WO2024130778A1 - 一种热稳定性提高的(2r,3r)-丁二醇脱氢酶突变体及其应用 - Google Patents

一种热稳定性提高的(2r,3r)-丁二醇脱氢酶突变体及其应用 Download PDF

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WO2024130778A1
WO2024130778A1 PCT/CN2022/143415 CN2022143415W WO2024130778A1 WO 2024130778 A1 WO2024130778 A1 WO 2024130778A1 CN 2022143415 W CN2022143415 W CN 2022143415W WO 2024130778 A1 WO2024130778 A1 WO 2024130778A1
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mutated
amino acid
asparagine
glycine
alanine
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于浩然
丁豪特
蒲中机
曹佳雯
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浙江大学杭州国际科创中心
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    • C12P7/00—Preparation of oxygen-containing organic compounds
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    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01004—R,R-butanediol dehydrogenase (1.1.1.4)
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    • C12R2001/19—Escherichia coli
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    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the invention relates to the field of genetic engineering, and in particular to a (2R, 3R)-butanediol dehydrogenase mutant with improved thermal stability and application thereof.
  • Acetoin chemical name 3-hydroxy-2-butanone, also known as methyl acetyl carbinol, is a smallest unit of ⁇ -hydroxy ketone. Because acetoin has a strong aroma of cream and fat, it is used as a food additive and widely used in the food processing industry; in addition, because acetoin has a high calorific value, it can be used as a high-energy fuel in the aerospace industry. Acetoin is also a good chemical synthesis raw material, which can be used in the synthesis of antifreeze, plasticizers, foaming agents, coatings, etc., and has a wide range of application value.
  • acetoin Since acetoin has a chiral carbon atom, it can form two configurations: R-acetoin (R-AC) and S-acetoin (S-AC). Homochiral acetoin has a higher product added value and can be used to synthesize optically active drugs.
  • acetoin can be used to synthesize 4-chloro-4,5-dimethyl-1,3-dioxolane-2-one, which is mainly used for the modification of penicillin antibiotics to improve the efficacy and reduce side effects; acetoin can also be used to synthesize ampicillin hydrochloride; acetoin can also be used to prepare nicotine derivatives of pyrrolidone or tetrahydropyrrole.
  • homochiral acetoin can also be used in the IT industry as an important component of liquid crystal materials.
  • acetoin by chemical synthesis There are three main processes for producing acetoin by chemical synthesis: partial hydrogenation reduction of diacetyl, selective oxidation of 2,3-butanediol, and hydrolysis of butanone.
  • the production of acetoin by chemical synthesis has the following main disadvantages: first, the yield and yield of the product are low; second, it is difficult to synthesize optically pure acetoin, which requires expensive catalysts and causes environmental pollution; third, the raw materials diacetyl and butanone are derived from petroleum resources. With the shortage of petroleum resources, the cost of raw materials has gradually increased, which is not in line with the current trend of sustainable development.
  • Microbial fermentation is the process of obtaining the target compound through a series of biochemical reactions through the catalytic action of the microorganism's own metabolism and the enzymes produced during the growth of the microorganism.
  • most research work focuses on the screening of wild strains producing acetoin and the optimization of fermentation medium or production process.
  • the production process of microbial fermentation is green and environmentally friendly, but there are many by-products in microbial fermentation, the fermentation process consumes a lot of energy, the yield is low, and none of the current fermentation strains can obtain a fermentation product with an optical purity of more than 99%, so it is not suitable for the production of optically pure acetoin products.
  • Enzymatic conversion methods can be roughly divided into two categories: whole-cell catalysis and in vitro enzyme catalysis.
  • 2,3-Butanediol can be oxidized to acetoin by (2R,3R)-butanediol dehydrogenase.
  • Xiao et al. have constructed a whole-cell catalytic system using (2R,3R)-butanediol dehydrogenase from Bacillus subtilis and NADH oxidase from Lactobacillus brevis, and the yield of acetoin reached 36.7 g/L.
  • a whole-cell catalytic system was constructed by (2R,3R)-butanediol dehydrogenase (BS-BDH) from Bacillus subtilis and NADH oxidase from Lactobacillus brevis.
  • BS-BDH can oxidize (2R,3R)-butanediol to acetoin.
  • the yield of acetoin reached 36.7 g/L, but the yield concentration is still low and cannot meet the needs of industrial production.
  • the whole-cell catalytic system constructed by Paenibacillus polymyxa can achieve a maximum yield of 72.38 g/L of acetoin.
  • the present invention designs a method to improve the thermal stability of BS-BDH based on the Rosetta_ddg calculation tool, transforms the gene by site-directed mutagenesis, and obtains a (2R, 3R)-butanediol dehydrogenase mutant with improved thermal stability, which provides a certain basis for industrial production.
  • the present invention uses Rosetta_ddg to calculate ⁇ G, that is, the difference between the wild-type Gibbs free energy and the mutant Gibbs free energy.
  • ⁇ G that is, the difference between the wild-type Gibbs free energy and the mutant Gibbs free energy.
  • a mutant with ⁇ G ⁇ 0, that is, a mutant with improved thermal stability shown in the calculation results, is selected and constructed through site-directed mutagenesis, and the thermal stability of BS-BDH is characterized by residual enzyme activity and half-life, and single-point mutations are combined to obtain a mutant with significantly improved thermal stability.
  • the present invention provides a (2R, 3R)-butanediol dehydrogenase mutant with improved thermal stability, which is obtained by subjecting a wild-type (2R, 3R)-butanediol dehydrogenase from Bacillus subtilis to a single-point mutation or a double-point mutation, wherein the amino acid sequence of the wild-type (2R, 3R)-butanediol dehydrogenase is shown in SEQ ID NO.2, and the specific single-point mutation is any one of the following:
  • the specific double point mutation is any of the following:
  • (k) amino acid position 61 is mutated from asparagine to glycine/amino acid position 258 is mutated from threonine to glycine;
  • the present invention also provides the use of the (2R, 3R)-butanediol dehydrogenase mutant in the production of 2,3-butanediol/acetoin.
  • the present invention also provides a gene encoding the (2R, 3R)-butanediol dehydrogenase mutant.
  • the invention also provides application of the gene in producing 2,3-butanediol/acetoin.
  • the present invention also provides an expression vector comprising the encoding gene.
  • the invention also provides application of the expression vector in producing 2,3-butanediol/acetoin.
  • the present invention also provides a genetically engineered bacterium for expressing the (2R, 3R)-butanediol dehydrogenase mutant.
  • the invention also provides application of the genetically engineered bacteria in producing 2,3-butanediol/acetoin.
