WO2015168866A1 - Double-carbonyl reductase, coding gene of same, and application thereof - Google Patents

Double-carbonyl reductase, coding gene of same, and application thereof Download PDF

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WO2015168866A1
WO2015168866A1 PCT/CN2014/076900 CN2014076900W WO2015168866A1 WO 2015168866 A1 WO2015168866 A1 WO 2015168866A1 CN 2014076900 W CN2014076900 W CN 2014076900W WO 2015168866 A1 WO2015168866 A1 WO 2015168866A1
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pet
seq
amino acid
reductase
alkyl
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PCT/CN2014/076900
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French (fr)
Chinese (zh)
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洪浩
盖吉詹姆斯
陈朝勇
周炎
高峰
吕彤
郭莉娜
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凯莱英医药集团（天津）股份有限公司
凯莱英生命科学技术(天津)有限公司
天津凯莱英制药有限公司
凯莱英医药化学（阜新）技术有限公司
吉林凯莱英医药化学有限公司
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Priority to PCT/CN2014/076900 priority Critical patent/WO2015168866A1/en
Publication of WO2015168866A1 publication Critical patent/WO2015168866A1/en

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    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters

Definitions

  • the present invention relates to the field of biocatalytic synthesis, and in particular to a dicarbonyl reductase, a gene encoding the same, and an application thereof.
  • Chiral alcohols are an important class of intermediate compounds that are widely used in the synthesis of chiral drugs and other chiral fine chemicals.
  • 3R, 5S-dihydroxy-6-benzyloxy-hexanoic acid tert-butyl ester is an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key chiral intermediate for statins, a lipid-lowering drug, It is prepared by chemical synthesis and biocatalytic synthesis.
  • the present invention is directed to a biscarbonyl reductase, a gene encoding the same, and use thereof to simplify the synthesis step of a 3R, 5S-dihydroxy compound, reducing production pollution and production costs.
  • a biscarbonylreductase is provided.
  • the biscarbonylreductase has one of the following amino acid sequences: 1) the amino acid sequence of SEQ N0.1; 2) the amino acid sequence of SEQ N0.1 is substituted, and/or deleted, and/or one or more amino acids. Added and has ooo OH OH O
  • R 2 is stereoselectively reduced to a 1 ⁇ functional amino acid sequence derived from SEQ N0.1, and the amino acid sequence derived from SEQ N0.1 has 80% or more homology with SEQ N0.1, wherein a base, an alkyl group, a cycloalkyl group, an alkyl-substituted aryl group, a halogen-substituted aryl group, an arylalkylheterocyclyl group, a cyclic heteroalkyl group or a cyclic heteroalkylated alkyl group, and R 2 is selected from the group consisting of an alkyl group and a ring. Alkyl, 3 ⁇ 4 alkyl or 3 ⁇ 4 cycloalkyl.
  • a gene encoding the above biscarbonylreductase is provided. Further, the above coding gene has a deoxynucleotide sequence of one of the following: 1) a deoxynucleotide sequence of SEQ N0.2; 2) 80% homologous to the deoxynucleotide sequence of SEQ N0.2 And encoded protein with ooo OH OH O
  • a deoxynucleotide sequence which stereoselectively reduces 1 , OR 2 to an OR 2 function is provided.
  • a recombinant expression vector comprising the above-described double-carbonyl reductase-encoding gene is provided.
  • a transgenic cell line comprising the gene encoding the above biscarbonylreductase is provided.
  • a transgenic recombinant strain comprising the gene encoding the above biscarbonylreductase is provided.
  • a diketone substrate can be reduced in one step to prepare a single optical purity 3R, 5S-dihydroxy compound, which is applied to the synthesis of As a tidal intermediate, a 3R, 5S-dihydroxy compound having an ee value of 99% and a de value of about 93% is obtained, which simplifies the synthesis step, reduces production pollution and production cost, and is suitable for large-scale industrial production.
  • DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS It should be noted that the embodiments in the present application and the features in the embodiments may be combined with each other without conflict.
  • the carbonyl reductase gene of the present invention is derived from Mycobacterium fortuitum subsp. fortuitum DSM 46621.
  • the carbonyl reductase has the function of a dicarbonyl reductase.
  • a biscarbonyl reductase is provided.
  • the bis-carbonyl reductase has an amino acid sequence: 1) the amino acid sequence of SEQ N0.1 (SEQ N0.1 is:
  • DKR dicarbonyloreductase
  • a diketone substrate can be reduced in one step to prepare a single optical purity 3R, 5S-dihydroxy compound, which is applied to a synthetic statin intermediate. It simplifies the synthesis step, reduces production pollution and production costs, and is suitable for large-scale industrial production.
  • the deoxynucleotide sequence of SEQ N0.2 has 80% homology and the encoded protein has o o o OH OH O
  • a deoxynucleotide sequence in which the OR 2 is stereoselectively reduced to the R 2 function - is provided.
  • the vector may be pET-22b (+), pET-22b (+), pET-3a (+), pET-3d (+), pET-l la (+), pET-12a (+), pET-14b (+), pET-15b ( + ), pET-16b (+), pET-17b (+), pET-19b ( + ), pET-20b ( + ), pET-21a ( + ), pET-23a ( + ), pET-23b ( + ), pET-24a ( + ), pET-25b ( + ), pET-26b ( + ), pET-27b ( + ), pET-28a ( + ), pET-29a ( + ), pET-30a ( + ), pET-31b ( + ), pET-32a ( + ), pET-35b ( + ), pET-38b ( + ), pET-39b ( + ), pET-40b ( + ) , p
  • the expression vector is overexpressed in E. coli.
  • the expressed amount of dicarbonyl reductase exhibits a molecular weight of about 30KD on SDS-PAGE. Under 30°C and pH6.0, it can be reduced in one step to obtain 3R with higher optical purity. , 5S-dihydroxy compound.
  • transgenic cell lines and transgenic recombinant bacteria including the above-described coding genes are also within the scope of the present invention. According to an exemplary embodiment of the present invention, there is provided a biscarbonyl reductase as described above
  • the enzyme can reduce the diketone substrate in one step to prepare a single optical purity 3R, 5S-dihydroxy compound, which can be applied to the synthesis of statin intermediates.
  • the dicarbonyl reductase is used in the preparation of tert-butyl 3R, 5S-dihydroxy-6-benzyloxy-hexanoate, wherein
  • Example (1) Cloning and expression of a dicarbonyl reductase derived from Mycobacterium fortuitum subsp. fortuitum DSM 46621 To facilitate expression and identification of the double-carbonyl reductase gene (DKR), at the 5' and 3' ends of the oligonucleotide primers Compatible restriction sites were designed.
  • the primer pairs are as follows: Upstream primer SEQ ID N0.3 : 5'- GGAATTCCATATGACTAACTCTGTAAAAACGGTGACTGTTC-3 '; Downstream primer SEQ ID N0.4: 5' - CCGCTCGAGGTTATATTTGTAGAAACCTTCACCAGAAGCC-3 Amplification
  • coli competent DH5 (x strain, coated on solid LB medium containing 50 ⁇ ⁇ / ⁇ 1 containing ampicillin) (Luria-Bertani medium), culture overnight at 37 ° C. Pick a single colony on the above medium and inoculate it in LB liquid medium containing 50 ⁇ ⁇ / ⁇ 1 containing ampicillin, shake culture at 37 °C overnight, and pass through the plasmid. extracting, after PCR and restriction analysis, the correct cloning vector pET-22b (+) - DKR transformed into E. coli BL21 (DE3), a coating containing 50 ⁇ ⁇ / ⁇ 1 LB medium containing ampicillin LB broth, 37 ° C overnight.
  • the cells are disrupted by a sonicator, 4. C, the supernatant and the precipitate were obtained by centrifugation at 12,000 rpm/min for 20 min, and the supernatant was subjected to SDS-PAGE detection using a vertical electrophoresis apparatus. As a result, it was found that the amount of the expression of the biscarbonylreductase was the highest when induced at 30 ° C for 8 hours.
  • Ooo uses a commercially available starting material or a readily prepared ketone compound ⁇ 0 « 2 as a starting material, wherein R1 is selected from an aromatic group; and R 2 is selected from an alkyl group.
  • the bishydroxy product is represented by the following chemical formula ooo
  • Rl v ⁇ k/kA OR2 wherein R1 is selected from an aryl group; and R 2 is selected from an alkyl group.
  • the nuclear magnetic data of the obtained product are as follows: 400 Hz, CDC13: 57.29 (m, 5H), 4.54 (s, 2H), 4.22 (m, lH), 4.07 (m, 1H), 3.45 to 3.40 (m, 4H), 2.4 l (d, 2H), 1.65 (t, 2H), 1.43 (S, 9H).
  • the organic phase was dried, filtered, and concentrated to give a crude product.
  • the dihydroxy group was 65.0%, the yield was 90%, the ee value was more than 90-99%, and the de value was 80-95%.
  • Example 2 A biscarbonyl reductase (SEQ ID NO. 5) having a sequence homology greater than 90% in Example 1, which is a mutant of SEQ NO.
  • SEQ ID NO. 5 A biscarbonyl reductase having a sequence homology greater than 90% in Example 1, which is a mutant of SEQ NO.
  • compatible restriction sites were designed at the 5' and 3' ends of the oligonucleotide primers.
  • the primer pair is as follows: upstream primer SEQ ID N0.6: 5 '-GGAATTCCATATGACCGAACTGAAACAAATCACC-3 '; downstream primer SEQ ID N0.7: 5 '-CCGCTCGAGACCTTTGTAGTTGTAAAAGCCGTCAC-3 '.
  • Amplified gene sequence SEQ ID NO. 5:
  • amino acid sequence is (SEQ ID NO. 8: MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYD
  • pET-22b (+) can also be pET-3a (+) with Nde l and Xho l respectively.
