological asymmetric reduction reaction has many advantages, such as high selectivity, mild conditions, and environmental friendliness, and is the most promising method for preparing chiral compounds.
  • the coenzyme NAD (P) H used is very expensive. For the sustainability and efficiency of the biological asymmetric reduction reaction, as well as to reduce the cost of the biological asymmetric reduction reaction, coenzyme regeneration is required.
  • the existing coenzyme regeneration methods include enzymatic regeneration, electrochemical regeneration and photochemical regeneration.
  • enzymatic regeneration has fast reaction speed, high selectivity, good compatibility between the regeneration system and the synthesis system, and the process is easy to control, etc.
  • Advantages has greater industrial application potential.
  • isopropanol and ethanol have good redox potential ( ⁇ G'-7.4kcal ⁇ mol –1 ) and are inexpensive. Therefore, alcohol dehydrogenase (ADH) is used to catalyze the oxidation of isopropanol and ethanol for coenzyme
  • ADH alcohol dehydrogenase
  • the auxiliary product acetaldehyde produced by the oxidation of ethanol by alcohol dehydrogenase is toxic, and the acetic acid produced by further catalytic oxidation will lower the pH of the reaction system, which is not conducive to the reaction; isopropanol is dehydrogenated by alcohol
  • the auxiliary product acetone produced by enzyme-catalyzed oxidation is unstable and toxic, and the process of isopropyl alcohol being oxidized by alcohol dehydrogenase to produce acetone is a reversible reaction, so that alcohol dehydrogenase (ADH) is used to catalyze the oxidation of isopropanol.
  • ADH alcohol dehydrogenase
  • the process of coenzyme regeneration usually requires the addition of excess isopropanol to maintain the entire reaction process towards the target product synthesis. Therefore, there is an urgent need to find a new coenzyme regeneration method to overcome the defects of the above technology.
  • Lavandera I and Kroutil W used the alcohol dehydrogenase SyADH derived from Sphinobium Yanoikuyae to catalyze the oxidation of a secondary alcohol with electron-withdrawing groups (such as chlorine, fluorine, methoxy, etc.) acetone for coenzyme regeneration.
  • electron-withdrawing groups such as chlorine, fluorine, methoxy, etc.
  • auxiliary substrate Because this kind of auxiliary substrate has an electron-withdrawing group, it can form a hydrogen bond with the hydroxyl group in the auxiliary product to stabilize the auxiliary product ( ⁇ G'-11.1kcal / mol), making the reaction tend to be irreversible, called quasi-irreversible reaction (Quasi-irreversible reaction), therefore, using this system, with the assistance of only 1.5 times the substrate equivalent of 1-chloroacetone, 30g / L 2-octanol can be completely oxidized in 24 hours of reaction (Lavandera I., et.al., Org Lett., 2018, 10, 2155-2158).
  • 1,4-butanediol can be used as an intelligent auxiliary substrate, which is first reduced to 4-hydroxybutyraldehyde during the catalytic oxidation process under the action of alcohol dehydrogenase, and then spontaneously converted to 2-hydroxytetrahydrofuran , And finally further irreversibly reduced to the final co-product ⁇ -butyrolactone (GBL), the whole process of ⁇ G 'is -8.2kcal ⁇ mol –1 , and two molecules of coenzyme NADH are released, therefore, 1,4-butan Glycol can be used as a smart cosubstrate for the regeneration of coenzymes, and further research on this process later found that the co-product ⁇ -butyrolactone produced by this process has kinetic and thermodynamic stability Characteristics, can change the chemical balance of the Meerwein-Ponndorf-Verley reaction to reduce the excessive addition of auxiliary substrates (Kara S., et al., Green Chem
  • the present invention provides an alcohol dehydrogenase mutant and its application in coenzyme regeneration.
  • This alcohol dehydrogenase mutant is obtained by mutating the 84th valine and / or 127th tyrosine of the alcohol dehydrogenase whose starting amino acid sequence is shown in SEQ ID No.
  • Hydrogenase mutants have a high catalytic activity for a variety of alcohol secondary substrates (such as 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol) and can catalyze these alcohols
  • the coenzyme-like substrate undergoes coenzyme NADPH regeneration; compared with the wild-type alcohol dehydrogenase KpADH, this alcohol dehydrogenase mutant has higher activity and catalytic efficiency, and its k cat to the co-substrate 1,4-butanediol
  • the value can be up to 75.9min -1 , which is 7.3 times that of wild-type KpADH, the value of k cat / K m can be up to 2009min –1 ⁇ M –1 , which is 14.7 times that of wild-type KpADH, and the minimum value of K m can be 11.3mM, Compared with the wild-type alcohol dehydrogenas
  • the present invention provides a mutant of alcohol dehydrogenase.
  • the mutant is obtained by setting the amino acid sequence of the starting amino acid sequence as shown in SEQ ID No. 1 at the 84th valine and / or 127th position of the alcohol dehydrogenase It is obtained by mutating tyrosine.
  • the mutant is M1, M2, M3, M4 or M5;
  • the M1 is obtained by mutating the 84th valine of the alcohol dehydrogenase to isoleucine as shown in SEQ ID No. 1;
  • the M2 is obtained by mutating the 127th tyrosine of the alcohol dehydrogenase to cysteine as shown in SEQ ID No. 1;
  • the M3 is obtained by mutating the 127th tyrosine of the alcohol dehydrogenase as SEQ ID No. 1 to methionine;
  • the M4 is obtained by mutating the 84th valine of the alcohol dehydrogenase to isoleucine and the 127th tyrosine to cysteine by the amino acid sequence shown in SEQ ID No. 1.
  • the M5 is obtained by mutating the 84th valine of the alcohol dehydrogenase to isoleucine and the 127th tyrosine to methionine as shown in SEQ ID No. 1.
  • the amino acid sequence of the mutant is SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, or SEQ ID No. 6.
  • the present invention provides genes encoding the above mutants.
  • the present invention provides a recombinant plasmid carrying the above genes.
  • the recombinant plasmid vector is a pET vector.
  • the recombinant plasmid vector is pET28a (+).
  • the present invention provides a host cell carrying the above gene or the above recombinant plasmid.
  • the host cell is Escherichia coli.
  • the host cell is E. coli BL21 (DE3).
  • the present invention provides the application of the aforementioned mutant or the aforementioned gene or the aforementioned recombinant plasmid or the aforementioned host cell in coenzyme regeneration.
  • the present invention provides a method for producing an alcohol dehydrogenase mutant.
  • the method is to inoculate the above-mentioned host cells into LB medium, and culture at 30-40 ° C and 100-200 r ⁇ min -1 shaker to culture.