  • the present invention performs site-directed mutagenesis on the BS-BDH sequence to improve its thermal stability.
  • the thermal stability of 21 single-point mutants is improved, among which the thermal stability of the T258G mutant (T258G represents: the 258th amino acid is mutated from threonine to glycine; the same applies to the others.)
  • T258G represents: the 258th amino acid is mutated from threonine to glycine; the same applies to the others.
  • T22V mutant, Q112N mutant, R285K mutant, N61G mutant, A260M mutant and A230R mutant is significantly improved, and the residual enzyme activity is retained by more than 45% after heat treatment at 50°C for 20 minutes.
  • the present invention constructs double-point mutants based on single-point mutants, and the thermal stability of the double-point mutants is further improved, among which Q112N/A260M, A230R/N61G, and N61G/A260M have the most significant improvement effects.
  • the residual enzyme activities of A230R/N61G and N61G/A260M double-point mutants after heat treatment at 50°C for 20 minutes are 93.11% and 85.28%, respectively, and Q112N/A2 60M had the best thermal stability, with 98.09% of the residual enzyme activity retained after heat treatment at 50°C for 20 min, basically retaining all enzyme activity.
  • the method of the present invention effectively improves the thermal stability of (2R, 3R)-butanediol dehydrogenase derived from Bacillus subtilis, which is conducive to alleviating the dilemma that the (2R, 3R)-butanediol dehydrogenase cannot be industrially applied due to its poor thermal stability, thereby promoting the biocatalytic synthesis of acetoin.
  • the plasmid extraction kit used in the embodiments of the present invention was purchased from Hangzhou Aisheng Biotechnology Co., Ltd.; PrimeSTAR Max Premix, DpnI and competent kit were purchased from Beijing Baoriyi Co., Ltd.; E. coli BL21 (DE3) was purchased from Novagen; DNA marker, low molecular weight standard protein, and agarose gel electrophoresis reagent were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; BCA protein concentration determination kit was purchased from Hefei Lanjieke Technology Co., Ltd.; primer synthesis, sequence sequencing and full plasmid pET28a-BS-BDH were completed by Qingke Bioengineering Co., Ltd.
  • T258G represents: the 258th amino acid is mutated from threonine to glycine; the same applies to the others.
  • the above single point mutants were constructed by site-directed mutagenesis, and the specific construction method is as follows:
  • BS-BDH whole plasmid PCR Using the nucleotide sequence shown in SEQ ID NO.1 as a template and the mutant primer sequences in Table 1, PCR was performed to obtain recombinant genes.
  • the recombinant genes are the nucleotide sequence shown in SEQ ID NO.1, in which ACA mutates to GGT (T258G), ACG mutates to GTG (T22V), CAA mutates to AAC (Q112N), CGT mutates to AAA (R285K), AAT mutates to GGT (N61G), GCC mutates to ATG (A260M), GCA mutates to CGT (A230R), TTC mutates to CAT (F1 37H), CAA mutated to TGG (Q252W), TCT mutated to GTG (S130V), GAC mutated to AAC (D295N), AAC mutated to GGT (N8G), GGG mutated to GAA (G330
  • the PCR amplification system is shown in Table 2:
  • the remaining plasmid template was digested with DpnI enzyme.
  • the digestion system is as shown in Table 4:
  • Digestion conditions 37°C for 1 h, 70°C for 15 min.
  • LB medium components peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L; LB solid medium components are based on LB liquid medium components plus 15g/L agar; LB medium needs to be sterilized at 121°C for 20min, and the added concentration before use is 100 ⁇ L/mL.
  • Example 2 The mutants sequenced successfully in Example 1 were streaked and isolated, and single colonies were picked and placed in 5 mL of LB liquid culture medium containing 100 ⁇ L/mL kanamycin, and cultured in a shaker at 37° C. and 220 rpm for 18 h to obtain seed solution.
  • the resuspended bacterial liquid was placed on ice for ultrasonic disruption.
  • the parameters of ultrasonic disruption were: total ultrasonic time 15min, working time 3s, intermittent time 7s, and disruption power 60%.
  • the disrupted bacterial liquid was centrifuged at 4°C12000rpm for 30min to obtain the supernatant (i.e., crude enzyme solution) for subsequent purification.
  • the crude enzyme solution obtained in the previous step was passed through the nickel column.
  • the target protein containing the His tag and some impurity proteins will specifically bind to the nickel column.
  • the collected target protein was ultrafiltered in a centrifuge at 4°C and 4000rpm until the imidazole concentration dropped below 5mM.
  • the final liquid obtained was the pure enzyme solution of the single-point mutant.
  • the reaction is a reversible reaction, with (2R,3R)-butanediol as the substrate and NAD+ as the coenzyme.
  • the substrates NADH and acetoin are generated. Since NADH has an absorbance value at 340nm, the absorbance value at 340nm can be measured to characterize the oxidase activity of BS-BDH.
  • the enzyme activity unit is defined as the amount of enzyme required to reduce 1 ⁇ mol of NAD+ per minute at room temperature.
  • the specific activity of the enzyme is defined as the enzyme activity per unit protein U/mg.
  • Enzyme concentration determination method Mix reagent A and reagent B of the BCA kit at a volume ratio of 50:1, add 200 ⁇ L of mixing reagent to each well of a 96-well plate, and add 20 ⁇ L of enzyme solution to mix. Place the 96-well plate at 37°C for 30 minutes, and detect the absorbance at a wavelength of 562 nm to obtain the enzyme concentration.
  • the enzyme reaction system is 200 ⁇ L, containing 12.5mM (2R, 3R)-butanediol and 0.50mM NAD + .
  • the enzymatic reaction starts immediately after adding a certain amount of enzyme solution.
  • the enzyme activity can be calculated by calculating the NADH generation rate based on the change in the absorbance value of the reaction solution at 340nm. Three groups of parallel experiments were set up for BS-BDH and mutants.
  • thermal stability of BS-BDH and mutants The enzyme solution was kept in a 50°C water bath for 20 minutes, and the enzyme solution after heat treatment was taken out and tested according to the above enzyme activity determination method.
  • the thermal stability of the enzyme can be characterized by the residual enzyme activity (enzyme activity after heat treatment/enzyme activity before heat treatment). Three groups of BS-BDH and mutants were set up for parallel experiments.