  • coli DH5 (competent state of x strain, plated in a solid LB culture dish containing 50 ⁇ ⁇ / ⁇ 1 containing ampicillin, and cultured overnight at 37 ° C. Single colonies on the above medium were inoculated to contain 50 ⁇ ⁇ / ⁇ 1 ampicillin-containing LB liquid medium, cultured at 37 °C overnight, after plasmid extraction, PCR identification and double enzyme digestion, the correct cloning vector pET22b ( + ) -DKR was transformed into E. coli BL21 In (DE3), it was applied to LB medium containing 50 ⁇ ⁇ / ⁇ 1 containing ampicillin, and cultured at 37 ° C overnight.
  • the bacterial solution was taken out, and the cells were collected by centrifugation at 8000 rpm/min for 10 min.
  • the cells were disrupted by a sonicator, centrifuged at 12000 rpm/min for 20 min to obtain a supernatant and a precipitate, and the supernatant was subjected to SDS-PAGE detection using a vertical electrophoresis apparatus.
  • SDS-PAGE detection using a vertical electrophoresis apparatus.
  • coli BL21 (DE3) containing recombinant plasmid was induced at a final concentration of IPTG of 0.02 mM for 8 h at 37 ° C, 8 h at 30 ° C and 16 h at 25 ° C for 16 h. Expression, and a negative control without IPTG inducer was also established. After the induction of expression was completed, the cells at three temperatures were centrifuged at 8000 rpm/min for 10 min to collect the cells.
  • the cells were disrupted with a sonicator, 4 ° C, 12000 rpm / min
  • the supernatant and the precipitate were obtained by centrifugation for 20 min, and the supernatant was subjected to SDS-PAGE detection using a vertical electrophoresis apparatus.
  • SDS-PAGE detection using a vertical electrophoresis apparatus.
  • the raw material which has been commercialized on the market or the easily prepared ketone compound 1 ⁇ 2 is used as a starting material, wherein it is selected from an aromatic group; and R 2 is selected from an alkyl group.
  • the bishydroxy product is expressed by the following chemical formula: Kl ⁇ u ⁇ / ⁇ OR2 , wherein is selected from an aromatic group; and R 2 is selected from an alkyl group.
  • the substance was uniformly dispersed in the buffer; 1 mg of NAD + , 4.12 mg of ammonium formate, 2 mg of coenzyme formate dehydrogenase and 4 mg of dicarbonyl reductase were added, the system pH was 6.0, and the temperature was maintained at 30 ⁇ 3 ° C for 15-24 h; The reaction mixture was quenched with ethyl acetate. EtOAc (EtOAc m. %, yield 90%, ee value 92.1%, de value 78.9%.
  • the nuclear magnetic data of the obtained product are as follows: 400 Hz, CDC13: 57.29 (m, 5H), 4.54 (s, 2H), 4.22 (m, lH), 4.07 (m, 1H), 3.45 to 3.40 (m, 4H), 2.4 l (d, 2H), 1.65 (t, 2H), 1.43 (S, 9H).
  • Example 3 A bis-reductase having a sequence homology of greater than 90% in Example 1 (SEQ ID N0.9); the gene is a mutant of SEQ NO.
  • amino acid sequence is (SEQ ID NO. 12: MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDIN
  • coli DH5 (competent state of x strain, and applied to solid LB medium containing 50 ⁇ ⁇ / ⁇ 1 containing ampicillin, and cultured overnight at 37 ° C. Single colonies on the above medium were inoculated to contain 50 ⁇ ⁇ / ⁇ 1 ampicillin-containing LB liquid medium, cultured at 37 °C overnight, after plasmid extraction, PCR identification and double enzyme digestion, the correct cloning vector pET22b ( + ) -DKR was transformed into E. coli BL21 In (DE3), it was applied to LB medium containing 50 ⁇ ⁇ / ⁇ 1 containing ampicillin, and cultured at 37 ° C overnight.
  • the bacterial solution was taken out, and the cells were collected by centrifugation at 8000 rpm/min for 10 min.
  • the cells were disrupted by a sonicator, centrifuged at 12000 rpm/min for 20 min to obtain a supernatant and a precipitate, and the supernatant was subjected to SDS-PAGE detection using a vertical electrophoresis apparatus.
  • SDS-PAGE detection using a vertical electrophoresis apparatus.
  • R 2 is selected from an alkyl group.
  • the bishydroxy product is represented by the following chemical formula
  • R 2 wherein is selected from an aryl group; and R 2 is selected from an alkyl group.
  • the nuclear magnetic data of the obtained product are as follows: 400Hz, CDC13: 57.29 (m, 5H), 4.54 (s, 2H), 4.22 (m, lH), 4.07 ( m, 1H), 3.45 ⁇ 3.40 (m, 4H), 2.4 l(d, 2H), 1.65 (t, 2H), 1.43 (S, 9H).
  • Example 4 A biscarbonyl reductase having a sequence homology of greater than 90% in Example 1 (SEQ ID NO: 13); the gene is a mutant of SEQ NO.
  • amino acid sequence is (SEQ ID NO. 16: MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDIN
  • the ligation reaction was carried out by DNA ligase, and the ligation product was transformed into E. coli DH5 (competent state of x strain, and plated in solid LB medium containing 50 ⁇ ⁇ / ⁇ 1 containing ampicillin, and cultured overnight at 37 ° C. A single colony on the medium was inoculated into LB liquid medium containing 50 ⁇ ⁇ / ⁇ 1 containing ampicillin, and cultured overnight at 37 ° C with shaking. After plasmid extraction, PCR identification and double enzyme digestion, the correct cloning vector pET22b ( + ) -DKR was transformed into E.
  • coli BL21 (DE3), plated in LB medium containing 50 ⁇ ⁇ / ⁇ 1 containing ampicillin, and cultured overnight at 37 ° C. Single colonies in the above medium were picked and inoculated into 5 ml.
  • the correct expression vector was identified by colony PCR to induce expression, and the above bacterial solution was transferred to 5 ml of LB liquid medium containing 50 ⁇ ⁇ / ⁇ 1 containing ampicillin.
  • IPTG IPTG was added to a final concentration of 0.02 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM and 1.0 mM at 18 ° C. Inducing expression, A negative control without IPTG inducer was established. After induction for 16 hours, the bacterial solution was taken out, and the cells were collected by centrifugation at 8000 rpm/min for 10 min. The cells were disrupted by sonication, centrifuged at 12000 rpm/min for 20 min to obtain the supernatant.
  • the supernatant was precipitated by SDS-PAGE with a vertical electrophoresis apparatus. It was found that the maximum amount of biscarbonyl reductase was induced at a final concentration of 0.02 mM IPTG. In order to increase the expression of biscarbonyl reductase, it would contain recombination.
  • the plasmid Escherichia coli BL21 (DE3) was induced at a final concentration of IPTG of 0.02 mM for 8 h at 37 ° C, 8 h at 30 ° C and 16 h at 25 ° C, and a negative control without IPTG inducer was also established.
  • the three cells were centrifuged at 8000 rpm/min for 10 min to collect the cells.
  • the cells were disrupted with a sonicator and centrifuged at 12000 rpm/min for 20 min to obtain the supernatant and precipitate.
  • the supernatant was subjected to SDS-PAGE detection by a vertical electrophoresis apparatus. It was found that the amount of the expression of the dicarbonyl reductase was the highest when induced at 25 ° C for 16 h.
  • the raw materials which have been commercialized on the market or the easily prepared ketones were selected.
  • R 2 is selected from an alkyl group.
  • the bishydroxy product is represented by the following chemical formula
  • R 2 wherein is selected from an aryl group; and R 2 is selected from an alkyl group.
  • amino acid sequence is (SEQ ID NO. 20: MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDIN
  • the bacterial solution was taken out, and the cells were collected by centrifugation at 8000 rpm/min for 10 min.
  • the cells were disrupted by a sonicator, centrifuged at 12000 rpm/min for 20 min to obtain a supernatant and a precipitate, and the supernatant was subjected to SDS-PAGE detection using a vertical electrophoresis apparatus.
  • SDS-PAGE detection using a vertical electrophoresis apparatus.
  • coli BL21 (DE3) containing recombinant plasmid was induced at a final concentration of IPTG of 0.02 mM for 8 h at 37 ° C, 8 h at 30 ° C and 16 h at 25 ° C for 16 h. Expression, and a negative control without IPTG inducer was also established. After the induction of expression was completed, the cells at three temperatures were centrifuged at 8000 rpm/min for 10 min to collect the cells. The cells were disrupted by a sonicator, centrifuged at 12000 rpm/min for 20 min to obtain a supernatant and a precipitate, and the supernatant was subjected to SDS-PAGE detection using a vertical electrophoresis apparatus.
  • R 2 is selected from an alkyl group.
  • the bishydroxy product is represented by the following chemical formula
  • Which is selected from an aromatic group; is selected from the group consisting of alkyl c to a 10 ml reaction bottle, and added O.Olg main raw material " ⁇ /" ⁇ , 0.04 ml of polyethylene glycol PEG-400, after the raw material is dissolved, 0.86 is added.

Abstract

Disclosed are a double-carbonyl reductase, a coding gene of same, and an application thereof. The double-carbonyl reductase is provided with one of the following amino acid sequences: 1) the amino acid sequence of SEQ NO. 1; or, 2) an amino acid sequence having a function of stereoselectively reducing formula (I) into formula (II) and derived from SEQ NO. 1 by means of substitution and/or deletion and/or addition of one or multiple amino acids in the amino acid sequence of SEQ NO. 1, where the amino acid sequence derived from SEQ NO. 1 and SEQ NO. 1 have a sequence similarity of 80% or more, R1 is selected from an aryl, an alkyl, a cycloalkyl, an alkyl-substituted aryl, a halogen-substituted aryl, an aralkyl heterocyclyl, a cyclic heteroalkyl or a cyclic heteroalkyl, and R2 is selected from an alkyl, a cycloalkyl, a haloalkyl or a halocycloalkyl. Employment of the double-carbonyl reductase of the present invention allows for one-step reduction of a dione substrate to prepare 3R,5S-dihydroxy compounds of a single optical purity.