  • the absorbance OD 600 in the solution reaches 0.5 ⁇ 1.0, then add isopropyl- ⁇ -D-hexagalactopyranoside (IPTG) to the culture solution to induce 5 ⁇ 10h at 16 ⁇ 30 °C to obtain alcohol dehydrogenase mutant.
  • IPTG isopropyl- ⁇ -D-hexagalactopyranoside
  • the LB medium is an LB medium containing 0-100 ⁇ g / mL kanamycin sulfate.
  • the amount of the isopropyl- ⁇ -D-hexagalactopyranoside (IPTG) added to the culture solution is 0.05 to 1.0 mmol / L.
  • the invention provides a method for producing sodium D-phenylalanine.
  • the method is to use sodium phenylpyruvate and amino acid dehydrogenase DAADH D94A and NADP + and (NH 4 ) 2 SO 4 or NH 4 Cl or CH 3
  • One of COONH 4 as the reaction system, using the above alcohol dehydrogenase mutant and 1,4-butanediol or 1,5-pentanediol or 1,6-hexanediol or isopropanol or 2
  • One of 3-butanediol is used as a coenzyme circulation system, using a buffer solution as a buffer system, and the asymmetric reduction reaction is carried out under the conditions of 30 to 35 ° C and pH 7 to 9 for 1 to 24 hours to obtain sodium D-phenylalanine .
  • the amount of sodium phenylpyruvate added to the reaction system is 10-200 mmol / L.
  • the addition amount of the amino acid dehydrogenase DAADH D94A in the reaction system is 0.5-5 kU / L.
  • the addition amount of the NADP + in the reaction system is 0.1-1.0 mmol / L.
  • the amount of (NH 4 ) 2 SO 4 added in the reaction system is 20-200 mmol / L.
  • the addition amount of the 1,4-butanediol in the reaction system is 5 to 100 mmol / L.
  • the addition amount of the alcohol dehydrogenase mutant in the reaction system is 0.3 to 3 kU / L.
  • the buffer solution is phosphate buffer solution, glycine-sodium hydroxide buffer solution or Tris-HCl buffer solution.
  • the buffer solution is a phosphate buffer solution.
  • the concentration of the phosphate buffer is 10 to 200 mmol / L.
  • the alcohol dehydrogenase mutant of the present invention has a high level of various alcohol auxiliary substrates (such as 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol)
  • the vitality can catalyze the regeneration of these coenzyme substrates NADPH;
  • the alcohol dehydrogenase mutant of the present invention has higher activity and catalytic efficiency, and its k cat value for the auxiliary substrate 1,4-butanediol is up to 75.9min -1 , which is 7.3 times that of wild-type KpADH.
  • the value of k cat / K m can reach 2009min –1 ⁇ M –1 , which is 14.7 times that of wild-type KpADH.
  • the minimum value of K m can be 11.3 mM, which is higher than that of wild-type alcohol.
  • the dehydrogenase KpADH has been significantly reduced, therefore, the alcohol dehydrogenase mutant of the present invention has higher potential industrial application value;
  • the alcohol dehydrogenase mutant of the present invention is coupled to the amino acid dehydrogenase DAADH D94A to catalyze the production of sodium phenylpyruvate to sodium D-phenylalanine with a product production rate of> 99% and an ee value of> 99.9%. Therefore, The alcohol dehydrogenase mutant of the present invention has high-efficiency coenzyme regeneration ability, can be used in amino acid dehydrogenase reaction, and has high industrial promotion value.
  • Figure 1 Whole plasmid PCR nucleic acid electrophoresis of wild-type and alcohol dehydrogenase mutants M1-M5;
  • Figure 2 Gradient elution protein electrophoresis of alcohol dehydrogenase mutants M1 ⁇ M3;
  • FIG. 4 Reaction schematic diagram of enzyme-coupled cofactor regeneration system
  • Figure 5 Chiral chromatogram of the product of enzyme-coupled cofactor regeneration reaction catalyzing the production of D-phenylalanine by sodium phenylpyruvate;
  • Figure 6 Gas chromatogram of ⁇ -butyrolactone coproduct in the enzyme-coupled cofactor regeneration reaction.
  • Alcohol dehydrogenase enzyme activity detection method
  • the total reaction system is 200 ⁇ L, including: 1.0 mM NADP + , 200 mM substrate 1,4-butanediol, glycine sodium hydroxide buffer (Gly-NaOH, 100 mM, pH 9.5)), mix well, and keep at 30 °C for 2 minutes , Add an appropriate amount of enzyme solution to detect the change of light absorption value at 340nm.
  • Enzyme activity (U) EW ⁇ V ⁇ 10 3 / (6220 ⁇ l)
  • EW is the change in absorbance at 340 nm in 1 minute
  • V is the volume of the reaction solution in mL
  • 6220 is the molar extinction coefficient of NAD (P) H in L / (mol ⁇ cm)
  • l is the light Distance, unit is cm.
  • One activity unit (U) corresponds to the amount of enzyme required to catalyze the oxidation of 1 ⁇ mol of NADP + per minute under the above conditions.
  • V max and K m values can be calculated from the intercept of the double reciprocal curve and the X and Y axes;
  • the k cat value can be further calculated according to the formula.
  • AS is the molar concentration of sodium D-phenylalanine obtained by liquid chromatography
  • AR is the molar concentration of sodium L-phenylalanine obtained by liquid chromatography.
  • the auxiliary product ⁇ -butyrolactone was detected by gas phase, using a gas column CP-Chirasil-Dex CB (25m ⁇ 0.25mm ⁇ 0.25 ⁇ m, Agilent Technologies Co. Ltd), the column temperature was kept at 70 °C for 5min, and then at 20 °C / The rate of min is increased to 200 ° C and held for 5 minutes. The temperature of the vaporization chamber and the detector are both set at 250 ° C. The retention time of ⁇ -butyrolactone is 7.92min.
  • the gas phase diagram is shown in Figure 6.