  • the protein obtained above was tested for oxidase activity using a microplate reader, and the measurement data results are shown in Table 5. It can be seen from Table 5 that the thermal stability of the 21 single-point mutants is improved to a certain extent. Only the T258G mutant, Q112N mutant, A230R mutant, N8G mutant and L135M mutant have improved stability while their oxidase activity is slightly decreased compared with the wild-type BS-BDH. The most obvious decrease is the L135M mutant, and its activity is only reduced to 78% of the oxidase activity of BS-BDH. Except for the five mutants mentioned above, the oxidative activity of the other mutants remained basically unchanged.
  • the oxidase activity of the T22V mutant, N61G mutant, Q252W mutant, S130V mutant, T263V mutant and A230R mutant was 3-4 times higher than that of the wild-type BS-BDH, among which the T22V mutant (4.232U/mg) and S130V mutant (3.915U/mg) showed the most obvious increase, both increased by 4.17 times.
  • the thermal stability of (2R,3R)-butanediol dehydrogenase was characterized by heat treatment at 50°C for 20min.
  • the characterization results are shown in Table 5.
  • the residual enzyme activity of wild-type BS-BDH after heat treatment at 50°C for 20min was only 15.40%, but the residual enzyme activity of single-point mutants was higher than 15.40%. The results showed that the thermal stability of single-point mutants was improved to a certain extent.
  • the present invention further combines single-point mutants to obtain double-point mutants with more obvious effects.
  • the thermal stability of the T258G mutant, the T22V mutant, the Q112N mutant, the R285K mutant, the N61G mutant, the A260M mutant and the A230R mutant is significantly improved, so the seven single-point mutants are further combined to obtain mutants with better thermal stability.
  • Plasmid template extraction method AxyPrep plasmid extraction kit was used for plasmid extraction. Take 4 mL of single-point mutant culture medium that has been cultured for more than 10 hours, centrifuge at 12000 rpm for 1 minute, discard the supernatant, collect the bacteria in a 2 mL centrifuge tube, add 250 ⁇ L Buffer S1 to the tube to resuspend, then add 250 ⁇ L Buffer S2 lysis solution for lysis, the duration should not exceed 5 minutes, then add 350 ⁇ L Buffer S3 to neutralize the lysis solution; centrifuge at 12000 rpm for 10 minutes, take the supernatant in the preparation tube, centrifuge for 1 minute, let the plasmid adhere to the preparation tube, and then use 500 ⁇ L and 700 ⁇ L of Buffer W1 and Buffer W2 respectively to centrifuge and wash to remove impurities, and finally use 70 ⁇ L of water to elute the plasmid to obtain the template plasmid.
  • plasmids of T258G mutant, T22V mutant, Q112N mutant, R285K mutant, N61G mutant and A260M mutant were extracted and used as templates to construct double-point mutants using the primers in Table 1.
  • the construction process was consistent with the single-point mutant construction method in Example 1.
  • the constructed E. coli vector of the double-point mutant was used to express and purify the double-point mutant protein in the same manner as in Example 2 to obtain a double-point mutant pure enzyme solution for subsequent thermal stability verification.
  • the obtained double point mutants were characterized for thermal stability according to Example 3. Through the thermal stability characterization, 14 double point mutants with further improved thermal stability compared with the single point mutants were obtained.
  • the genes encoding the 14 double point mutants were GCA mutated to CGT (A230R) and GCC mutated to ATG (A260M), GCA mutated to CGT (A230R) and AAT mutated to GGT (N61G), CAA mutated to AAC (Q112N) and GCA mutated to CGT (A230R), GCA mutated to CGT (A230R) and CGT mutated to AAA (R285K), GCA mutated to CGT (A230R) and ACA mutated to GGT (T258G), AAT mutated to GGT (N61G) and GCC mutated to ATG (A260M), CAA mutated to AAC (Q112N) and GCA mutated to CGT (A230R), CC mut
  • the oxidase activity of the double-point mutants was further improved on the basis of the single-point mutants, which was basically increased by 3-5 times compared with the wild-type oxidase activity (1.013U/mg).
  • Q112N/R285K had the most obvious improvement effect, with an oxidase activity of 5.086U/mg, and an oxidase activity increase of 5 times.
  • the thermal stability of the 14 double-point mutants was further improved compared with the single-point mutants, among which Q112N/A260M, A230R/N61G, and N61G/A260M had the most significant improvement.
  • Half-life determination method keep the enzyme solution in a 45°C water bath, take samples every 30 minutes, and test according to the above enzyme activity determination method.
  • the half-life can be calculated based on the relationship between the logarithm of the residual enzyme activity and time.
  • the half-life of the double point mutants is significantly improved compared with the wild type.
  • the half-life of A230R/N61G is 129.32 min
  • the half-life of N61G/A260M is 149.46 min
  • the half-life of Q112N/A260M is 176.82 min.
  • the half-lives are 3.58 times, 4.13 times, and 4.89 times the half-life of the wild-type BS-BDH (36.16 min), respectively.
  • the thermal stability of the double point mutants has been significantly improved.