Description

双羰基还原酶、 其编码基因及应用 技术领域 本发明涉及生物催化合成技术领域， 具体而言， 涉及一种双羰基还原酶、 其编码 基因及应用。 背景技术 手性醇是一类重要的中间体化合物， 广泛应用于手性药物和其他手性精细化学品 的合成。 3R, 5S-二羟基 -6-苄氧基-己酸叔丁酯是 3-羟基 -3-甲基戊二酰辅酶 A还原酶的 抑制剂他汀类降血脂药物的关键手性中间体， 可以通过化学合成和生物催化合成的方 法制得。 然而， 通过化学合成 3R， 5S-双羟基产物存在下列问题： 一般需用手性金属 催化剂， 生产成本高； 产物的光学纯度较难达到要求； 大量使用有机溶剂， 造成环境 污染严重。 采用生物催化合成这类化合物是一种有效的方法， 即将含有羰基的底物通过特定 的酶催化还原为相应的手性醇。 Wolberg等利用细菌 Jacto6ac//¾y 6re ^将一种 β， δ- 双羰基底物中的 δ-羰基还原成 δ-羟基， ee为 98.1%; 去氧核糖 -5-磷酸醛缩酶（DERA) 能够以乙醛和氯乙醛为底物， 通过两步醛缩反应同时引入两个手性中心得到 3R， 5S- 双羟基产物， ee>99.9%, de=96.6%,但去氧核糖 -5-磷酸醛缩酶催化反应生产成本较高。 Guo等利用 Acinetobacter species SCI 3874还原制备得到 3R， 5S-二羟基 -6-苄氧基 -己酸 酯， 但 de=63.3%。也有报道腈基水解酶通过对 3-羟基戊二腈去对称化得到单羟基中间 体 R-4氰基 -3-羟基丁酸， 再经过多步反应得到 3R， 5S-二羟基 -6-苄氧基-己酸酯。 可 见， 通过生物催化方法合成 3R， 5S-双羟基产物的现有技术也仍存在一些技术问题： 腈水解酶反应路线较长难度较大， 成本较高； Wolberg等利用细菌 Jactotoc/to 6re ^ 合成方法中需用其他方法引入另一个手性中心； 利用去氧核糖 -5-磷酸醛缩酶（DERA) 合成方法中需要用酶量较大， 底物抑制作用很强并且初始反应原料为易燃易爆试剂， 工业化生产难度较高。 发明内容 本发明旨在提供一种双羰基还原酶、 其编码基因及应用， 以简化 3R， 5S-二羟基化 合物的合成步骤， 降低生产污染和生产成本。 为了实现上述目的， 根据本发明的一个方面， 提供了一种双羰基还原酶。 该双羰 基还原酶具有下列之一的氨基酸序列： 1 ) SEQ N0.1的氨基酸序列； 2) 将 SEQ N0.1 的氨基酸序列经过一个或几个氨基酸的取代、 和 /或缺失、 和 /或添加且具有将 o o o OH OH O TECHNICAL FIELD The present invention relates to the field of biocatalytic synthesis, and in particular to a dicarbonyl reductase, a gene encoding the same, and an application thereof. BACKGROUND OF THE INVENTION Chiral alcohols are an important class of intermediate compounds that are widely used in the synthesis of chiral drugs and other chiral fine chemicals. 3R, 5S-dihydroxy-6-benzyloxy-hexanoic acid tert-butyl ester is an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key chiral intermediate for statins, a lipid-lowering drug, It is prepared by chemical synthesis and biocatalytic synthesis. However, by chemically synthesizing the 3R, 5S-dihydroxy product, the following problems exist: Generally, a chiral metal catalyst is required, and the production cost is high; the optical purity of the product is difficult to meet the requirements; and the organic solvent is used in a large amount, causing serious environmental pollution. The biocatalytic synthesis of such compounds is an effective method for the catalytic reduction of a substrate containing a carbonyl group to the corresponding chiral alcohol by a specific enzyme. Wolberg et al. used the bacteria Jacto6ac//3⁄4y 6re ^ to reduce the δ-carbonyl group in a β, δ-biscarbonyl base to a δ-hydroxy group with an ee of 98.1%; deoxyribose-5-phosphate aldolase (DERA) It is possible to use acetaldehyde and chloroacetaldehyde as substrates to simultaneously introduce two chiral centers by two-step aldol reaction to obtain 3R, 5S-dihydroxy product, ee>99.9%, de=96.6%, but deoxyribose-5 - Phosphate aldolase catalytic reaction is costly to produce. Guo et al. used Acinetobacter species SCI 3874 to reduce the preparation of 3R, 5S-dihydroxy-6-benzyloxy-hexanoate, but de=63.3%. It has also been reported that a nitrile hydrolase can be desymmetrically 3-hydroxyglutaronitrile to obtain a monohydroxy intermediate R-4 cyano-3-hydroxybutyric acid, which is then subjected to a multi-step reaction to obtain 3R, 5S-dihydroxy-6-benzyl. Oxy-hexanoate. It can be seen that the prior art of synthesizing 3R, 5S-dihydroxy product by biocatalysis still has some technical problems: The nitrilase reaction route is relatively difficult and costly; Wolberg et al. utilizes the bacteria Jactotoc/to 6re ^ synthesis In the method, another method is needed to introduce another chiral center; the deoxyribose-5-phosphate aldolase (DERA) synthesis method requires a large amount of enzyme, the substrate inhibition is strong, and the initial reaction raw material is flammable. Explosive reagents, industrial production is more difficult. SUMMARY OF THE INVENTION The present invention is directed to a biscarbonyl reductase, a gene encoding the same, and use thereof to simplify the synthesis step of a 3R, 5S-dihydroxy compound, reducing production pollution and production costs. In order to achieve the above object, according to one aspect of the present invention, a biscarbonylreductase is provided. The biscarbonylreductase has one of the following amino acid sequences: 1) the amino acid sequence of SEQ N0.1; 2) the amino acid sequence of SEQ N0.1 is substituted, and/or deleted, and/or one or more amino acids. Added and has ooo OH OH O
Ri\ 、 Ri\,
R2立体选择性地还原为 1 ^ 功能的由 SEQ N0.1衍生 的氨基酸序列， 且 SEQ N0.1衍生的氨基酸序列与 SEQ N0.1具有 80%以上的同源性， 其中， 选自芳香基、 烷基、 环烷基、 烷基取代的芳香基、 卤素取代的芳香基、 芳烷 杂环基、 环状杂烷基或环状杂烷化烷基， R2选自烷基、 环烷基、 ¾烷基或 ¾环烷基。 根据本发明的另一个方面， 提供一种上述双羰基还原酶的编码基因。 进一步地， 上述编码基因， 具有下述之一的脱氧核苷酸序列： 1 ) SEQ N0.2的脱 氧核苷酸序列； 2)与 SEQ N0.2的脱氧核苷酸序列具有 80%同源性且编码的蛋白质具 o o o OH OH O R 2 is stereoselectively reduced to a 1 ^ functional amino acid sequence derived from SEQ N0.1, and the amino acid sequence derived from SEQ N0.1 has 80% or more homology with SEQ N0.1, wherein a base, an alkyl group, a cycloalkyl group, an alkyl-substituted aryl group, a halogen-substituted aryl group, an arylalkylheterocyclyl group, a cyclic heteroalkyl group or a cyclic heteroalkylated alkyl group, and R 2 is selected from the group consisting of an alkyl group and a ring. Alkyl, 3⁄4 alkyl or 3⁄4 cycloalkyl. According to another aspect of the present invention, a gene encoding the above biscarbonylreductase is provided. Further, the above coding gene has a deoxynucleotide sequence of one of the following: 1) a deoxynucleotide sequence of SEQ N0.2; 2) 80% homologous to the deoxynucleotide sequence of SEQ N0.2 And encoded protein with ooo OH OH O
Ri\ 、  Ri\,
有将 1 、OR2立体选择性还原为 OR2功能的脱氧核苷酸序 列。 根据本发明的再一个方面， 提供一种含有上述双羰基还原酶的编码基因的重组表 达载体。 根据本发明的又一个方面， 提供一种含有上述双羰基还原酶的编码基因的转基因 细胞系。 根据本发明的再一个方面， 提供一种含有上述双羰基还原酶的编码基因的转基因 重组菌。 There is a deoxynucleotide sequence which stereoselectively reduces 1 , OR 2 to an OR 2 function. According to still another aspect of the present invention, a recombinant expression vector comprising the above-described double-carbonyl reductase-encoding gene is provided. According to still another aspect of the present invention, a transgenic cell line comprising the gene encoding the above biscarbonylreductase is provided. According to still another aspect of the present invention, a transgenic recombinant strain comprising the gene encoding the above biscarbonylreductase is provided.