  • Example 1 Construction of recombinant vector containing recombinant M1 ⁇ M5 gene encoding alcohol dehydrogenase and recombinant bacteria
  • the full plasmid PCR method was used to determine the amino acid sequence as shown in SEQ ID No. 1 at position 84 of alcohol dehydrogenase , 127 amino acid residues were subjected to site-directed saturation mutations to obtain recombinant vectors containing M1, M2, and M3. Then, using the M1 recombinant plasmid as a template, site-directed saturation mutations were performed on the 127th position of M1 to obtain M4, M5 using the whole plasmid PCR method Recombinant vector;
  • the primers involved are as follows (all described in the 5'-3 'direction, underlined mutation sites):
  • V84-F whose amino acid sequence is shown in SEQ ID No. 7: GCTTCACCA nnk AACTTCGGC
  • V84-R whose amino acid sequence is shown in SEQ ID No. 8: GCCGAAGTT mnn TGGTGAAGC
  • amino acid sequence is shown as SEQ ID No. 9 Y127-F: ACTGCTTCT nnk GCTTCAATT
  • amino acid sequence is shown in SEQ ID No. 10 Y127-R: AATTGAAGC mnn AGAAGCAGT
  • V84I / Y127C-F whose amino acid sequence is shown in SEQ ID No. 11: ACTGCTTCT tgt GCTTCAATT
  • V84I / Y127C-R whose amino acid sequence is shown in SEQ ID No. 12: AATTGAAGC aca AGAAGCAGT
  • V84I / Y127M-F whose amino acid sequence is shown in SEQ ID No. 13: ACTGCTTCT atg GCTTCAATT
  • V84I / Y127M-R whose amino acid sequence is shown in SEQ ID No. 14: AATTGAAGC cat AGAAGCAGT
  • the PCR reaction system (50 ⁇ L) is: KOD enzyme (2.5 U / mL) 1.0 ⁇ L, template (5-50 ng) 1.0 ⁇ L, dNTP 4.0 ⁇ L, 10 ⁇ reaction buffer 5.0 ⁇ L, and 1.0 ⁇ L each of the upstream and downstream primers, ddH 2 O make up to 50 ⁇ L;
  • the PCR amplification procedure is: (1) Denaturation at 94 °C for 3min, (2) Denaturation at 94 °C for 30sec, (3) Annealing at 54 °C for 30sec, (4) Extension at 72 °C for 150sec, repeat steps (2) to (4) for 10- After 15 cycles, the final extension was 72 ° C for 10 min, and the PCR amplification product was stored at 4 ° C.
  • DpnI restriction enzyme was added to the reaction mixture and incubated at 37 ° C for 1 h.
  • the 10 ⁇ L digested PCR reaction solution was transferred into 50 ⁇ LE.coli BL21 (DE3) competent cells by CaCl 2 thermal conversion method, and It was evenly spread on LB agar plates containing 50 ⁇ g / ml kanamycin sulfate and cultured upside down at 37 ° C for 12 hours to obtain recombinant bacteria containing recombinant vectors.
  • Example 2 Screening of recombinant bacteria containing recombinant vectors
  • Example 1 the recombinant bacteria into which the alcohol dehydrogenase mutants M1 to M3 are introduced are obtained, and the recombinant bacteria plates are cultured upside down to obtain colonies;
  • the supernatant was collected by centrifugation to determine the specific activity of the alcohol dehydrogenase mutant expressed by the recombinant bacteria to 1,4-butanediol, and the alcohol dehydrogenase mutant with improved specific activity was selected and sequenced to verify Recombinant strains containing recombinant vectors that successfully overexpressed alcohol dehydrogenase mutants M1 to M3 (M1 to M3 whole plasmid PCR nucleic acid electrophoresis results are shown in Figure 1).
  • M4 and M5 can be obtained by directly using the recombinant plasmid containing M1 as a template and performing site-directed mutation on the 127th position of M1 by using the whole plasmid PCR method (the results of whole plasmid PCR nucleic acid electrophoresis of M4 and M5 are shown in Figure 1).
  • the recombinant bacteria containing the recombinant vector obtained in Example 1 were inoculated into an LB medium containing kanamycin sulfate (50 ⁇ g / mL) at a transfer rate of 2%, and cultured at 37 ° C in a shaker of 200 r ⁇ min -1 .
  • the column used for purification is a nickel affinity column HisTrap FF crude, which is completed by affinity chromatography using the histidine tag on the recombinant protein.
  • the specific steps are as follows:
  • First use solution A to equilibrate the nickel column load the crude enzyme solution, continue to use solution A to elute the breakthrough peak, and after equilibration, perform gradient washing with solution B (20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4) After desorption, the recombinant protein bound to the nickel column was eluted to obtain purified alcohol dehydrogenase mutants M1 to M5. After purification, the alcohol dehydrogenase protein was concentrated and replaced with PBS (100 mM) using a 10 kDa ultrafiltration tube. , PH 7.0) in the buffer and set aside.
  • the purified alcohol dehydrogenase mutants M1-M5 were assayed for viability and SDS-PAGE analysis.
  • mutant M1 plays an important role in the oxidation of 1,4-butanediol, its k cat value is 75.9 min -1 , which is 7.3 times that of wild-type KpADH, and k cat / K m value is 620 min –1 ⁇ M –1 , which is 4.53 times that of wild-type KpADH; the K m values of M2 and M3 to 1,4-butanediol are 38.1 mM and 12.1 mM, respectively, and the catalytic rates k cat are 27.8 mM and 16.8 mM, It can be seen that the Y127 site significantly reduces the K m value, which has a positive effect on the binding of the substrate 1,4-butanediol; the mutant M5 has the highest k cat / K m value of 2009min –1 ⁇ M –1 is 14.7 times that of WT.
  • Example 5 Kinetic analysis of alcohol dehydrogenase mutants M1 ⁇ M5 on intelligent auxiliary substrates 1,5-pentanediol and 1,6-benzdiol
  • the mutant M3 had a K m value of 1,5-pentanediol decreased from 74.4 mM to 14.3 mM compared to wild-type KpADH, and a K m value of 1,6-hexanediol was changed from 18.5 mM to wild-type KpADH. Reduced to 6.48mM, mainly due to the improvement of substrate affinity;
  • the catalytic efficiency k cat / K m of M3 to 1,5-pentanediol and 1,6-hexanediol is 4.36 and 2.69 times that of wild-type KpADH, respectively;
  • the double mutant M5 K m 1,5- pentanediol and 1,6-hexanediol is 8.21mM and 6.52mM, much lower than the K m value (74.4mM and 18.5 mM) compared to the wild-type KpADH
  • the catalytic efficiency k cat / K m is 2353min –1 ⁇ M –1 and 12032min –1 ⁇ M –1 respectively , which is 3.14 and 5.3 times that of wild-type KpADH. It can be seen that the double mutant M5 is a catalytic oxidation diol
  • the substrate regenerates promising mutants of NADPH.
  • Example 6 Alcohol dehydrogenase mutants M1, M2, M5 coupled amino acid dehydrogenase DAADH D94A catalyzes the production of sodium phenylpyruvate to sodium D-phenylalanine
  • Amino acid dehydrogenase DAADH D94A from Ureibacillus thermosphaericus can catalyze the production of sodium phenylpyruvate from the substrate sodium phenylalanine, D-phenylalanine is an important hand-shaped component of nateglinide for the treatment of type II diabetes , But the catalytic reaction requires the addition of expensive coenzyme NADPH.
  • the following uses the alcohol dehydrogenase mutants M1, M2, M5 obtained in Example 3 to regenerate NADPH to construct an enzyme-coupled cofactor regeneration reaction to generate sodium D-phenylalanine.