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Abstract

提供一种热稳定性提高的(2R,3R)-丁二醇脱氢酶突变体及其应用。通过对BS-BDH序列进行了定点突变改造，提高了其热稳定性，与野生型相比，21 个单点突变体其热稳定性得到了提高。并在单点突变体的基础上构建双点突变体，双点突变体的热稳定性得到了进一步提升。所述方法有效提高了枯草芽孢杆菌来源的(2R,3R)-丁二醇脱氢酶的热稳定性，有利于缓解(2R,3R)-丁二醇脱氢酶热稳定性差而导致无法进行工业化应用的困境，从而推进乙偶姻的生物催化法合成。

Description

一种热稳定性提高的(2R,3R)-丁二醇脱氢酶突变体及其应用 技术领域
本发明涉及基因工程领域，具体涉及一种热稳定性提高的(2R,3R)-丁二醇脱氢酶突变体及其应用。
背景技术
乙偶姻，化学名3-羟基-2-丁酮，又叫甲基乙酰甲醇，是一种最小单元的α-羟基酮。由于乙偶姻具有强烈的奶油、脂肪香气，因此，乙偶姻被用作食品添加剂广泛应用于食品加工行业；此外，由于乙偶姻具有较高的热值，可作为高能燃料用于航天工业。乙偶姻还是很好的化学合成原料，可用于防冻剂、增塑剂、起泡剂、涂料等合成，具有广泛的应用价值。
由于乙偶姻具有一个手性碳原子，可以形成R-乙偶姻(R-Acetoin，R-AC)和S-乙偶姻(S-Acetoin，S-AC)两种构型，纯手性乙偶姻具有更高的产品附加值，可用于合成具有光学活性的药物，例如，乙偶姻可以用于合成4-氯-4,5-二甲基-1,3-二氧杂环戊烷-2-酮，主要用于青霉素类抗生素药物的修饰，从而提高药效、减轻副作用；乙偶姻还可以用于合成伦氨苄西林盐酸盐；乙偶姻还可用于制备吡咯烷酮或四氢吡咯的烟碱衍生物。此外，纯手性乙偶姻还可用于IT行业，作为液晶材料的重要成分。
乙偶姻的生产方法主要有三种：化学合成法、微生物发酵法和酶转化法。
化学合成法生成乙偶姻主要有三种工艺：丁二酮部分加氢还原、2,3-丁二醇选择性氧化、丁酮水解工艺。通过化学合成法生产乙偶姻主要有以下缺点：第一、产品的收率和得率较低；第二，合成光学纯乙偶姻难度较大，需要耗费昂贵催化剂，且会造成环境污染；第三、原料丁二酮和丁酮来源于石油资源，随着石油资源的短缺，原料的成本逐步上升，不符合当前的可持续发展潮流。
微生物发酵法，即通过微生物的自身代谢和微生物生长过程中产生 的酶进行的催化作用，经过一系列生化反应获得目的化合物。目前，由于基因工程改造菌株生产乙偶姻的产量较低，大多数研究工作集中于生产乙偶姻野生菌株的筛选和发酵培养基或生产工艺的优化。微生物发酵法生产过程绿色环保，但是微生物发酵中副产物较多，发酵过程耗能多，得率低，而且目前的发酵菌株无一能够获得光学纯度99％以上的发酵产物，因此不适合生产光学纯的乙偶姻产品。
酶转化法大致可以分为两类：全细胞催化和体外酶催化。2,3-丁二醇能够在(2R,3R)-丁二醇脱氢酶的氧化作用下得到乙偶姻，肖等人已经通过枯草芽孢杆菌来源的(2R,3R)-丁二醇脱氢酶和短乳杆菌来源的NADH氧化酶构建全细胞催化体系，乙偶姻的产量达到36.7g/L。
通过枯草芽孢杆菌来源的(2R,3R)-丁二醇脱氢酶(BS-BDH)和短乳杆菌来源的NADH氧化酶构建全细胞催化体系，BS-BDH能够将(2R,3R)-丁二醇氧化生成乙偶姻，乙偶姻的产量达到36.7g/L，但是该产量浓度依旧较低，无法满足工业生产需求。而通过多粘类芽孢杆菌构建的全细胞催化体系，乙偶姻的最高产量可以达到72.38g/L。但枯草芽孢杆菌来源的(2R,3R)-丁二醇脱氢酶的酶活高于多粘类芽孢杆菌，仅仅其热稳定性较差，因此，可能是由于热稳定性较差的原因导致其产量较低。提高BS-BDH的热稳定性有利于全细胞催化或酶催化生产乙偶姻，降低生产成本并提高产量。
发明内容
为了解决BS-BDH热稳定性不高、难以进行工业化应用的技术问题，本发明基于Rosetta_ddg计算工具，对BS-BDH的热稳定性提高进行设计，通过定点突变对该基因进行改造，得到了热稳定性提高的(2R,3R)-丁二醇脱氢酶突变体，为工业化生产提供了一定的依据。
为了解决BS-BDH热稳定性不高的问题，本发明利用Rosetta_ddg进行ΔΔG计算，即野生型吉布斯自由能与突变体吉布斯自由能的差值。选取ΔΔG＜0的突变体，即计算结果上显示热稳定性提高的突变体，通过定点突变构建，并通过残留酶活及半衰期对BS-BDH热稳定性进行表征，并对单点突变进行组合，以获得热稳定性显著提高的突变体。
具体的技术方案如下：
本发明提供了一种热稳定性提高的(2R,3R)-丁二醇脱氢酶突变体，是由来自枯草芽孢杆菌(Bacillus subtilis)的野生型(2R,3R)-丁二醇脱氢酶进行单点突变或双点突变而得，所述野生型(2R,3R)-丁二醇脱氢酶的氨基酸序列如SEQ ID NO.2所示，具体单点突变为以下任意一种：
(1)第258位氨基酸由苏氨酸突变为甘氨酸；
(2)第22位氨基酸由苏氨酸突变为缬氨酸；
(3)第112位氨基酸由谷氨酰胺突变为天冬酰胺；
(4)第285位氨基酸由精氨酸突变为赖氨酸；
(5)第61位氨基酸由天冬酰胺突变为甘氨酸；
(6)第260位氨基酸由丙氨酸突变为甲硫氨酸；
(7)第230位氨基酸由丙氨酸突变为精氨酸；
(8)第137位氨基酸由苯丙氨酸突变为组氨酸；
(9)第252位氨基酸由谷氨酰胺突变为色氨酸；
(10)第130位氨基酸由丝氨酸突变为缬氨酸；
(11)第295位氨基酸由天冬氨酸突变为天冬酰胺；
(12)第8位氨基酸由天冬酰胺突变为甘氨酸；
(13)第330位氨基酸由甘氨酸突变为谷氨酸；
(14)第145位氨基酸由酪氨酸突变为苯丙氨酸；
(15)第276位氨基酸由组氨酸突变为天冬酰胺；
(16)第263位氨基酸由苏氨酸突变为异亮氨酸；
(17)第132位氨基酸由天冬氨酸突变为脯氨酸；
(18)第260位氨基酸由丙氨酸突变为亮氨酸；
(19)第263位氨基酸由苏氨酸突变为缬氨酸；
(20)第135位氨基酸由亮氨酸突变为甲硫氨酸；
(21)第154位氨基酸由丝氨酸突变为丙氨酸；
具体的双点突变为以下任意一种：
(a)第230位氨基酸由丙氨酸突变为精氨酸/第260位氨基酸由丙氨酸突变为甲硫氨酸；
(b)第230位氨基酸由丙氨酸突变为精氨酸/第61位氨基酸由天冬酰胺突变为甘氨酸；
(c)第112位氨基酸由谷氨酰胺突变为天冬酰胺/第230位氨基酸由 丙氨酸突变为精氨酸；
(d)第230位氨基酸由丙氨酸突变为精氨酸/第285位氨基酸由精氨酸突变为赖氨酸；
(e)第230位氨基酸由丙氨酸突变为精氨酸/第258位氨基酸由苏氨酸突变为甘氨酸；
(f)第61位氨基酸由天冬酰胺突变为甘氨酸/第260位氨基酸由丙氨酸突变为甲硫氨酸；