o o o 根据本发明的又一个方面，提供一种上述双羰基还原酶在将 、OR2立 ooo According to another aspect of the present invention, there is provided one of the aforementioned carbonyl reductase in the dual, OR 2 Li
OH OH O  OH OH O
体选择性还原为 OR2中的应用。 步地， 双羰基还原酶在制备 3R， 5S-二羟基 -6-苄氧基-己酸叔丁酯中的应用, Bulk selective reduction is an application in OR 2 . Step by step, the application of dicarbonyl reductase in the preparation of tert-butyl 3R, 5S-dihydroxy-6-benzyloxy-hexanoate,
Figure imgf000004_0001
采用本发明的双羰基还原酶 （Diketoreductase, DKR) 为生物催化剂， 可以一步 还原二酮底物， 制备得到单一光学纯度的 3R， 5S-二羟基化合物， 将其应用到合成他 汀类药物中间体， 得到 ee值为 99%， de值为 93%左右的 3R， 5S-二羟基化合物， 简化 了合成步骤， 降低了生产污染和生产成本， 适合于大规模工业化生产。 具体实施方式 需要说明的是， 在不冲突的情况下， 本申请中的实施例及实施例中的特征可以相 互组合。 下面将结合实施例来详细说明本发明。 本发明的羰基还原酶基因来源于 Mycobacterium fortuitum subsp. fortuitum DSM 46621该羰基还原酶具有双羰基还原酶的功能。 根据本发明一种典型的实施方式， 提供一种双羰基还原酶。 该双羰基还原酶具有 一的氨基酸序列： 1 ) SEQ N0.1 的氨基酸序列 （ SEQ N0.1 为：
Figure imgf000004_0001
Using the dicarbonyloreductase (DKR) of the present invention as a biocatalyst, a diketone substrate can be reduced in one step to prepare a single optical purity 3R, 5S-dihydroxy compound, which is applied to the synthesis of As a tidal intermediate, a 3R, 5S-dihydroxy compound having an ee value of 99% and a de value of about 93% is obtained, which simplifies the synthesis step, reduces production pollution and production cost, and is suitable for large-scale industrial production. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS It should be noted that the embodiments in the present application and the features in the embodiments may be combined with each other without conflict. The invention will be described in detail below with reference to the embodiments. The carbonyl reductase gene of the present invention is derived from Mycobacterium fortuitum subsp. fortuitum DSM 46621. The carbonyl reductase has the function of a dicarbonyl reductase. According to an exemplary embodiment of the invention, a biscarbonyl reductase is provided. The bis-carbonyl reductase has an amino acid sequence: 1) the amino acid sequence of SEQ N0.1 (SEQ N0.1 is:
Figure imgf000005_0001
Figure imgf000005_0001
YN) ; 2 ) 将 SEQ N0.1的氨基酸序列经过一个或几个氨基酸的取代、 和 /或缺失、 和 /  YN) ; 2) subjecting the amino acid sequence of SEQ N0.1 to one or several amino acid substitutions, and/or deletions, and /
O O O OH OH O  O O O OH OH O
或添加且具有将 1^^^^^^2立体选择性还原为 K1^U^/^八 OR2功能的由Or added and has a stereoselective reduction of 1 ^^^^^^ 2 to K1 ^ U ^/^ eight OR 2 function
SEQ NO. l衍生的氨基酸序列， 其中， 选自芳香基， R2选自烷基。 采用本发明的双 羰基还原酶 （Diketoreductase, DKR) 为生物催化剂， 可以一步还原二酮底物， 制备 得到单一光学纯度的 3R， 5S-二羟基化合物， 将其应用到合成他汀类药物中间体， 简 化了合成步骤， 降低了生产污染和生产成本， 适合于大规模工业化生产。 根据本发明一种典型的实施方式， 提供一种上述双羰基还原酶的编码基因， 该编 码基因具有下述之一的脱氧核苷酸序列： 1 ) SEQ N0.2的脱氧核苷酸序列（SEQ N0.2 为 ： The amino acid sequence derived from SEQ NO. 1, wherein is selected from an aryl group, and R2 is selected from an alkyl group. By using the dicarbonyloreductase (DKR) of the present invention as a biocatalyst, a diketone substrate can be reduced in one step to prepare a single optical purity 3R, 5S-dihydroxy compound, which is applied to a synthetic statin intermediate. It simplifies the synthesis step, reduces production pollution and production costs, and is suitable for large-scale industrial production. According to an exemplary embodiment of the present invention, there is provided a gene encoding the above biscarbonylreductase having a deoxynucleotide sequence of one of the following: 1) a deoxynucleotide sequence of SEQ N0.2 ( SEQ N0.2 is:
'； 2 ) 与'; 2 ) with
SEQ N0.2 的脱氧核苷酸序列具有 80%同源性且编码的蛋白质具有将 o o o OH OH O The deoxynucleotide sequence of SEQ N0.2 has 80% homology and the encoded protein has o o o OH OH O
、OR2立体选择性还原为 R2功能的脱氧核苷酸序列- 根据本发明一种典型的实施方式， 提供一种含有上述双羰基还原酶的编码基因的 重组表达载体。 该载体可以是 pET-22b (+)、 pET-22b (+)、 pET-3a ( + )、 pET-3d (+)、 pET-l la (+)、 pET-12a ( + )、 pET-14b (+)、 pET-15b ( + )、 pET-16b (+)、 pET-17b (+)、 pET-19b ( + )、 pET-20b ( + )、 pET-21a ( + )、 pET-23a ( + )、 pET-23b ( + )、 pET-24a ( + )、 pET-25b ( + )、 pET-26b ( + )、 pET-27b ( + )、 pET-28a ( + )、 pET-29a ( + )、 pET-30a ( + )、 pET-31b ( + )、 pET-32a ( + )、 pET-35b ( + )、 pET-38b ( + )、 pET-39b ( + )、 pET-40b ( + )、 pET-41a ( + )、 pET-41b ( + )、 pET-42a ( + )、 pET-43a ( + )、 pET-43b ( + )、 pET-44a ( + )、 pET-49b ( + )、 pQE2、 pQE9、 pQE30、 pQE31、 pQE32 pQE40、 pQE70、 pQE80、 pRSET-A、 pRSET-B、 pRSET-C、 pGEX-5X-l pGEX-6p-l pGEX-6p-2 pBV220 pBV221 pBV222 pTrc99A、 pTwinK pEZZ18、 pKK232-18 pUC-18 或 pUC-19。 表达载体在大肠杆菌 中过量表达， 经过量表达的双羰基还原酶在 SDS-PAGE上呈现的分子量约为 30KD, 在 30°C， pH6.0条件下， 可以一步还原得到光学纯度较高的 3R， 5S-双羟基化合物。 当然， 包括上述编码基因的转基因细胞系、 转基因重组菌也在本发明的保护范围 之内。 根据本发明一种典型的实施方式， 提供一种上述双羰基还原酶在将 A deoxynucleotide sequence in which the OR 2 is stereoselectively reduced to the R 2 function - According to an exemplary embodiment of the present invention, a recombinant expression vector containing the above-described double-carbonyl reductase-encoding gene is provided. The vector may be pET-22b (+), pET-22b (+), pET-3a (+), pET-3d (+), pET-l la (+), pET-12a (+), pET-14b (+), pET-15b ( + ), pET-16b (+), pET-17b (+), pET-19b ( + ), pET-20b ( + ), pET-21a ( + ), pET-23a ( + ), pET-23b ( + ), pET-24a ( + ), pET-25b ( + ), pET-26b ( + ), pET-27b ( + ), pET-28a ( + ), pET-29a ( + ), pET-30a ( + ), pET-31b ( + ), pET-32a ( + ), pET-35b ( + ), pET-38b ( + ), pET-39b ( + ), pET-40b ( + ) , pET-41a ( + ), pET-41b ( + ), pET-42a ( + ), pET-43a ( + ), pET-43b ( + ), pET-44a ( + ), pET-49b ( + ), pQE2, pQE9, pQE30, pQE31, pQE32 pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-l pGEX-6p-l pGEX-6p-2 pBV220 pBV221 pBV222 pTrc99A, pTwinK pEZZ18, pKK232-18 pUC-18 or pUC-19. The expression vector is overexpressed in E. coli. The expressed amount of dicarbonyl reductase exhibits a molecular weight of about 30KD on SDS-PAGE. Under 30°C and pH6.0, it can be reduced in one step to obtain 3R with higher optical purity. , 5S-dihydroxy compound. Of course, transgenic cell lines and transgenic recombinant bacteria including the above-described coding genes are also within the scope of the present invention. According to an exemplary embodiment of the present invention, there is provided a biscarbonyl reductase as described above
OH OH O  OH OH O
、OR2立体选择性还原为 K lvu^\/^/ VOR冲的应用。 该酶可以一步还原 二酮底物， 制备得到单一光学纯度的 3R， 5S-二羟基化合物， 可以应用到合成他汀类药物中间体。 优选的， 双羰基还原酶在制备 3R， 5S-二羟基 -6-苄氧基-己酸叔丁酯中应用， 其中， , OR 2 stereoselective reduction to K l v u ^ / / ^ / V OR punch application. The enzyme can reduce the diketone substrate in one step to prepare a single optical purity 3R, 5S-dihydroxy compound, which can be applied to the synthesis of statin intermediates. Preferably, the dicarbonyl reductase is used in the preparation of tert-butyl 3R, 5S-dihydroxy-6-benzyloxy-hexanoate, wherein
Figure imgf000006_0001