  • the specific steps are as follows:
  • Alcohol dehydrogenase mutants M1, M2, M5 or wild KpADH pure enzyme protein concentration 1.5kU / L) or glucose dehydrogenase GDH (1.5kU / L), DAADH D94A (5kU / L), substrate sodium phenylpyruvate (50mM), auxiliary substrate 1,4-butanediol (50mM), (NH 4 ) 2 SO 4 (100mM);
  • the biocatalytic system was reacted under the conditions of 30 ° C and 180r ⁇ min -1 , and the generation rate of sodium D-phenylalanine in the reaction was measured according to the time course.
  • the test results are shown in Table 3.
  • Example 7 Alcohol dehydrogenase mutant M5 coupled amino acid dehydrogenase DAADH D94A catalyzes the production of sodium phenylpyruvate to sodium D-phenylalanine
  • the optimal alcohol dehydrogenase mutant M5 measured in Example 6 was used to regenerate NADPH to construct an enzyme-coupled cofactor regeneration system and reduce the addition amount of the co-substrate 1,4-butanediol to 0.5-fold equivalent, specifically The steps are as follows (the regeneration system reaction is shown in Figure 4):
  • the biocatalytic systems A and B were reacted under the conditions of 30 °C and 180r ⁇ min -1 , and samples were taken according to time course to determine the production rate of sodium D-phenylalanine and co-product ⁇ -butyrolactone in the reaction. Table 4.

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Abstract

一种醇脱氢酶突变体及其在辅酶再生中的应用，属于酶工程以及生物工程技术领域。醇脱氢酶突变体是通过将出发氨基酸序列如SEQ ID No.1所示的醇脱氢酶的第84位缬氨酸和/或第127位酪氨酸进行突变得到的；此醇脱氢酶突变体对多种醇类辅底物均具有较高的活力，可以催化这些醇类辅底物进行辅酶NADPH再生；且与野生型醇脱氢酶KpADH相比，其活性、催化效率更高，对辅底物1,4-丁二醇的k cat值最高可达75.9min -1，k cat/K m值可达2009min –1·M –1，K m值最低可为11.3mM，此醇脱氢酶突变体具有更高的工业应用价值。

Description

一种醇脱氢酶突变体及其在辅酶再生中的应用 技术领域
本发明提供了一种醇脱氢酶突变体及其在辅酶再生中的应用，属于酶工程以及生物工程技术领域。
背景技术
依赖辅酶NAD(P)H的生物不对称还原反应具有选择性高、条件温和、环境友好等众多优点，是最具应用开发潜力的手性化合物制备方法。但是，其所使用的辅酶NAD(P)H价格十分昂贵，为了生物不对称还原反应的可持续性和高效性，也为了降低生物不对称还原反应的成本，需要进行辅酶再生。
现在，已经有的辅酶再生方法有酶法再生、电化学法再生和光化学法再生等，其中，酶法再生因为反应速度快、选择性高、再生体系与合成体系兼容性好及过程容易控制等优点，具有较大的工业应用潜力。
而酶法辅酶再生中，异丙醇和乙醇具有良好的氧化还原潜力(ΔG’-7.4kcal·mol –1)且价格低廉，因此，利用醇脱氢酶(ADH)催化氧化异丙醇和乙醇进行辅酶再生的方法具有价格低廉、原子利用率高等优点。
但是，此方法中，乙醇被醇脱氢酶催化氧化生成的辅产物乙醛具有毒性，进一步催化氧化生成的醋酸会降低反应体系的pH值，不利于反应的进行；异丙醇被醇脱氢酶催化氧化生成的辅产物丙酮则不稳定且具有毒性，且由于异丙醇被醇脱氢酶催化氧化生成丙酮的过程是可逆反应，使得利用醇脱氢酶(ADH)催化氧化异丙醇进行辅酶再生的过程通常需要加入过量的异丙醇才能维持整个反应过程朝向目标产物合成的方向进行。因此，急需找到一种新的辅酶再生方法以克服上述技术的缺陷。
目前，已经有部分针对新型辅酶再生方法的研究取得了一定的进展。例如，2008年，Lavandera I和Kroutil W等利用以Sphingobium yanoikuyae来源的醇脱氢酶SyADH催化带有吸电子基团(如氯、氟、甲氧基等)丙酮的仲醇氧化进行辅酶再生。由于这类辅底物带有吸电子基团，可与辅产物中的羟基形成氢键作用而将辅产物稳定(ΔG’-11.1kcal/mol)，使得反应趋于不可逆，称为准不可逆反应(Quasi-irreversible reaction)，因此，利用该体系，在仅1.5倍底物当量1-氯丙酮协助下，反应24h可将30g/L 2-辛醇完全氧化(Lavandera I.,et al.,Org Lett.,2018,10,2155-2158)。
Hollmann等人则发现：1,4-丁二醇可作为智能辅底物，在醇脱氢酶的作用下催化氧化过程中先被还原生成4-羟基丁醛，再自发转化为2-羟基四氢呋喃，最后进一步不可逆地被还原生成最终辅产物γ-丁内酯(GBL)，整个过程的ΔG’为-8.2kcal·mol –1，并释放出两分子的辅酶NADH，因此，1,4-丁二醇可以作为一种智能辅底物(smart cosubstrate)用于辅酶再生，且后期在针对此过程的进一步研究中还发现，此过程产生的辅产物γ-丁内酯具有动力学和热力学稳定的特点，能够改变Meerwein-Ponndorf-Verley反应的化学平衡从而减少辅底物的过量添加(Kara S.,et al.,Green Chem.2013,15,330-335)。