(g)第112位氨基酸由谷氨酰胺突变为天冬酰胺/第260位氨基酸由丙氨酸突变为甲硫氨酸；
(h)第285位氨基酸由精氨酸突变为赖氨酸/第260位氨基酸由丙氨酸突变为甲硫氨酸；
(i)第112位氨基酸由谷氨酰胺突变为天冬酰胺/第61位氨基酸由天冬酰胺突变为甘氨酸；
(j)第61位氨基酸由天冬酰胺突变为甘氨酸/第285位氨基酸由精氨酸突变为赖氨酸；
(k)第61位氨基酸由天冬酰胺突变为甘氨酸/第258位氨基酸由苏氨酸突变为甘氨酸；
(l)第112位氨基酸由谷氨酰胺突变为天冬酰胺/第285位氨基酸由精氨酸突变为赖氨酸；
(m)第112位氨基酸由谷氨酰胺突变为天冬酰胺/第258位氨基酸由苏氨酸突变为甘氨酸；
(n)第285位氨基酸由精氨酸突变为赖氨酸/第258位氨基酸由苏氨酸突变为甘氨酸。
本发明还提供了所述(2R,3R)-丁二醇脱氢酶突变体在生产2,3-丁二醇/乙偶姻中的应用。
本发明还提供了编码所述(2R,3R)-丁二醇脱氢酶突变体的基因。
本发明还提供了所述的基因在生产2,3-丁二醇/乙偶姻中的应用。
本发明还提供了一种包含所述编码基因的表达载体。
本发明还提供了所述表达载体在生产2,3-丁二醇/乙偶姻中的应用。
本发明还提供了一种表达所述(2R,3R)-丁二醇脱氢酶突变体的基因工程菌。
本发明还提供了所述基因工程菌在生产2,3-丁二醇/乙偶姻中的应用。
本发明的有益效果：
本发明对BS-BDH序列进行了定点突变改造，提高了其热稳定性，与野生型相比，21个单点突变体其热稳定性得到了提高，其中T258G突变体(T258G代表：第258位氨基酸由苏氨酸突变为甘氨酸；其他同理。)、T22V突变体、Q112N突变体、R285K突变体、N61G突变体、A260M突变体以及A230R突变体的热稳定性提升显著，在50℃下热处理20min后其残留酶活保留45％以上。
此外，本发明在单点突变体的基础上构建双点突变体，双点突变体的热稳定性得到了进一步提升，其中Q112N/A260M、A230R/N61G、N61G/A260M提升效果最为显著，A230R/N61G、N61G/A260M双点突变体在50℃热处理20min后残留酶活分别保留了93.11％和85.28％，Q112N/A260M热稳定性最佳，在50℃热处理20min后残留酶活保留98.09％，基本保留所有酶活，Q112N/A260M(176.82min)、A230R/N61G(129.32min)、N61G/A260M(149.46min)半衰期分别为野生型BS-BDH半衰期(36.16min)的4.89倍、3.58倍、4.13倍。
本发明方法有效提高了枯草芽孢杆菌来源的(2R,3R)-丁二醇脱氢酶的热稳定性，有利于缓解(2R,3R)-丁二醇脱氢酶热稳定性差而导致无法进行工业化应用的困境，从而推进乙偶姻的生物催化法合成。
具体实施方式
上游基因工程所用试剂：本发明实施例中使用的质粒提取试剂盒购自杭州爱思进生物技术有限公司；PrimeSTAR Max Premix、DpnI及感受态试剂盒购自北京宝日医有限公司；E.coli BL21(DE3)购自Novagen公司；DNA marker、低分子量标准蛋白、琼脂糖凝胶电泳试剂购自北京全式金生物技术有限公司；BCA蛋白浓度测定试剂盒购自合肥兰杰柯科技有限公司；引物合成、序列测序及全质粒pET28a-BS-BDH由擎科生物工程股份有限公司完成。
实施例1
BS-BDH单点突变体的大肠杆菌表达载体的构建。
通过Rosetta_ddg计算工具计算，得到21个热稳定性在计算结果上提高的单点突变体：T258G、T22V、Q112N、R285K、N61G、A260M、A230R、F137H、Q252W、S130V、D295N、N8G、G330E、Y145F、H276N、T263I、D132P、A260L、T263V、L135M、S154A，其中，突变体简写代表如下：T258G代表：第258位氨基酸由苏氨酸突变为甘氨酸；其他同理。通过定点突变的方法构建以上单点突变体，具体构建方法如下：
(1)BS-BDH全质粒PCR：以SEQ ID NO.1所示的核苷酸序列为模板，使用表1中突变体引物序列，分别进行PCR得到重组基因，所述重组基因为SEQ ID NO.1所示的核苷酸序列中的ACA突变为GGT(T258G)、ACG突变为GTG(T22V)、CAA突变为AAC(Q112N)、CGT突变为AAA(R285K)、AAT突变为GGT(N61G)、GCC突变为ATG(A260M)、GCA突变为CGT(A230R)、TTC突变为CAT(F137H)、CAA突变为TGG(Q252W)、TCT突变为GTG(S130V)、GAC突变为AAC(D295N)、AAC突变为GGT(N8G)、GGG突变为GAA(G330E)、TAT突变为TTT(Y145F)、CAT突变为AAC(H276N)、ACC突变为ATT(T263I)、GAT突变为CCG(D132P)、GCC突变为CTG(A260L)、ACC突变为GTG(T263V)、CTT突变为ATG(L135M)、TCT突变为GCG(S154A)。
表1突变体引物序列
引物 序列
T258G-F agtccactggtattgccggtgaaaccg
T258G-R cggcaataccagtggactggatggct
T22V-F gccaaaagtggagccgggaaaagtaaag
T22V-R cggctccacttttggctcttcgatatgttc
Q112N-F ccttgatgaaaacatgggattcctcggct
Q112N-R gaatcccatgttttcatcaaggttgtaggcg
R285K-F taatcaaagaaaaaacagtaaaaggaattatcggatac
R285K-R cttttactgttttttctttgattacgatatcgttcg
N61G-F ccattaacaggtgaaacggcacctgtca
N61G-R gccgtttcacctgttaatgggtgcggtttg
A260M-F ctacaattatgggtgaaaccgtcatcg
A260M-R tttcacccataattgtagtggactggatgg
A230R-F ctgagattcgtgaacgtacaggaggcg
A230R-R tacgttcacgaatctcagcgactacatcg
F137H-F gagcttttgcataaacttcctgatgaattatcatatg
F137H-R ggaagtttatgcaaaagctcttcatccacag
Q252W-F tgttacgatgggccatccagtccacta
Q252W-R ggatggcccatcgtaacaccactggg