下面将结合实施例进一步说明本发明的有益效果。 实施例 ( 1 )来源于 Mycobacterium fortuitum subsp. fortuitum DSM 46621的双羰基还原酶 的克隆与表达 为了便于双羰基还原酶基因 （DKR) 的表达以及鉴定， 在寡聚核苷酸引物的 5'和 3'末端设计了兼容的限制性酶切位点。 其引物对如下： 上游引物 SEQ ID N0.3 : 5'- GGAATTCCATATGACTAACTCTGTAAAAACGGTGACTGTTC-3 '； 下游引物 SEQ ID N0.4: 5' - CCGCTCGAGGTTATATTTGTAGAAACCTTCACCAGAAGCC-3 扩增得基
Figure imgf000006_0001
Advantageous effects of the present invention will be further described below in conjunction with the examples. Example (1) Cloning and expression of a dicarbonyl reductase derived from Mycobacterium fortuitum subsp. fortuitum DSM 46621 To facilitate expression and identification of the double-carbonyl reductase gene (DKR), at the 5' and 3' ends of the oligonucleotide primers Compatible restriction sites were designed. The primer pairs are as follows: Upstream primer SEQ ID N0.3 : 5'- GGAATTCCATATGACTAACTCTGTAAAAACGGTGACTGTTC-3 '; Downstream primer SEQ ID N0.4: 5' - CCGCTCGAGGTTATATTTGTAGAAACCTTCACCAGAAGCC-3 Amplification
Figure imgf000007_0001
其 氨 基 酸 序 列 为 （ SEQ ID NO. l ： MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDIN
Figure imgf000007_0001
Its amino acid sequence is (SEQ ID NO. l : MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDIN
DEVLEAAKARFEKLAETYA EVPDARDGKAREALGRLTLTSDLKAAVADADLVIEA
Figure imgf000007_0002
DEVLEAAKARFEKLAETYA EVPDARDGKAREALGRLTLTSDLKAAVADADLVIEA
Figure imgf000007_0002
ENYIDKGKLGLASGEGFYKYN )。 双羰基还原酶基因全合成后， 连接到 pUC57载体上， 用 Nde l和 Xho l分别将目 的基因和 pET22b (+) (pET-22b (+)也可以为 pET-3a (+)， pET-3d (+)， pET-l la ( + )， pET-12a ( + )， pET-14b (+)， pET-15b ( + )， pET-16b (+)， pET-17b (+)， pET-19b (+)， pET-20b ( + )， pET-21a ( + )， pET-23a ( + )， pET-23b ( + )， pET-24a ( + )， pET-25b ( + )， pET-26b ( + )， pET-27b ( + )， pET-28a ( + )， pET-29a ( + )， pET-30a ( + )， pET-31b ( + )， pET-32a ( + )， pET-35b ( + )， pET-38b ( + )， pET-39b ( + )， pET-40b ( + )， pET-41a ( + )， pET-41b ( + )， pET-42a ( + )， pET-43a ( + )， pET-43b ( + )， pET-44a ( + )， pET-49b ( + )， pQE2, pQE9, pQE30, pQE31 , pQE32, pQE40, pQE70 , pQE80, pRSET-A, pRSET-B , pRSET-C, pGEX-5X-l , pGEX-6p-l , pGEX-6p-2, pBV220 , pBV221 , pBV222, pTrc99A， pTwinl , pEZZ 18， pKK232-18 , pUC-18 , pUC-19 )质粒同时进行酶切， T4 DNA连接 酶进行连接反应， 将连接产物转化到大肠杆菌感受态 DH5(x 菌株中， 涂布于含有 50μ§/ιη1含氨苄青霉素的固体 LB培养基 （Luria-Bertani培养基） 中， 37°C培养过夜。 挑取上述培养基上的单菌落接种于含有 50μ§/ιη1含氨苄青霉素的 LB液体培养基中， 37 °C振荡培养过夜， 经过质粒提取， PCR鉴定和双酶切鉴定后， 将正确的克隆载体 pET-22b ( + ) - DKR转化到大肠杆菌 BL21 (DE3 ) 中， 涂布于含有 50μ§/ιη1含氨苄青 霉素的 LB 培养基中， 37°C培养过夜。 挑取上述培养基中上的单菌落接种于 5ml 含 50μ§/ιη1含氨苄青霉素的 LB液体培养基中， 经过菌落 PCR (聚合酶链式反应）鉴定正 确的表达载体进行诱导表达， 将上述菌液转接于 5ml含 50μ§/ιη1含氨苄青霉素的 LB 液体培养基中， 37°C振荡培养至 OD6(K^0.6时， 加入 IPTG (异丙基硫代半乳糖苷） 至 终浓度分别为 0.02mM， 0.05mM， O. lmM, 0.2mM, 0.4mM， 0.6mM， 0.8mM禾 P l .OmM, 在 18 °C下进行诱导表达， 并设立不加 IPTG诱导剂的阴性对照。 诱导 16h后， 取出菌 液， 8000rpm/min离心 lOmin收集菌体。菌体用超声破碎仪破碎细胞， 4°C， 12000rpm/min 离心 20min获得上清液和沉淀， 上清液用垂直电泳仪进行 SDS-PAGE检测。 结果发现 在终浓度为 0.02mM IPTG浓度时诱导表达双羰基还原酶的量最多。为提高双羰基还原 酶的表达量， 将含有重组质粒的大肠杆菌 BL21 (DE3 ) 在 IPTG终浓度为 0.02mM， 分别于 37°C诱导 8h，30°C诱导 8h以及 25 °C诱导 16h条件下表达，并同时设立不加 IPTG 诱导剂的阴性对照。 诱导表达结束后， 分别将三个温度的诱导菌体于 8000rpm/min离 心 lOmin收集菌体。 菌体用超声破碎仪破碎细胞， 4。C， 12000rpm/min离心 20min获 得上清液和沉淀， 上清液用垂直电泳仪进行 SDS-PAGE检测。 结果发现在 30°C诱导 8h时表达双羰基还原酶的量最多。 ENYIDKGKLGLASGEGFYKYN). After the total synthesis of the dicarbonyl reductase gene, it is ligated to the pUC57 vector, and the target gene and pET22b (+) (pET-22b (+) can also be pET-3a (+), pET-3d, respectively, using Nde l and Xho l. (+), pET-l la ( + ), pET-12a ( + ), pET-14b (+), pET-15b ( + ), pET-16b (+), pET-17b (+), pET-19b (+), pET-20b ( + ), pET-21a ( + ), pET-23a ( + ), pET-23b ( + ), pET-24a ( + ), pET-25b ( + ), pET-26b ( + ), pET-27b ( + ), pET-28a ( + ), pET-29a ( + ), pET-30a ( + ), pET-31b ( + ), pET-32a ( + ), pET-35b ( + ), pET-38b ( + ), pET-39b ( + ), pET-40b ( + ), pET-41a ( + ), pET-41b ( + ), pET -42a ( + ), pET-43a ( + ), pET-43b ( + ), pET-44a ( + ), pET-49b ( + ), pQE2, pQE9, pQE30, pQE31 , pQE32, pQE40, pQE70 , pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-l, pGEX-6p-l, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwinl, pEZZ 18, pKK232-18, pUC-18 , pUC-19 ) plasmid was digested simultaneously, T4 DNA ligase was ligated, and the ligation product was transformed into E. coli competent DH5 (x strain, coated on solid LB medium containing 50 μ § /ιη1 containing ampicillin) (Luria-Bertani medium), culture overnight at 37 ° C. Pick a single colony on the above medium and inoculate it in LB liquid medium containing 50 μ § /ιη1 containing ampicillin, shake culture at 37 °C overnight, and pass through the plasmid. extracting, after PCR and restriction analysis, the correct cloning vector pET-22b (+) - DKR transformed into E. coli BL21 (DE3), a coating containing 50μ § / ιη1 LB medium containing ampicillin LB broth, 37 ° C overnight. Picked said medium containing a single colony was inoculated into 5ml 50μ § / ιη1 containing ampicillin and identified through colony expressing the correct PCR (polymerase chain reaction) The vector was induced to express, and the above bacterial solution was transferred to 5 ml of LB liquid medium containing 50 μ § /ιη1 containing ampicillin, and cultured at 37 ° C to OD 6 (when K ^ 0.6, IPTG (isopropyl thio) was added. Galactosides) to a final concentration of 0.02 mM, 0.05 mM, O. lmM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, P l .OmM, induced expression at 18 ° C, and no IPTG was established. Negative control of inducer. After induction for 16h, the bacterial solution was taken out, and the cells were collected by centrifugation at 8000 rpm/min for 10 min. The cells were disrupted by sonication, centrifuged at 12000 rpm/min for 20 min to obtain supernatant and precipitate, and the supernatant was obtained. The liquid was subjected to SDS-PAGE detection using a vertical electrophoresis apparatus. As a result, it was found that the amount of expression of the biscarbonyl reductase was most induced at a final concentration of 0.02 mM IPTG. In order to increase the expression level of dicarbonyl reductase, E. coli BL21 (DE3) containing recombinant plasmid was induced at a final concentration of IPTG of 0.02 mM for 8 h at 37 ° C, 8 h at 30 ° C and 16 h at 25 ° C for 16 h. Expression and simultaneous establishment of a negative control without IPTG inducer. After the induction of expression was completed, the cells at three temperatures were centrifuged at 8000 rpm/min for 10 min to collect the cells. The cells are disrupted by a sonicator, 4. C, the supernatant and the precipitate were obtained by centrifugation at 12,000 rpm/min for 20 min, and the supernatant was subjected to SDS-PAGE detection using a vertical electrophoresis apparatus. As a result, it was found that the amount of the expression of the biscarbonylreductase was the highest when induced at 30 ° C for 8 hours.