不过，上述研究依旧存在酶的催化效率低、底物对酶催化有明显的抑制作用等缺陷，因此，针对新的辅酶再生方法的研究仍需进一步展开。
发明内容
为解决上述问题，本发明提供了一种醇脱氢酶突变体及其在辅酶再生中的应用。此醇脱氢酶突变体是通过将出发氨基酸序列如SEQ ID No.1所示的醇脱氢酶的第84位缬氨酸和/或第127位酪氨酸进行突变得到的；此醇脱氢酶突变体对多种醇类辅底物(如1,4-丁二醇、1,5-戊二醇、1,6-己二醇)均具有较高的催化活力，可以催化这些醇类辅底物进行辅酶NADPH再生；与野生型醇脱氢酶KpADH相比，此醇脱氢酶突变体的活性、催化效率更高，其对辅底物1,4-丁二醇的k cat值最高可达75.9min -1，是野生型KpADH的7.3倍，k cat/K m值可达2009min –1·M –1，是野生型KpADH的14.7倍，K m值最低可为11.3mM，较野生型醇脱氢酶KpADH 有了显著的降低，因此，此醇脱氢酶突变体具有更高的工业应用价值。
本发明的技术方案如下：
本发明提供了一种醇脱氢酶的突变体，所述突变体是通过将出发氨基酸序列如SEQ ID No.1所示的醇脱氢酶的第84位缬氨酸和/或第127位酪氨酸进行突变得到的。
在本发明的一种实施方式中，所述突变体为M1、M2、M3、M4或M5；
所述M1是通过将出发氨基酸序列如SEQ ID No.1所示醇脱氢酶的第84位缬氨酸突变为异亮氨酸得到的；
所述M2是通过将出发氨基酸序列如SEQ ID No.1所示醇脱氢酶的第127位酪氨酸突变为半胱氨酸得到的；
所述M3是通过将出发氨基酸序列如SEQ ID No.1所示醇脱氢酶的第127位酪氨酸突变为甲硫氨酸得到的；
所述M4是通过将出发氨基酸序列如SEQ ID No.1所示醇脱氢酶的第84位缬氨酸突变为异亮氨酸以及第127位酪氨酸突变为半胱氨酸得到的；
所述M5是通过将出发氨基酸序列如SEQ ID No.1所示醇脱氢酶的第84位缬氨酸突变为异亮氨酸以及第127位酪氨酸突变为甲硫氨酸得到的。
在本发明的一种实施方式中，所述突变体的氨基酸序列为SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5或SEQ ID No.6。
本发明提供了编码上述突变体的基因。
本发明提供了携带上述基因的重组质粒。
在本发明的一种实施方式中，所述重组质粒载体为pET载体。
在本发明的一种实施方式中，所述重组质粒载体为pET28a(+)。
本发明提供了携带上述基因或上述重组质粒的宿主细胞。
在本发明的一种实施方式中，所述宿主细胞为大肠杆菌(Escherichia coli)。
在本发明的一种实施方式中，所述宿主细胞为大肠杆菌E.coli BL21(DE3)。
本发明提供了上述突变体或上述基因或上述重组质粒或上述宿主细胞在辅酶再生方面的应用。
本发明提供了一种生产醇脱氢酶突变体的方法，所述方法为先将上述宿主细胞接种至LB培养基中，于30～40℃、100～200r·min -1摇床培养至培养液中吸光度OD 600达到0.5～1.0，然后在培养液中加入异丙基-β-D-六代呋喃半乳糖苷(IPTG)于16～30℃下进行诱导5～10h，得到醇脱氢酶突变体。
在本发明的一种实施方式中，所述LB培养基为含有0-100μg/mL硫酸卡那霉素的LB培养基。
在本发明的一种实施方式中，所述异丙基-β-D-六代呋喃半乳糖苷(IPTG)在培养液中的添加量为0.05～1.0mmol/L。
本发明提供了一种生产D-苯丙氨酸钠的方法，所述方法为以苯丙酮酸钠和氨基酸脱氢酶DAADH D94A和NADP +以及(NH 4) 2SO 4或NH 4Cl或CH 3COONH 4中的一种作为反应体系，以上述醇脱氢酶突变体以及1,4-丁二醇或1,5-戊二醇或1,6-己二醇或异丙醇或2,3-丁二醇中的一种作为辅酶循环系统，以缓冲液作为缓冲体系，于30～35℃、pH 7～9的条件下不对称还原反应1～24h，得到D-苯丙氨酸钠。
在本发明的一种实施方式中，所述苯丙酮酸钠在反应体系中的添加量为10-200mmol/L。
在本发明的一种实施方式中，所述氨基酸脱氢酶DAADH D94A在反应体系中的添加量为0.5～5kU/L。
在本发明的一种实施方式中，所述NADP +在反应体系中的添加量为0.1～1.0mmol/L。
在本发明的一种实施方式中，所述(NH 4) 2SO 4在反应体系中的添加量为20～200mmol/L。
在本发明的一种实施方式中，所述1,4-丁二醇在反应体系中的添加量为5～100mmol/L。
在本发明的一种实施方式中，所述醇脱氢酶突变体在反应体系中的添加量为0.3～3kU/L。
在本发明的一种实施方式中，所述缓冲液为磷酸盐缓冲液、甘氨酸-氢氧化钠缓冲液或Tris-HCl缓冲液。
在本发明的一种实施方式中，所述缓冲液为磷酸盐缓冲液。
在本发明的一种实施方式中，所述磷酸盐缓冲液的浓度为10～200mmol/L。
有益效果：
(1)本发明的醇脱氢酶突变体对多种醇类辅底物(如1,4-丁二醇、1,5-戊二醇、1,6-己二醇)均具有较高的活力，可以催化这些醇类辅底物进行辅酶NADPH再生；
(2)与野生型醇脱氢酶KpADH相比，本发明的醇脱氢酶突变体的活性、催化效率更高，其对辅底物1,4-丁二醇的k cat值最高可达75.9min -1，是野生型KpADH的7.3倍，k cat/K m值可达2009min –1·M –1，是野生型KpADH的14.7倍，K m值最低可为11.3mM，较野生型醇脱氢酶KpADH有了显著的降低，因此，本发明的醇脱氢酶突变体具有更高的潜在工业应用价值；
(3)利用本发明的醇脱氢酶突变体偶联氨基酸脱氢酶DAADH D94A催化苯丙酮酸钠生成D-苯丙氨酸钠，产物生成率＞99％，ee值＞99.9％，因此，本发明的醇脱氢酶突变体具有高效的辅酶再生能力，且能够在氨基酸脱氢酶反应中得到应用，具有很高的工业推广价值。
附图说明
图1：野生型和醇脱氢酶突变体M1～M5的全质粒PCR核酸电泳图；
图2：醇脱氢酶突变体M1～M3梯度洗脱蛋白电泳图；
图3：醇脱氢酶突变体M4～M5梯度洗脱蛋白电泳图；
图4：酶偶联型辅因子再生体系反应示意图；
图5：酶偶联型辅因子再生反应催化苯丙酮酸钠生成D-苯丙氨酸产物手性色谱图；
图6：酶偶联型辅因子再生反应中γ-丁内酯辅产物气相色谱图。
具体实施方式