S130V-F aatacgtcgtggtggatgaagagcttttgtt
S130V-R catccaccacgacgtattcagagaaaccg
D295N-F gataccgcaacatcttcccggctgtattg
D295N-R ggaagatgttgcggtatccgataattcc
N8G-F gatggcatggtcaaaaggatatccgtattgaac
N8G-R ccttttgaccatgccatcttgctgcc
G330E-F ggcttcgaagctcttattaaagagaaaagcc
G330E-R agagcttcgaagccttcctcgatc
Y145F-F tgaattatcatttgaacaaggcgcgctc
Y145F-R gccttgttcaaatgataattcatcaggaagtttgaac
H276N-F ctgaaatcaacccgaacgatatcgtaatcaa
H276N-R cgttcgggttgatttcagcacctttttccc
T263I-F cggtgaaattgtcatcgtcagcatttgg
T263I-R gatgacaatttcaccggcaattgtagtg
D132P-F tctctgtgccggaagagcttttgttcaaacttc
D132P-R gctcttccggcacagagacgtattcagag
A260L-F ctacaattctgggtgaaaccgtcatcg
A260L-R tttcacccagaattgtagtggactggatgg
T263V-F cggtgaagtggtcatcgtcagcatttgg
T263V-R gatgaccacttcaccggcaattgtagtg
L135M-F ggatgaagagatgttgttcaaacttcctgatgaattat
L135M-R tttgaacaacatctcttcatccacagagac
S154A-F ttgaacctgcggcagttgctctatacgc
S154A-R caactgccgcaggttcaacgagcgcg
PCR扩增体系如下表2：
表2 PCR扩增体系
成分 体积/μL
PrimeSTAR Max DNA Polymerase 25
正向引物 0.5
反向引物 0.5
pET28a-BS-BDH 1
ddH 2O 补足至50
PCR扩增条件如下表3：共计30个循环：
表3 PCR扩增程序
Figure PCTCN2022143415-appb-000001
(2)模板消化：
利用DpnI酶消化剩余的质粒模板，消化体系如下表4：
表4 DpnI酶消化体系
成分 体系/μL
DpnI 1
Quick Cut Buffer 4
DNA 50
消化条件：37℃下1h，70℃下15min。
(3)感受态细胞转化及筛选：
取10μL PCR产物于100μL E.coli BL21(DE3)感受态细胞，置冰上孵育30min；孵育30min后，将离心管于42℃水浴锅中热激90s，立即取出置冰面孵育3min。于无菌操作台内向感受态离心管内加入500μL LB培养基，并置于37℃摇床中220rpm下恒温培养60min。将复苏后的菌体12000rpm离心60s，于无菌操作台内倒去约400μL的上清，将剩余液体与菌体混匀后，用一次性涂布棒将其均匀涂抹于含卡那霉素的LB固体培养基上，倒置37℃培养箱，过夜培养。每个固体培养基上挑取3-4个单菌落进行测序验证。获得上述表1内的21个突变体。
实施例2
BS-BDH单点突变体蛋白表达及纯化。
LB培养基组成成分：蛋白胨10g/L，酵母提取物5g/L，氯化钠10g/L；LB固体培养基的组成成分在LB液体培养基组成成分基础上添加15g/L琼脂；LB培养基需121℃灭菌20min，使用前添加浓度为100μL/mL。
将实施例1中测序成功的突变体进行划线分离，挑取单菌落至5mL含有100μL/mL卡那霉素的LB液体培养基中，37℃220rpm摇床中培养18h以获得种子液。
将500μL种子液转接至200mL含有100μL/mL卡那霉素的LB液体培养基中，于37℃220rpm摇床中培养至OD 600＝0.6-0.8(约3-4h)，加入200μL 1mol/L IPTG，于18℃220rpm摇床中低温诱导18h。
将诱导表达后的菌体收集100mL，4℃4000rpm离心10min，弃掉上清，加入10mL pH 8.0 Tris-HCl缓冲液进行重悬。
重悬的菌体液置于冰面上进行超声破碎，超声破碎的参数为：超声总时15min，工作时间3s，间歇时间7s，破碎功率60％。将破碎后的菌液在4℃12000rpm离心30min，获取上清液(即粗酶液)，用于后续纯化。用十倍柱体积的3mM咪唑Tris-HCl缓冲液(3mM咪唑，20mM、pH＝8.0 Tris-HCl，500mM NaCl)平衡镍柱。将上一步处理获得的粗酶液流过镍柱，此过程含有His标签的目标蛋白和部分杂蛋白会与镍柱特异性结合。采用十倍柱体积的20mM咪唑Tris-HCl缓冲液(20mM咪唑，20mM、pH＝8.0Tris-HCl，500mM NaCl)洗脱镍柱中的杂蛋白。最终用500mM咪唑Tris-HCl缓冲液(500mM咪唑，20mM、pH＝8.0Tris-HCl，500mM NaCl)洗脱目标蛋白。将收集到的目标蛋白于4℃、4000rpm离心机内进行超滤，直至咪唑浓度降至5mM以下。最终得到的液体即为单点突变体的纯酶液。
实施例3
BS-BDH及其突变体的热稳定性测定。
用下述反应进行酶学特性鉴定：
Figure PCTCN2022143415-appb-000002
该反应为可逆反应，以(2R,3R)-丁二醇为底物，NAD+为辅酶，在BS-BDH催化作用下，生成底物NADH和乙偶姻，由于NADH在340nm处具有吸光值，因此可以通过测定340nm处的吸光值，从而表征BS-BDH的氧化酶活。
BS-BDH及突变体酶活力定义：酶活力单位定义为在室温下，每分钟还原1μmol的NAD+所需的酶量为一个酶活力单位U。酶比活力定义为单位蛋白的酶活U/mg。
酶浓度测定方法：BCA试剂盒试剂A与试剂B以50∶1体积比进行混匀，每个96孔板孔内添加200μL混匀试剂，添加20μL酶液混匀。将96孔板置于37℃环境中30min，在562nm波长下检测吸光值，即可得到酶浓度。
BS-BDH及突变体酶活力测定方法：酶反应体系为200μL，含有12.5mM(2R,3R)-丁二醇，0.50mM NAD +，酶促反应在加入一定量的酶液后立即开始。根据反应液在340nm处吸光值的变化，计算NADH生成速率，即可计算酶活力。BS-BDH及突变体设置三组平行进行实验。