( 2 )来源于 Mycobacterium fortuitum subsp. fortuitum DSM 46621的双羰基还原酶 的活性鉴定 (2) Identification of the activity of biscarbonyl reductase from Mycobacterium fortuitum subsp. fortuitum DSM 46621
o o o 选用已经在市场上商业化的原料或者容易制备的酮类化合物 ^^^^^0«2 为初始原料， 其中 R1选自芳香基； R2选自烷基。 所述的双羟基产物由以下化学通式 o o o Ooo uses a commercially available starting material or a readily prepared ketone compound ^^^^^0« 2 as a starting material, wherein R1 is selected from an aromatic group; and R 2 is selected from an alkyl group. The bishydroxy product is represented by the following chemical formula ooo
表达： Rlv^ k/kAOR2， 其中 Rl选自芳香基； R2选自烷基。 向 500ml 反应瓶中， 加入 10g
Figure imgf000009_0001
， 40ml 聚乙二醇 PEG-400 (聚乙二醇 400)， 原料溶解后， 加入 360ml磷酸盐缓冲液（100mM, pH=6.0)， 底物均匀分散于缓冲液中； 加酮还原酶： 加入 0.6g NAD+ (烟酰胺腺嘌吟二核苷酸）， 118g D-葡萄糖， 0.6g辅酶葡萄糖脱氢酶和 lg双羰基还原酶，体系 pH=6.0，并于 30±3 °C 保温 40h; 用 400ml乙酸乙酯停止反应， 用 250g硅藻土过滤， 400ml乙酸乙酯萃取两 次， 静置分液， 有机相经干燥， 过滤， 浓缩得到粗品， 再经柱层析纯化得到 4g纯度较 高的产品
Figure imgf000009_0002
纯度 98.0%， 收率 50%， ee值 99%， de值 93%。 所得产品的核磁数据如下： 400Hz， CDC13: 57.29 (m, 5H),4.54(s,2H), 4.22(m,lH), 4.07 (m, 1H) ,3.45~3.40(m,4H), 2.4 l(d, 2H), 1.65 (t,2H) , 1.43(S,9H)。 所得产品质谱数据如下： MW=310± 1。 向 500ml 反应瓶中， 加入 lOg
Figure imgf000009_0003
， 40ml 聚乙二醇 PEG-400, 原料溶解后， 加入 360ml磷酸盐缓冲液 （ lOOmM, pH=6.0)， 底物均匀分散 于缓冲液中； 加酮还原酶： 加入 0.3g NAD+， 41.2g 甲酸铵， 0.5g辅酶甲酸脱氢酶和 lg双羰基还原酶， 体系 pH=6.0， 并于 30±3 °C保温 17h; 用 400ml乙酸乙酯停止反应， 用 250g硅藻土过滤， 400ml乙酸乙酯萃取两次， 静置分液， 有机相经干燥， 过滤， 浓 缩得到粗品，二羟基 65.0 % ,收率 90%， ee值大于 90-99%， de值 80-95%。 所得产品质谱数据为： MW=282±1 向 500ml 反应瓶中， 加入 10g
Figure imgf000009_0004
， 40ml 聚乙二醇 PEG-400, 原料溶解后， 加入 360ml磷酸盐缓冲液 (lOOmM, pH=6.0)， 底物均匀分散于 缓冲液中； 加酮还原酶： 加入 0.3g\NAD+， 41.2g甲酸铵， 0.5g辅酶甲酸脱氢酶和 lg 双羰基还原酶， 体系 pH=6.0， 并于 30±3 °C保温 17h; 用 400ml乙酸乙酯停止反应， 用 250g硅藻土过滤， 400ml乙酸乙酯萃取两次， 静置分液， 有机相经干燥， 过滤， 浓缩 Expression: Rl v^ k/kA OR2 , wherein R1 is selected from an aryl group; and R 2 is selected from an alkyl group. Into a 500ml reaction bottle, add 10g
Figure imgf000009_0001
, 40ml polyethylene glycol PEG-400 (polyethylene glycol 400), after the raw material is dissolved, add 360ml phosphate buffer (100mM, pH=6.0), the substrate is uniformly dispersed in the buffer; ketone reductase: add 0.6g NAD+ (nicotinamide adenine dinucleotide), 118g D-glucose, 0.6g coenzyme glucose dehydrogenase and lg double carbonyl reductase, system pH=6.0, and kept at 30±3 °C for 40h; The reaction was stopped with 400 ml of ethyl acetate, filtered with EtOAc (250 g), EtOAc (EtOAc)jjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj product
Figure imgf000009_0002
The purity was 98.0%, the yield was 50%, the ee value was 99%, and the de value was 93%. The nuclear magnetic data of the obtained product are as follows: 400 Hz, CDC13: 57.29 (m, 5H), 4.54 (s, 2H), 4.22 (m, lH), 4.07 (m, 1H), 3.45 to 3.40 (m, 4H), 2.4 l (d, 2H), 1.65 (t, 2H), 1.43 (S, 9H). The mass spectrum data of the obtained product was as follows: MW = 310 ± 1. Into a 500ml reaction bottle, add lOg
Figure imgf000009_0003
40ml polyethylene glycol PEG-400, after the raw material is dissolved, add 360ml phosphate buffer (100 mM, pH=6.0), the substrate is uniformly dispersed in the buffer; add ketone reductase: add 0.3g NAD+, 41.2g formic acid Ammonium, 0.5 g of coenzyme formate dehydrogenase and lg bis-carbonyl reductase, system pH = 6.0, and incubated at 30 ± 3 ° C for 17 h; stop with 400 ml of ethyl acetate, filter with 250 g of diatomaceous earth, 400 ml of ethyl acetate The mixture was extracted twice, and the mixture was allowed to stand for separation. The organic phase was dried, filtered, and concentrated to give a crude product. The dihydroxy group was 65.0%, the yield was 90%, the ee value was more than 90-99%, and the de value was 80-95%. The mass spectrum data of the obtained product was: MW=282±1 In a 500 ml reaction flask, 10 g was added.
Figure imgf000009_0004
40ml polyethylene glycol PEG-400, after the raw material is dissolved, add 360ml phosphate buffer (100mM, pH=6.0), the substrate is evenly dispersed in the buffer; add ketone reductase: add 0.3g\NAD+, 41.2g Ammonium formate, 0.5 g of coenzyme formate dehydrogenase and lg dicarbonyl reductase, system pH=6.0, and incubated at 30±3 °C for 17 h; stop the reaction with 400 ml of ethyl acetate, filter with 250 g of diatomaceous earth, 400 ml of acetic acid The ester is extracted twice, and the mixture is allowed to stand for separation. The organic phase is dried, filtered and concentrated.
OH OH O  OH OH O
得到粗品，二羟基 、0z产品 50%，纯度 98.0 %，收率 90 %， ee值 90-99%， de值 80-95%。 所得产品质谱数据如下： MW=268±L 向 500ml反应瓶中， 加入 10g
Figure imgf000010_0001
, 40ml聚乙二醇 PEG-400, 原料溶解后， 加入 360ml磷酸盐缓冲液 ClOOmM, pH=6.0)， 底物均匀分散于 缓冲液中； 加酮还原酶： 加入 0.3g\NAD+， 41.2g甲酸铵， 0.5g辅酶甲酸脱氢酶和 3g 双羰基还原酶， 体系 pH=6.0， 并于 30±3 °C保温 17h; 用 400ml乙酸乙酯停止反应， 用 250g硅藻土过滤， 400ml乙酸乙酯萃取两次， 静置分液， 有机相经干燥， 过滤， 浓缩 Obtained crude product, dihydroxy, 0 z product 50%, purity 98.0%, yield 90%, ee value 90-99%, de value 80-95%. The mass spectrum data of the obtained product are as follows: MW=268±L Into a 500ml reaction flask, add 10g
Figure imgf000010_0001
40ml polyethylene glycol PEG-400, after the raw material is dissolved, add 360ml phosphate buffer ClOOmM, pH=6.0), the substrate is uniformly dispersed in the buffer; add ketone reductase: add 0.3g\NAD+, 41.2g formic acid Ammonium, 0.5 g of coenzyme formate dehydrogenase and 3 g of dicarbonyl reductase, system pH = 6.0, and incubated at 30 ± 3 ° C for 17 h; stop with 400 ml of ethyl acetate, filter with 250 g of diatomaceous earth, 400 ml of ethyl acetate Extract twice, stand still, organic phase dried, filtered, concentrated
OH OH O  OH OH O
得到粗品， 二羟基 H "5^1^1^0"^产品 60%， 纯度 98.0 %， 收率 90%， ee值 90-99%, de值 80-95%。 所得产品质谱数据如下： MW=324±1。 The crude product was obtained, dihydroxy H " 5 ^ 1 ^ 1 ^ 0 "^ product 60%, purity 98.0%, yield 90%, ee value 90-99%, de value 80-95%. The mass spectrum data of the obtained product was as follows: MW = 324 ± 1.
实施例 2 与实施例 1中序列同源性大于 90%的双羰基还原酶（SEQ ID NO.5 ), 该基因为 SEQ NO.l的突变体。 为了便于双羰基还原酶基因的表达以及鉴定， 在寡聚核苷酸引物的 5'和 3'末端设 计了兼容的限制性酶切位点。 其引物对如下： 上游引物 SEQ ID N0.6 : 5 ' -GGAATTCCATATGACCGAACTGAAACAAATCACC-3 '； 下游引物 SEQ ID N0.7: 5 ' -CCGCTCGAGACCTTTGTAGTTGTAAAAGCCGTCAC-3 '。 扩增得基因序列 （SEQ ID NO.5 ： Example 2 A biscarbonyl reductase (SEQ ID NO. 5) having a sequence homology greater than 90% in Example 1, which is a mutant of SEQ NO. To facilitate expression and identification of the dicarbonyl reductase gene, compatible restriction sites were designed at the 5' and 3' ends of the oligonucleotide primers. The primer pair is as follows: upstream primer SEQ ID N0.6: 5 '-GGAATTCCATATGACCGAACTGAAACAAATCACC-3 '; downstream primer SEQ ID N0.7: 5 '-CCGCTCGAGACCTTTGTAGTTGTAAAAGCCGTCAC-3 '. Amplified gene sequence (SEQ ID NO. 5:
其 氨 基 酸 序 列 为 （ SEQ ID NO.8 ： MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYD Its amino acid sequence is (SEQ ID NO. 8: MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYD
Figure imgf000011_0001
Figure imgf000011_0001