下面结合具体实施例，对本发明进行进一步的阐述，下述实施例中所涉及的引物、载体以及宿主细胞均只用于对本发明进行解释说明，不对本发明有任何限定作用。
下述实施例中涉及的检测方法如下：
醇脱氢酶酶活检测方法：
总反应体系为200μL，包括：1.0mM NADP +、200mM底物1,4-丁二醇、甘氨酸氢氧化钠缓冲液(Gly-NaOH、l00mM、pH 9.5))，充分混匀，30℃保温2min，加入适量的酶液，检测340nm下光吸收值的变化。
用下式计算得到酶活力：
酶活力(U)＝EW×V×10 3/(6220×l)
式中，EW为1分钟内340nm处吸光度的变化；V为反应液的体积，单位为mL；6220为NAD(P)H的摩尔消光系数，单位为L/(mol·cm)；l为光程距离，单位为cm。
1个活力单位(U)对应于上述条件下每分钟催化氧化lμmol NADP +所需的酶量。
k cat值、K m值、k cat/K m值的检测方法：
动力学测定按照酶活测定的方法，10μL不同浓度的醇类底物，1mM NADP +，10μL适当浓度酶，170μLpH 9.5的甘氨酸-氢氧化钠缓冲液，每个样品做三个平行，测定在30℃下OD 340的变化，计算不同浓度下的比活力，进一步计算得米氏常数(K m)和最大反应速率(V max)及催化速率(k cat)，计算公式如下所示：
Figure PCTCN2018118368-appb-000001
由双倒数曲线与X轴和Y轴的截距可以计算出V max和K m值；
Figure PCTCN2018118368-appb-000002
根据公式进一步可以计算得到k cat值。
产物D-苯丙氨酸钠的生成率检测方法：
反应处理后的样品过膜后待检测，液相检测使用Astec CHIROBIOTICTM T手型柱(150mm×4.6mm×5μm,Sigma Technologies Co.Ltd)，流动相为甲醇：超纯水＝70：30，流速为0.5mL·min -1，柱温箱设定为30℃，紫外吸收波长为210nm，产物D-苯丙氨酸钠的保留时间为8.49min,液相图谱如图5所示。
产物D-苯丙氨酸钠的光学纯度检测方法：
光学纯度ee的测定方法：
Figure PCTCN2018118368-appb-000003
式中，A S为液相色谱获得的D-苯丙氨酸钠的摩尔浓度；A R为液相色谱获得的L-苯丙氨酸钠的摩尔浓度。
辅产物γ-丁内酯的生成率检测方法：
辅产物γ-丁内酯由气相检测，使用气相柱为CP-Chirasil-Dex CB(25m×0.25mm×0.25μm,Agilent Technologies Co.Ltd)，柱温于70℃保持5min，然后以20℃/min的速率升温至200℃并保持5min，汽化室和检测器的温度都设定温250℃，γ-丁内酯的保留时间为7.92min，气相图谱如图6所示。
实施例1：含有编码醇脱氢酶突变体M1～M5基因的重组载体及重组菌的构建
具体步骤如下：
以实验室保藏的pET28a-KpADH重组质粒为模板(记载于公开号为CN105936909A的专利申请中)，采用全质粒PCR方法对氨基酸序列如SEQ ID No.1所示的醇脱氢酶的第84位、127位氨基酸残基进行定点饱和突变得到含有M1、M2、M3的重组载体，然后进一步以M1重组质粒为模板，采用全质粒PCR方法对M1的第127位进行定点饱和突变得到含有M4、M5的重组载体；
涉及的引物如下(均按5’-3’方向描述，下划线代表突变位点)：
M1：
氨基酸序列如SEQ ID No.7所示的V84-F：GCTTCACCA nnkAACTTCGGC
氨基酸序列如SEQ ID No.8所示的V84-R：GCCGAAGTT mnnTGGTGAAGC
M2、M3：
氨基酸序列如SEQ ID No.9所示的Y127-F：ACTGCTTCT nnkGCTTCAATT
氨基酸序列如SEQ ID No.10所示的Y127-R：AATTGAAGC mnnAGAAGCAGT
M4：
氨基酸序列如SEQ ID No.11所示的V84I/Y127C-F：ACTGCTTCT tgtGCTTCAATT
氨基酸序列如SEQ ID No.12所示的V84I/Y127C-R：AATTGAAGC acaAGAAGCAGT
M5：
氨基酸序列如SEQ ID No.13所示的V84I/Y127M-F：ACTGCTTCT atgGCTTCAATT
氨基酸序列如SEQ ID No.14所示的V84I/Y127M-R：AATTGAAGC catAGAAGCAGT
其中，PCR反应体系(50μL)为：KOD酶(2.5U/mL)l.0μL、模板(5-50ng)l.0μL、dNTP 4.0μL、10×reaction buffer 5.0μL、上下游引物各1.0μL，ddH 2O补足至50μL；
PCR扩增程序为：(1)94℃变性3min，(2)94℃变性30sec，(3)54℃退火30sec，(4)72℃延伸150sec，重复步骤(2)～(4)进行10-15个循环，最后72℃延伸10min，4℃ 保存PCR扩增产物。
PCR结束后，添加DpnI限制性内切酶于反应混合物中并置于37℃孵育1h，用CaCl 2热转化法将10μL消化后PCR反应液转入50μLE.coli BL21(DE3)感受态细胞，并均匀涂布于含有50μg/ml硫酸卡那霉素的LB琼脂平板，37℃倒置培养12h，得到含有重组载体的重组菌。
实施例2：含有重组载体的重组菌的筛选
具体步骤如下：
根据实施例1得到引入了醇脱氢酶突变体M1～M3的重组菌，并将重组菌平板倒置培养，得到菌落；
在96深孔板内每孔加入300μL含卡那霉素(Kan)的LB液体培养基，再把实施例1中平板上的单菌落用牙签逐一挑出到深孔板，预留两个孔放母本作对照，留两个孔不接种做空白对照，放置到摇床37℃，120r·min –1过夜培养；
第2天将培养得到的重组菌转接到另一块加了600μL已加kan的液体培养基的96深孔板内，原板每孔加入70μL浓度为30％的甘油，-80℃保菌，转接后的深孔板放入摇床，37℃，120r·min –1震荡培养2h后加入诱导剂IPTG(终浓度为0.2mmol·L –1)，温度设为30℃震荡培养5h，然后4℃，4000r·min –1离心10min，倒掉上清液，-80℃冷冻1h以上，然后每孔加入200μL破碎缓冲液(pH 7.5，10mmol·L -1Tris-HCl、750mg·L -1的溶菌酶、10mg·L –1的DNase)摇床37℃，120r·min –1震荡1h。
离心取上清测定重组菌表达得到的醇脱氢酶突变体对1,4-丁二醇的比活力，挑选出比活力提高的醇脱氢酶突变体，并对得到其进行测序验证，得到成功过表达了醇脱氢酶突变体M1～M3的含有重组载体的重组菌(M1～M3的全质粒PCR核酸电泳结果如图1)。