BS-BDH及突变体的热稳定性表征：将酶液于50℃水浴锅中进行保温处理，保温时长20min，取出热处理后的酶液，按照上述酶活力测定方法进行检测，通过残留酶活(热处理后酶活/热处理前酶活)即可表征酶的热 稳定性。BS-BDH及突变体设置三组平行进行实验。
表5 BS-BDH及其单点突变体测定结果
突变体 酶活(U/mg) 残留酶活(％)
BS-BDH 1.013±0.092 15.40±0.44
T258G 0.903±0.037 55.95±2.24
T22V 4.232±0.058 81.00±4.46
Q112N 0.947±0.014 57.20±1.37
R285K 1.125±0.061 48.48±1.59
N61G 3.867±0.044 47.75±0.86
A260M 2.344±0.017 47.50±3.76
A230R 0.810±0.016 45.82±0.67
F137H 2.535±0.030 42.69±0.90
Q252W 4.225±0.105 40.45±1.93
S130V 3.915±0.075 37.61±0.63
D295N 2.359±0.065 35.61±1.95
N8G 0.867±0.012 33.93±1.49
G330E 1.239±0.034 33.33±1.57
Y145F 0.968±0.010 27.47±0.48
H276N 1.020±0.032 26.59±0.83
T263I 1.378±0.103 23.95±4.16
D132P 1.220±0.134 23.24±0.57
A260L 1.592±0.107 22.25±0.72
T263V 3.974±0.113 21.81±1.06
L135M 0.790±0.011 18.11±0.49
S154A 1.236±0.174 17.65±0.00
将上述获得的蛋白利用酶标仪进行氧化酶活检测，测定数据结果如表5所示，从表5可以看出，21个单点突变体其热稳定性均有一定的提高，仅T258G突变体、Q112N突变体、A230R突变体、N8G突变体以及L135M突变体在稳定性提高的同时其氧化酶活相较于野生型BS-BDH略有下降，下降最为明显的是L135M突变体，其活性也仅仅下降至BS-BDH氧化酶活的78％。除上述的五个突变体，其余突变体其氧化活性基本保持不变，其中T22V突变体、N61G突变体、Q252W突变体、S130V突变体、T263V突变体以及A230R突变体的氧化酶活相较于野生型BS-BDH提高了3-4倍，其中T22V突变体(4.232U/mg)和S130V突变体(3.915U/mg)提升最为明显，均提高了4.17倍。
通过50℃热处理20min对(2R,3R)-丁二醇脱氢酶的热稳定性进行表征，表征结果如表5所示，野生型BS-BDH在50℃下热处理20min后其残留酶活仅为15.40％，但单点突变体残留酶活均高于15.40％，该结果表明单点突变体其热稳定性均得到了一定的提高。由表5可以看到，在21 个突变体中，T258G突变体、T22V突变体、Q112N突变体、R285K突变体、N61G突变体、A260M突变体以及A230R突变体的热稳定性提升显著，在50℃下热处理20min后其残留酶活保留45％以上，T22V突变体其热稳定性提升最为显著，在50℃下热处理20min后其残留酶活保留81％。
实施例4
双点突变体的大肠杆菌表达载体构建及双点突变体的热稳定性测定。
为了进一步提高热稳定性的改造效果，本发明在单点突变体的基础上进一步进行组合，以获得效果更加明显的双点突变体。单点突变体中，T258G突变体、T22V突变体、Q112N突变体、R285K突变体、N61G突变体、A260M突变体以及A230R突变体热稳定性提升显著，因此将该七个单点突变体进一步组合，获得热稳定更佳的突变体。
质粒模板提取方法：质粒提取使用AxyPrep质粒提取试剂盒。取4mL培养10h以上的单点突变体培养液，12000rpm离心1min，弃上清，收集菌体于2mL离心管中，向管中加入250μL Buffer S1重悬，随后加入250μL Buffer S2裂解液进行裂解，时长不宜超过5min，随即加入350μL Buffer S3中和裂解液；12000rpm离心10min，取上清于制备管中，离心1min，使质粒黏附于制备管中，随及利用Buffer W1、Buffer W2各500μL、700μL离心清洗，去除杂质，最终使用70μL水进行质粒洗脱，获得模板质粒。
利用上述质粒提取方法，对T258G突变体、T22V突变体、Q112N突变体、R285K突变体、N61G突变体以及A260M突变体进行质粒提取，并作为模板，利用表1内的引物进行双点突变体构建，构建过程与实施例1中的单点突变体构建方法一致。
将构建完成的双点突变体的大肠杆菌载体按照实施例2相同方法进行双点突变体蛋白表达及纯化，获得双点突变体纯酶液，以便后续进行热稳定性验证。
将获得的双点突变体按照实施例3进行热稳定性表征，通过热稳定性表征，获得14个在热稳定性上相较于单点突变体进一步提高的双点突变体，编码14个双点突变体的基因分别为SEQ ID NO.1所示的核苷酸序列中的GCA突变为CGT(A230R)和GCC突变为ATG(A260M)、GCA 突变为CGT(A230R)和AAT突变为GGT(N61G)、CAA突变为AAC(Q112N)和GCA突变为CGT(A230R)、GCA突变为CGT(A230R)和CGT突变为AAA(R285K)、GCA突变为CGT(A230R)和ACA突变为GGT(T258G)、AAT突变为GGT(N61G)和GCC突变为ATG(A260M)、CAA突变为AAC(Q112N)和GCC突变为ATG(A260M)、CGT突变为AAA(R285K)和GCC突变为ATG(A260M)、CAA突变为AAC(Q112N)和AAT突变为GGT(N61G)、AAT突变为GGT(N61G)和CGT突变为AAA(R285K)、AAT突变为GGT(N61G)和ACA突变为GGT(T258G)、CGT突变为AAA(R285K)和ACA突变为GGT(T258G)。
双点突变体的氧化酶活及热稳定测定结果如表6所示。
表6 BS-BDH及其双点突变体测定结果
突变体 酶活(U/mg) 残留酶活(％)
BS-BDH 1.013±0.092 15.40±0.44
A230R/A260M 4.380±0.065 71.11±0.75
A230R/N61G 2.939±0.028 93.11±0.39
Q112N/A230R 3.708±0.019 73.82±1.04
A230R/R285K 3.855±0.043 61.49±2.69