WLKENYIDKGKLGLASGEGFYKYN) 双羰基还原酶基因全合成后， 连接到 pUC57载体上， 用 Nde l和 Xho l分别将目 的基因和 pET-22b (+) (pET-22b (+) 也可以为 pET-3a (+)， pET-3d (+)， pET-l la ( + )， pET-12a ( + )， pET-14b ( + )， pET-15b ( + )， pET-16b ( + )， pET-17b ( + )， pET-19b ( + )， pET-20b ( + )， pET-21a ( + )， pET-23a ( + )， pET-23b ( + )， pET-24a ( + )， pET-25b ( + )， pET-26b ( + )， pET-27b ( + )， pET-28a ( + )， pET-29a ( + )， pET-30a ( + )， pET-31b ( + )， pET-32a ( + )， pET-35b ( + )， pET-38b ( + )， pET-39b ( + )， pET-40b ( + )， pET-41a ( + )， pET-41b ( + )， pET-42a ( + )， pET-43a ( + )， pET-43b ( + )， pET-44a ( + )， pET-49b ( + )， pQE2, pQE9, pQE30, pQE31 , pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-l , pGEX-6p-l , pGEX-6p-2, pBV220, pBV221 , pBV222, pTrc99A, pTwinl , pEZZ18， pKK232-18, pUC-18, pUC-19) 质粒同时进行酶切， T4 DNA连接酶进行连接反应， 将连接产物转化到大肠杆菌 DH5(x菌株的感受态中， 涂布 于含有 50μ§/ιη1含氨苄青霉素的固体 LB培养皿中， 37°C培养过夜。 挑取上述培养基 上的单菌落接种于含有 50μ§/ιη1含氨苄青霉素的 LB液体培养基中， 37°C振荡培养过 夜， 经过质粒提取， PCR鉴定和双酶切鉴定后， 将正确的克隆载体 pET22b ( + ) -DKR 转化到大肠杆菌 BL21 (DE3 )中， 涂布于含有 50μ§/ιη1含氨苄青霉素的 LB培养基中， 37°C培养过夜。 挑取上述培养基中上的单菌落接种于 5ml含 50μ§/ιη1含氨苄青霉素的 LB液体培养基中， 经过菌落 PCR鉴定正确的表达载体进行诱导表达， 将上述菌液转 接于 5ml含 50μ§/ιη1含氨苄青霉素的 LB液体培养基中， 37°C振荡培养至 OD6QQ=0.6时， 加入 IPTG至终浓度分别为 0.02mM， 0.05mM， O. lmM, 0.2mM, 0.4mM， 0.6mM， 0.8mM 禾口 1.0mM， 在 18°C下进行诱导表达， 并设立不加 IPTG诱导剂的阴性对照。 诱导 16h 后， 取出菌液， 8000rpm/min离心 lOmin收集菌体。菌体用超声破碎仪破碎细胞， 4°C， 12000rpm/min离心 20min获得上清液和沉淀，上清液用垂直电泳仪进行 SDS-PAGE检 测。结果发现在终浓度为 0.02mM IPTG浓度时诱导表达双羰基还原酶的量最多。为提 高双羰基还原酶的表达量， 将含有重组质粒的大肠杆菌 BL21 (DE3 ) 在 IPTG终浓度 为 0.02mM， 分别于 37°C诱导 8h， 30°C诱导 8h以及 25 °C诱导 16h条件下表达， 并同 时设立不加 IPTG诱导剂的阴性对照。 诱导表达结束后， 分别将三个温度的诱导菌体 于 8000rpm/min离心 lOmin收集菌体。菌体用超声破碎仪破碎细胞， 4°C , 12000rpm/min 离心 20min获得上清液和沉淀， 上清液用垂直电泳仪进行 SDS-PAGE检测。 结果发现 在 25°C诱导 16h时表达双羰基还原酶的量最多。选用已经在市场上商业化的原料或者 容易制备的酮类化合物 1^^^^^^2 为初始原料， 其中 选自芳香基； R2选自 烷基。 所述的双羟基产物由以下化学通式表达： Kl^u^/^OR2， 其中 选自芳 香基； R2选自烷基。 向 10ml反应瓶中， 加入 O.Olg主原料^^^^^/^ ^， 0.04ml聚乙二醇 PEG-400, 原料溶解后， 加入 0.86ml磷酸盐缓冲液 ClOOmM, pH=6.0)， 底物均匀分散于 缓冲液中； 加入 lmg NAD+， 4.12mg甲酸铵， 2mg辅酶甲酸脱氢酶和 4mg双羰基还原 酶， 体系 pH=6.0， 并于 30±3 °C保温 15-24h; 用 lml乙酸乙酯停止反应， 用 0.25g硅藻 土过滤， lml 乙酸乙酯萃取两次， 静置分液， 有机相经干燥， 过滤， 浓缩得到粗品，
Figure imgf000012_0001
%， 收率 90%， ee值 92.1%， de值 78.9%。 所得产品的核磁数据如下： 400Hz， CDC13: 57.29 (m, 5H),4.54(s,2H), 4.22(m,lH), 4.07 (m, 1H) ,3.45~3.40(m,4H), 2.4 l(d, 2H), 1.65 (t,2H) , 1.43(S,9H)。 所得产品质谱数据如下： MW=310±1。 实施例 3 与实施例 1中序列同源性大于 90%的双幾基还原酶 ( SEQ ID N0.9 ); 该基因为 SEQ NO.l的突变体。 为了便于双羰基还原酶基因的表达以及鉴定， 在寡聚核苷酸引物的 5'和 3'末端设 计了兼容的限制性酶切位点。 其引物对如下： 上游引物 SEQ ID NO.10 : 5 ' -GGAATTCCATATGACCGAACTGAAACAAATCACC-3 '； 下游弓 |物 SEQ ID NO. ll： 5 ' -CCGCTCGAGACCTTTGTAGTTGTAAAAGCCGTCAC-3 '。 扩增得基因序列 （SEQ ID N0.9 ： WLKENYIDKGKLGLASGEGFYKYN) After the total synthesis of the dicarbonyl reductase gene, it is ligated to the pUC57 vector, and the target gene and pET-22b (+) (pET-22b (+) can also be pET-3a (+) with Nde l and Xho l respectively. , pET-3d (+), pET-l la ( + ), pET-12a ( + ), pET-14b ( + ), pET-15b ( + ), pET-16b ( + ), pET-17b ( + ) , pET-19b ( + ), pET-20b ( + ), pET-21a ( + ), pET-23a ( + ), pET-23b ( + ), pET-24a ( + ), pET-25b ( + ), pET-26b ( + ), pET-27b ( + ), pET-28a ( + ), pET-29a ( + ), pET-30a ( + ), pET-31b ( + ), pET-32a ( + ), pET -35b ( + ), pET-38b ( + ), pET-39b ( + ), pET-40b ( + ), pET-41a ( + ), pET-41b ( + ), pET-42a ( + ), pET- 43a ( + ), pET-43b ( + ), pET-44a ( + ), pET-49b ( + ), pQE2, pQE9, pQE30, pQE31 , pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-l, pGEX-6p-l, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwinl, pEZZ18, pKK232-18, pUC-18, pUC-19) Cut, T4 DNA ligase for ligation, will be produced The material was transformed into E. coli DH5 (competent state of x strain, plated in a solid LB culture dish containing 50 μ § /ιη1 containing ampicillin, and cultured overnight at 37 ° C. Single colonies on the above medium were inoculated to contain 50μ § /ιη1 ampicillin-containing LB liquid medium, cultured at 37 °C overnight, after plasmid extraction, PCR identification and double enzyme digestion, the correct cloning vector pET22b ( + ) -DKR was transformed into E. coli BL21 In (DE3), it was applied to LB medium containing 50 μ § /ιη1 containing ampicillin, and cultured at 37 ° C overnight. A single colony in the above medium was picked and inoculated into 5 ml of LB liquid medium containing 50 μ § /ιη1 containing ampicillin, and the correct expression vector was identified by colony PCR to induce expression, and the above bacterial solution was transferred to 5 ml containing 50 μ. § /ιη1 ampicillin-containing LB liquid medium, shake culture at 37 °C until OD 6QQ = 0.6, IPTG was added to a final concentration of 0.02 mM, 0.05 mM, O. lmM, 0.2 mM, 0.4 mM, 0.6 mM , 0.8 mM and 1.0 mM, induced expression at 18 ° C, and a negative control without IPTG inducer was established. After induction for 16 h, the bacterial solution was taken out, and the cells were collected by centrifugation at 8000 rpm/min for 10 min. The cells were disrupted by a sonicator, centrifuged at 12000 rpm/min for 20 min to obtain a supernatant and a precipitate, and the supernatant was subjected to SDS-PAGE detection using a vertical electrophoresis apparatus. As a result, it was found that the amount of expression of the biscarbonyl reductase was most induced at a final concentration of 0.02 mM IPTG. In order to increase the expression level of dicarbonyl reductase, E. coli BL21 (DE3) containing recombinant plasmid was induced at a final concentration of IPTG of 0.02 mM for 8 h at 37 ° C, 8 h at 30 ° C and 16 h at 25 ° C for 16 h. Expression, and a negative control without IPTG inducer was also established. After the induction of expression was completed, the cells at three temperatures were centrifuged at 8000 rpm/min for 10 min to collect the cells. The cells were disrupted with a sonicator, 4 ° C, 12000 rpm / min The supernatant and the precipitate were obtained by centrifugation for 20 min, and the supernatant was subjected to SDS-PAGE detection using a vertical electrophoresis apparatus. As a result, it was found that the amount of expression of the biscarbonylreductase was the highest when induced at 25 ° C for 16 hours. The raw material which has been commercialized on the market or the easily prepared ketone compound 1 ^^^^^^ 2 is used as a starting material, wherein it is selected from an aromatic group; and R 2 is selected from an alkyl group. The bishydroxy product is expressed by the following chemical formula: Kl ^ u ^ / ^ OR2 , wherein is selected from an aromatic group; and R 2 is selected from an alkyl group. To a 10 ml reaction flask, O.Olg main raw material ^^^^^/^ ^, 0.04 ml polyethylene glycol PEG-400 was added, and after the raw material was dissolved, 0.86 ml of phosphate buffer solution COOmM, pH=6.0) was added. The substance was uniformly dispersed in the buffer; 1 mg of NAD + , 4.12 mg of ammonium formate, 2 mg of coenzyme formate dehydrogenase and 4 mg of dicarbonyl reductase were added, the system pH was 6.0, and the temperature was maintained at 30 ± 3 ° C for 15-24 h; The reaction mixture was quenched with ethyl acetate. EtOAc (EtOAc m.