M4、M5可通过直接以含有M1的重组质粒为模板，采用全质粒PCR方法对M1的第127位进行定点突变得到(M4、M5的全质粒PCR核酸电泳结果如图1)。
实施例3：醇脱氢酶突变体M1～M5的表达及纯化
1、表达
具体步骤如下：
将实施例1得到的含有重组载体的重组菌按2％的转接量接种至含有硫酸卡那霉素(50μg/mL)的LB培养基中，37℃，200r·min -1摇床培养，培养液的吸光度OD 600达到0.8时，加入0.2mM的异丙基-β-D-六代呋喃半乳糖苷(IPTG)进行诱导，诱导温度为25℃，诱导8h后，8000r·min -1离心10min获得了分别表达醇脱氢酶突变体M1～M5的重组菌菌体，将收集的菌体悬浮于磷酸钾缓冲液(100mM，pH 7.0)中，超声破碎，得到醇脱氢酶突变体M1～M5。
2、纯化
纯化所使用的柱子为镍亲和柱HisTrap FF crude，利用重组蛋白上的组氨酸标签进行亲和层析来完成，具体步骤如下：
首先使用A液将镍柱平衡，粗酶液上样，继续使用A液将穿透峰洗脱下来，待平衡后用B液(20mM磷酸钠、500mM NaCl、500mM咪唑，pH 7.4)进行梯度洗脱，将结合到镍柱上的重组蛋白洗脱下来，获得纯化后的醇脱氢酶突变体M1～M5，纯化后使用10kDa的超滤管将醇脱氢酶蛋白浓缩并置换到PBS(l00mM，pH 7.0)缓冲液中，备用。
对纯化后的醇脱氢酶突变体M1～M5进行活力测定以及SDS-PAGE分析。
检测结果为：如图2和图3所示，经SDS-PAGE分析，镍柱纯化后，醇脱氢酶突变体M1～M5在40kDa左右显示单条带，且杂蛋白较少，说明柱纯化效果较好。
实施例4：醇脱氢酶突变体M1～M5对智能辅底物1,4-丁二醇的动力学分析
测定KpADH在不同底物浓度和辅酶浓度情况下的活力，并根据活力和底物浓度的倒数做出双倒数曲线，计算动力学参数，检测结果如表1。
由表1可知，突变体M1对于1,4-丁二醇的氧化起着重要的作用，其k cat值为75.9min -1，是野生型KpADH的7.3倍，k cat/K m值为620min –1·M –1，是野生型KpADH的4.53倍；M2和 M3对1,4-丁二醇的K m值分别为38.1mM和12.1mM，催化速率k cat分别为27.8mM和16.8mM，可以看出由于Y127位点显著地降低了K m值，其对底物1,4-丁二醇的结合起着积极的作用；突变体M5有最高的k cat/K m值为2009min –1·M –1，是WT的14.7倍。
表1醇脱氢酶突变体对1,4-丁二醇的动力学参数
Figure PCTCN2018118368-appb-000004
实施例5：醇脱氢酶突变体M1～M5对智能辅底物1,5-戊二醇和1,6-节二醇的动力学分析
测定KpADH在不同底物浓度和辅酶浓度情况下的活力，并根据活力和底物浓度的倒数做出双倒数曲线，计算动力学参数，检测结果如表2。
由表2可知，这两种碳链更长的二醇底物拥有比1,4-丁二醇更优秀的催化效率，其中，突变体M1对1,5-戊二醇和1,6-己二醇的K m值分别为42.5mM和12.3mM，k cat值分别为51.1min -1和64.7min -1，表明了M1与这两个二醇底物的结合能力和催化速率方面都有所提高；
突变体M3对1,5-戊二醇的K m值相比于野生型KpADH从74.4mM降低到14.3mM，对1,6-己二醇的K m值相比于野生型KpADH从18.5mM降低到6.48mM，这主要是由于底物亲和力方面的改善；
M3对1,5-戊二醇和1,6-己二醇的催化效率k cat/K m分别是野生型KpADH的4.36和2.69倍；
双突变M5对1,5-戊二醇和1,6-己二醇的K m值为8.21mM和6.52mM，远低于相比于野生型KpADH的K m值(74.4mM和18.5mM)，催化效率k cat/K m分别是2353min –1·M –1和12032min –1·M –1，是野生型KpADH的3.14和5.3倍，可以看出，双突变体M5是一种催化氧化二醇底物再生NADPH很有前景的突变体。
表2醇脱氢酶突变体对1,5-戊二醇和1,6-己二醇的动力学参数
Figure PCTCN2018118368-appb-000005
实施例6：醇脱氢酶突变体M1、M2、M5偶联氨基酸脱氢酶DAADH D94A催化苯丙酮酸钠生成D-苯丙氨酸钠
来源于Ureibacillus thermosphaericus的氨基酸脱氢酶DAADH D94A能够催化底物苯丙酮酸钠生成产物D-苯丙氨酸钠，D-苯丙氨酸是治疗Ⅱ型糖尿病药物那格列奈的重要手型成分，但 是催化反应中需要昂贵的辅酶NADPH的加入。
下面使用实施例3得到的醇脱氢酶突变体M1、M2、M5再生NADPH构建酶偶联型辅因子再生反应生成D-苯丙氨酸钠，具体步骤如下：
建立底物苯丙酮酸钠50mM的生物催化体系：醇脱氢酶突变体M1、M2、M5或野生KpADH纯酶(蛋白浓度1.5kU/L)或葡萄糖脱氢酶GDH(1.5kU/L)、DAADH D94A(5kU/L)、底物苯丙酮酸钠(50mM)、辅底物1,4-丁二醇(50mM)、(NH 4) 2SO 4(100mM)；
将生物催化体系在30℃和180r·min -1条件下反应，按时间进程取样测定反应中D-苯丙氨酸钠的生成率，检测结果如表3。
由表3可知，反应4h时野生型KpADH产物生成率仅有46.4％，突变体M1，M2再生NADPH反应中D-苯丙氨酸钠生成率分别提高到88.0％和89.0％，突变体M5于4h得到D-苯丙氨酸钠生成率99.9％，e.e.值>99.9％。
表3酶偶联型辅因子再生反应制备D-苯丙氨酸钠
Figure PCTCN2018118368-appb-000006
实施例7：醇脱氢酶突变体M5偶联氨基酸脱氢酶DAADH D94A催化苯丙酮酸钠生成D-苯丙氨酸钠
使用实施例6测得的最优醇脱氢酶突变体M5再生NADPH构建酶偶联型辅因子再生体系，并将辅底物1,4-丁二醇的添加量减少到0.5倍当量，具体步骤如下(再生体系反应如图4)：
建立底物苯丙酮酸钠100mM的生物催化体系A：醇脱氢酶突变体M5(1.5kU/L)、DAADH D94A(5kU/L)、底物苯丙酮酸钠(100mM)、辅底物1,4-丁二醇(50mM)、(NH 4) 2SO 4(200mM)；
建立底物苯丙酮酸钠200mM的生物催化体系B：醇脱氢酶突变体M5(蛋白浓度10mg/mL)、DAADH D94A(5kU/L)、底物苯丙酮酸钠(200mM)、辅底物1,4-丁二醇(100mM)、(NH 4) 2SO 4(200mM)；