A230R/T258G 4.800±0.069 65.08±0.79
N61G/A260M 4.231±0.032 85.28±1.00
Q112N/A260M 3.714±0.045 98.09±1.01
R285K/A260M 3.293±0.021 69.12±0.82
Q112N/N61G 3.859±0.023 74.81±0.81
N61G/R285K 4.522±0.076 60.58±0.00
N61G/T258G 4.457±0.102 72.38±0.79
Q112N/R285K 5.086±0.021 75.50±2.16
Q112N/T258G 4.152±0.011 74.47±0.96
R285K/T258G 4.499±0.047 69.66±0.87
由表6可以看出，双点突变体在单点突变体基础上氧化酶活有了进一步提升，相较于野生型氧化酶活(1.013U/mg)，基本提升了3-5倍，其中Q112N/R285K提升效果最为明显，其氧化酶活为5.086U/mg，氧化酶活 提升了5倍。此外，14个双点突变体热稳定性都相较于单点突变体有了进一步提升，其中Q112N/A260M、A230R/N61G、N61G/A260M提升效果最为显著，A230R/N61G、N61G/A260M双点突变体在50℃热处理20min后残留酶活分别保留了93.11％和85.28％，Q112N/A260M热稳定性最佳，在50℃热处理20min后残留酶活保留98.09％，基本保留所有酶活。
实施例5
BS-BDH及其双点突变体半衰期测定。
为了进一步具体了解提升效果，将对野生型BS-BDH及热稳定性提升最为显著的双点突变体(Q112N/A260M、A230R/N61G、N61G/A260M)进行半衰期测定。
半衰期的测定方法：将酶液于45℃水浴锅中进行保温处理，在每个30min取样，按照上述酶活力测定方法进行检测，通过残留酶活力对数值与时间的关系，即可计算获得半衰期。
表7 BS-BDH及双点突变体的半衰期测定
突变体 半衰期(min)
BS-BDH 36.16
A230R/N61G 129.32
N61G/A260M 149.46
Q112N/A260M 176.82
由表7可以看出，双点突变体相较于野生型半衰期有了明显提升，A230R/N61G的半衰期为129.32min，N61G/A260M的半衰期为149.46min，Q112N/A260M的半衰期为176.82min，半衰期分别为野生型BS-BDH半衰期(36.16min)的3.58倍、4.13倍、4.89倍，双点突变体的热稳定性得到了明显提高。

Claims (8)

  1. 一种热稳定性提高的(2R,3R)-丁二醇脱氢酶突变体，其特征在于，是由来自枯草芽孢杆菌(Bacillus subtilis)的野生型(2R,3R)-丁二醇脱氢酶进行单点突变或双点突变而得，所述野生型(2R,3R)-丁二醇脱氢酶的氨基酸序列如SEQ ID NO.2所示，具体单点突变为以下任意一种：
    (1)第258位氨基酸由苏氨酸突变为甘氨酸；
    (2)第22位氨基酸由苏氨酸突变为缬氨酸；
    (3)第112位氨基酸由谷氨酰胺突变为天冬酰胺；
    (4)第285位氨基酸由精氨酸突变为赖氨酸；
    (5)第61位氨基酸由天冬酰胺突变为甘氨酸；
    (6)第260位氨基酸由丙氨酸突变为甲硫氨酸；
    (7)第230位氨基酸由丙氨酸突变为精氨酸；
    (8)第137位氨基酸由苯丙氨酸突变为组氨酸；
    (9)第252位氨基酸由谷氨酰胺突变为色氨酸；
    (10)第130位氨基酸由丝氨酸突变为缬氨酸；
    (11)第295位氨基酸由天冬氨酸突变为天冬酰胺；
    (12)第8位氨基酸由天冬酰胺突变为甘氨酸；
    (13)第330位氨基酸由甘氨酸突变为谷氨酸；
    (14)第145位氨基酸由酪氨酸突变为苯丙氨酸；
    (15)第276位氨基酸由组氨酸突变为天冬酰胺；
    (16)第263位氨基酸由苏氨酸突变为异亮氨酸；
    (17)第132位氨基酸由天冬氨酸突变为脯氨酸；
    (18)第260位氨基酸由丙氨酸突变为亮氨酸；
    (19)第263位氨基酸由苏氨酸突变为缬氨酸；
    (20)第135位氨基酸由亮氨酸突变为甲硫氨酸；
    (21)第154位氨基酸由丝氨酸突变为丙氨酸；
    具体的双点突变为以下任意一种：
    (a)第230位氨基酸由丙氨酸突变为精氨酸/第260位氨基酸由丙氨酸突变为甲硫氨酸；
    (b)第230位氨基酸由丙氨酸突变为精氨酸/第61位氨基酸由天冬酰 胺突变为甘氨酸；
    (c)第112位氨基酸由谷氨酰胺突变为天冬酰胺/第230位氨基酸由丙氨酸突变为精氨酸；
    (d)第230位氨基酸由丙氨酸突变为精氨酸/第285位氨基酸由精氨酸突变为赖氨酸；
    (e)第230位氨基酸由丙氨酸突变为精氨酸/第258位氨基酸由苏氨酸突变为甘氨酸；
    (f)第61位氨基酸由天冬酰胺突变为甘氨酸/第260位氨基酸由丙氨酸突变为甲硫氨酸；
    (g)第112位氨基酸由谷氨酰胺突变为天冬酰胺/第260位氨基酸由丙氨酸突变为甲硫氨酸；
    (h)第285位氨基酸由精氨酸突变为赖氨酸/第260位氨基酸由丙氨酸突变为甲硫氨酸；
    (i)第112位氨基酸由谷氨酰胺突变为天冬酰胺/第61位氨基酸由天冬酰胺突变为甘氨酸；
    (j)第61位氨基酸由天冬酰胺突变为甘氨酸/第285位氨基酸由精氨酸突变为赖氨酸；
    (k)第61位氨基酸由天冬酰胺突变为甘氨酸/第258位氨基酸由苏氨酸突变为甘氨酸；
    (l)第112位氨基酸由谷氨酰胺突变为天冬酰胺/第285位氨基酸由精氨酸突变为赖氨酸；
    (m)第112位氨基酸由谷氨酰胺突变为天冬酰胺/第258位氨基酸由苏氨酸突变为甘氨酸；
    (n)第285位氨基酸由精氨酸突变为赖氨酸/第258位氨基酸由苏氨酸突变为甘氨酸。
  2. 如权利要求1所述(2R,3R)-丁二醇脱氢酶突变体在生产2,3-丁二醇/乙偶姻中的应用。
  3. 编码权利要求1所述(2R,3R)-丁二醇脱氢酶突变体的基因。
  4. 如权利要求3所述的基因在生产2,3-丁二醇/乙偶姻中的应用。
  5. 一种包含权利要求3所述编码基因的表达载体。
  6. 如权利要求5所述表达载体在生产2,3-丁二醇/乙偶姻中的应用。
  7. 一种表达权利要求1所述(2R,3R)-丁二醇脱氢酶突变体的基因工程菌。
  8. 如权利要求6所述基因工程菌在生产2,3-丁二醇/乙偶姻中的应用。
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