将生物催化体系A、B均在30℃和180r·min -1条件下反应，按时间进程取样测定反应中D-苯丙氨酸钠和辅产物γ-丁内酯的生成率，检测结果如表4。
由表4可知，100mM底物反应中，D-苯丙氨酸钠的生成率于2h达到了99.8％，在1h时γ-丁内酯生成率达到90.6％；底物苯丙酮酸钠的添加量进一步提高到200mM，辅底物1,4-丁二醇的添加量也为100mM，产物D-苯丙氨酸钠产物生成率于6h达到99.2％，辅产物γ-丁内酯生成率达到90.3％(D-苯丙氨酸钠产物手性色谱见图5，γ-丁内酯辅产物气相色谱见图6)。
表4使用M5再生NADPH偶联反应中D-苯丙氨酸的测定
Figure PCTCN2018118368-appb-000007
表5使用M5再生NADPH偶联反应中γ-丁内酯的测定
Figure PCTCN2018118368-appb-000008
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2018118368-appb-000009
Figure PCTCN2018118368-appb-000010
Figure PCTCN2018118368-appb-000011
Figure PCTCN2018118368-appb-000012
Figure PCTCN2018118368-appb-000013
Figure PCTCN2018118368-appb-000014
Figure PCTCN2018118368-appb-000015
Figure PCTCN2018118368-appb-000016
Figure PCTCN2018118368-appb-000017
Figure PCTCN2018118368-appb-000018

Claims (23)

  1. 一种醇脱氢酶的突变体，其特征在于，所述突变体是通过将出发氨基酸序列如SEQ ID No.1所示的醇脱氢酶的第84位缬氨酸和/或第127位酪氨酸进行突变得到的；
    所述突变体为M1、M2、M3、M4或M5；
    所述M1是通过将出发氨基酸序列如SEQ ID No.1所示醇脱氢酶的第84位缬氨酸突变为异亮氨酸得到的；
    所述M2是通过将出发氨基酸序列如SEQ ID No.1所示醇脱氢酶的第127位酪氨酸突变为半胱氨酸得到的；
    所述M3是通过将出发氨基酸序列如SEQ ID No.1所示醇脱氢酶的第127位酪氨酸突变为甲硫氨酸得到的；
    所述M4是通过将出发氨基酸序列如SEQ ID No.1所示醇脱氢酶的第84位缬氨酸突变为异亮氨酸以及第127位酪氨酸突变为半胱氨酸得到的；
    所述M5是通过将出发氨基酸序列如SEQ ID No.1所示醇脱氢酶的第84位缬氨酸突变为异亮氨酸以及第127位酪氨酸突变为甲硫氨酸得到的。
  2. 如权利要求1所述的一种醇脱氢酶的突变体，其特征在于，所述突变体的氨基酸序列为SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5或SEQ ID No.6。
  3. 编码权利要求1或2所述突变体的基因。
  4. 携带权利要求3所述基因的重组质粒。
  5. 如权利要求4所述的重组质粒，其特征在于，所述重组质粒载体为pET载体。
  6. 如权利要求4或5所述的重组质粒，其特征在于，所述重组质粒载体为pET28a(+)。
  7. 携带权利要求3所述基因或权利要求4-6任一所述重组质粒的宿主细胞。
  8. 如权利要求7所述的宿主细胞，其特征在于，所述宿主细胞为大肠杆菌(Escherichia coli)。
  9. 如权利要求7或8所述的宿主细胞，其特征在于，所述宿主细胞为大肠杆菌E.coli BL21(DE3)。
  10. 权利要求1或2所述的突变体或权利要求3所述的基因或权利要求4-6任一所述的重组质粒或权利要求7-9任一所述的宿主细胞在辅酶再生方面的应用。
  11. 一种生产醇脱氢酶突变体的方法，其特征在于，所述方法为先将权利要求7-9任一所述的宿主细胞接种至LB培养基中，于30～40℃、100～200r·min -1摇床培养至培养液中吸光度OD 600达到0.5～1.0，然后在培养液中加入异丙基-β-D-六代呋喃半乳糖苷(IPTG)于16～30℃下进行诱导5～10h，得到醇脱氢酶突变体。
  12. 如权利要求11所述的方法，其特征在于，所述LB培养基为含有0-100μg/mL硫酸卡那霉素的LB培养基。
  13. 如权利要求11或12所述的方法，其特征在于，所述异丙基-β-D-六代呋喃半乳糖苷(IPTG)在培养液中的添加量为0.05～1.0mmol/L。
  14. 一种生产D-苯丙氨酸钠的方法，其特征在于，所述方法为以苯丙酮酸钠和氨基酸脱氢酶DAADH D94A和NADP +以及(NH 4) 2SO 4或NH 4Cl或CH 3COONH 4中的一种作为反应体系，以权利要求1或2所述醇脱氢酶突变体以及1,4-丁二醇或1,5-戊二醇或1,6-己二醇或异丙醇或2,3-丁二醇中的一种作为辅酶循环系统，以缓冲液作为缓冲体系，于30～35℃、pH 7～9的条件下不对称还原反应1～24h，得到D-苯丙氨酸钠。
  15. 如权利要求14所述的方法，其特征在于，所述苯丙酮酸钠在反应体系中的添加量为10-200mmol/L。
  16. 如权利要求14或15所述的方法，其特征在于，所述氨基酸脱氢酶DAADH D94A在反应体系中的添加量为0.5～5kU/L。
  17. 如权利要求14-16任一所述的方法，其特征在于，所述NADP +在反应体系中的添加量为0.1～1.0mmol/L。
  18. 如权利要求14-17任一所述的方法，其特征在于，所述(NH 4) 2SO 4在反应体系中的添加 量为20～200mmol/L。
  19. 如权利要求14-18任一所述的方法，其特征在于，所述1,4-丁二醇在反应体系中的添加量为5～100mmol/L。
  20. 如权利要求14-19任一所述的方法，其特征在于，所述醇脱氢酶突变体在反应体系中的添加量为0.3～3kU/L。
  21. 如权利要求14-20任一所述的方法，其特征在于，所述缓冲液为磷酸盐缓冲液、甘氨酸-氢氧化钠缓冲液或Tris-HCl缓冲液。
  22. 如权利要求14-21任一所述的方法，其特征在于，所述缓冲液为磷酸盐缓冲液。
  23. 如权利要求14-22任一所述的方法，其特征在于，所述磷酸盐缓冲液的浓度为10～200mmol/L。
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