Figure imgf000012_0001
%, yield 90%, ee value 92.1%, de value 78.9%. The nuclear magnetic data of the obtained product are as follows: 400 Hz, CDC13: 57.29 (m, 5H), 4.54 (s, 2H), 4.22 (m, lH), 4.07 (m, 1H), 3.45 to 3.40 (m, 4H), 2.4 l (d, 2H), 1.65 (t, 2H), 1.43 (S, 9H). The mass spectrum data of the obtained product was as follows: MW = 310 ± 1. Example 3 A bis-reductase having a sequence homology of greater than 90% in Example 1 (SEQ ID N0.9); the gene is a mutant of SEQ NO. To facilitate expression and identification of the dicarbonyl reductase gene, compatible restriction sites were designed at the 5' and 3' ends of the oligonucleotide primers. The primer pairs are as follows: Upstream primer SEQ ID NO. 10: 5 '-GGAATTCCATATGACCGAACTGAAACAAATCACC-3 '; Downstream bow | SEQ ID NO. ll: 5 '-CCGCTCGAGACCTTTGTAGTTGTAAAAGCCGTCAC-3 '. Amplified gene sequence (SEQ ID N0.9:
其 氨 基 酸 序 列 为 （ SEQ ID NO.12 ： MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDINIts amino acid sequence is (SEQ ID NO. 12: MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDIN
DEVLEAAKARFEKLAETYA EVPDARDGKAREALGRLTLTSDLKAAVADADLVIEA
Figure imgf000013_0001
DEVLEAAKARFEKLAETYA EVPDARDGKAREALGRLTLTSDLKAAVADADLVIEA
Figure imgf000013_0001
ENYIDKGKLGLASGEGFYKYN) 双羰基还原酶基因全合成后， 连接到 pUC57载体上， 用 Nde l和 Xho l分别将目 的基因和 pET-22b (+) (pET-22b (+) 也可以为 pET-3a (+)， pET-3d (+)， pET-l la (+)， pET-12a (+)， pET-14b (+)， pET-15b (+)， pET-16b (+)， pET-17b (+)， pET-19b ( + )， pET-20b ( + )， pET-21a ( + )， pET-23a ( + )， pET-23b ( + )， pET-24a ( + )， pET-25b ( + )， pET-26b ( + )， pET-27b ( + )， pET-28a ( + )， pET-29a ( + )， pET-30a ( + )， pET-31b ( + )， pET-32a ( + )， pET-35b ( + )， pET-38b ( + )， pET-39b ( + )， pET-40b ( + )， pET-41a ( + )， pET-41b ( + )， pET-42a ( + )， pET-43a ( + )， pET-43b ( + )， pET-44a ( + )， pET-49b ( + )， pQE2, pQE9, pQE30, pQE31 , pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B， pRSET-C， pGEX-5X- 1， pGEX-6p- 1， pGEX-6p-2， pBV220， pBV221， pBV222， pTrc99A, pTwinl , pEZZ18， pKK232-18, pUC-18, pUC-19) 质粒同时进行酶切， T4 DNA连接酶进行连接反应， 将连接产物转化到大肠杆菌 DH5(x菌株的感受态中， 涂布 于含有 50μ§/ιη1含氨苄青霉素的固体 LB培养基中， 37°C培养过夜。 挑取上述培养基 上的单菌落接种于含有 50μ§/ιη1含氨苄青霉素的 LB液体培养基中， 37°C振荡培养过 夜， 经过质粒提取， PCR鉴定和双酶切鉴定后， 将正确的克隆载体 pET22b ( + ) -DKR 转化到大肠杆菌 BL21 (DE3 )中， 涂布于含有 50μ§/ιη1含氨苄青霉素的 LB培养基中， 37°C培养过夜。 挑取上述培养基中上的单菌落接种于 5ml含 50μ§/ιη1含氨苄青霉素的 LB液体培养基中， 经过菌落 PCR鉴定正确的表达载体进行诱导表达， 将上述菌液转 接于 5ml含 50μ§/ιη1含氨苄青霉素的 LB液体培养基中， 37°C振荡培养至 OD6QQ=0.6时， 加入 IPTG至终浓度分别为 0.02mM， 0.05mM， O. lmM, 0.2mM, 0.4mM， 0.6mM， 0.8mM 禾口 1.0mM， 在 18°C下进行诱导表达， 并设立不加 IPTG诱导剂的阴性对照。 诱导 16h 后， 取出菌液， 8000rpm/min离心 lOmin收集菌体。菌体用超声破碎仪破碎细胞， 4°C， 12000rpm/min离心 20min获得上清液和沉淀，上清液用垂直电泳仪进行 SDS-PAGE检 测。结果发现在终浓度为 0.02mM IPTG浓度时诱导表达双羰基还原酶的量最多。为提 高双羰基还原酶的表达量， 将含有重组质粒的大肠杆菌 BL21 (DE3 ) 在 IPTG终浓度 为 0.02mM， 分别于 37°C诱导 8h， 30°C诱导 8h以及 25°C诱导 16h条件下表达， 并同 时设立不加 IPTG诱导剂的阴性对照。 诱导表达结束后， 分别将三个温度的诱导菌体 于 8000rpm/min离心 lOmin收集菌体。菌体用超声破碎仪破碎细胞， 4°C , 12000rpm/min 离心 20min获得上清液和沉淀， 上清液用垂直电泳仪进行 SDS-PAGE检测。 结果发现 在 25°C诱导 16h时表达双羰基还原酶的量最多。 选用已经在市场上商业化的原料或者容易制备的酮类化合物 ENYIDKGKLGLASGEGFYKYN) After the total synthesis of the dicarbonyl reductase gene, it is ligated to the pUC57 vector, and the target gene and pET-22b (+) (pET-22b (+) can also be pET-3a (+) with Nde l and Xho l respectively. , pET-3d (+), pET-l la (+), pET-12a (+), pET-14b (+), pET-15b (+), pET-16b (+), pET-17b (+) , pET-19b ( + ), pET-20b ( + ), pET-21a ( + ), pET-23a ( + ), pET-23b ( + ), pET-24a ( + ), pET-25b ( + ), pET-26b ( + ), pET-27b ( + ), pET-28a ( + ), pET-29a ( + ), pET-30a ( + ), pET-31b ( + ), pET-32a ( + ), pET -35b ( + ), pET-38b ( + ), pET-39b ( + ), pET-40b ( + ), pET-41a ( + ), pET-41b ( + ), pET-42a ( + ), pET- 43a ( + ), pET-43b ( + ), pET-44a ( + ), pET-49b ( + ), pQE2, pQE9, pQE30, pQE31 , pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X- 1, pGEX-6p- 1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwinl, pEZZ18, pKK232-18, pUC-18, pUC-19) Cut, T4 DNA ligase for ligation, will be produced The material was transformed into E. coli DH5 (competent state of x strain, and applied to solid LB medium containing 50 μ § /ιη1 containing ampicillin, and cultured overnight at 37 ° C. Single colonies on the above medium were inoculated to contain 50μ § /ιη1 ampicillin-containing LB liquid medium, cultured at 37 °C overnight, after plasmid extraction, PCR identification and double enzyme digestion, the correct cloning vector pET22b ( + ) -DKR was transformed into E. coli BL21 In (DE3), it was applied to LB medium containing 50 μ § /ιη1 containing ampicillin, and cultured at 37 ° C overnight. A single colony in the above medium was picked and inoculated into 5 ml of LB liquid medium containing 50 μ § /ιη1 containing ampicillin, and the correct expression vector was identified by colony PCR to induce expression, and the above bacterial solution was transferred to 5 ml containing 50 μ. § /ιη1 ampicillin-containing LB liquid medium, shake culture at 37 ° C until OD 6QQ = 0.6, IPTG was added to a final concentration of 0.02 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM and 1.0 mM, respectively, and induced expression at 18 ° C, and no IPTG inducer was added. Negative control. After induction for 16 h, the bacterial solution was taken out, and the cells were collected by centrifugation at 8000 rpm/min for 10 min. The cells were disrupted by a sonicator, centrifuged at 12000 rpm/min for 20 min to obtain a supernatant and a precipitate, and the supernatant was subjected to SDS-PAGE detection using a vertical electrophoresis apparatus. As a result, it was found that the amount of expression of the biscarbonyl reductase was most induced at a final concentration of 0.02 mM IPTG. In order to increase the expression level of dicarbonyl reductase, E. coli BL21 (DE3) containing recombinant plasmid was induced at a final concentration of IPTG of 0.02 mM for 8 h at 37 ° C, 8 h at 30 ° C and 16 h at 25 ° C for 16 h. Expression, and a negative control without IPTG inducer was also established. After the induction of expression was completed, the cells at three temperatures were centrifuged at 8000 rpm/min for 10 min to collect the cells. The cells were disrupted by a sonicator, centrifuged at 12000 rpm/min for 20 min to obtain a supernatant and a precipitate, and the supernatant was subjected to SDS-PAGE detection using a vertical electrophoresis apparatus. As a result, it was found that the amount of expression of the biscarbonylreductase was the highest when induced at 25 ° C for 16 hours. Use raw materials that have been commercialized on the market or ketone compounds that are easy to prepare
为初始原料， 其中 选自芳香基； R2选自烷基。所述的双羟基产物由以下化学通式表 As a starting material, which is selected from an aromatic group; R 2 is selected from an alkyl group. The bishydroxy product is represented by the following chemical formula
OH OH O OH OH O
R2， 其中 选自芳香基； R2选自烷基。 向 10ml反应瓶中， 加入 O.Olg
Figure imgf000014_0001
0.04ml聚乙二醇 PEG-400, 原料溶解后， 加入 0.86ml磷酸盐缓冲液 ClOOmM, pH=6.0)， 底物均匀分散于 缓冲液中； 加入 lmg NAD+， 4.12mg甲酸铵， 2mg辅酶甲酸脱氢酶和 4mg双羰基还原 酶， 体系 pH=6.0， 并于 30±3 °C保温 15-24h; 用 lml乙酸乙酯停止反应， 用 0.25g硅藻 土过滤， lml 乙酸乙酯萃取两次， 静置分液， 有机相经干燥， 过滤， 浓缩得到粗品，
Figure imgf000014_0002
, 收率 90%， ee值 93.9%， de值 90.7%, 所得产品的核磁数据如下： 400Hz， CDC13: 57.29 (m, 5H),4.54(s,2H), 4.22(m,lH), 4.07 (m, 1H) ,3.45~3.40(m,4H), 2.4 l(d, 2H), 1.65 (t,2H) , 1.43(S,9H)。 所得产品质谱数据如下： MW=310±1。 实施例 4 ： 与实施例 1中序列同源性大于 90%的双羰基还原酶 ( SEQ 1D 0. 13 ); 该基因为 SEQ NO.l的突变体。 为了便于双羰基还原酶基因的表达以及鉴定， 在寡聚核苷酸引物的 5'和 3'末端设 计了兼容的限制性酶切位点。 其引物对如下： 上游引物 SEQ ID N0.14 : 5 ' -GGAATTCCATATGACCGAACTGAAACAAATCACC-3 '； 下游弓 |物 SEQ ID NO.15： 5 ' -CCGCTCGAGACCTTTGTAGTTGTAAAAGCCGTCAC-3 '。 扩增得基因序列 （SEQ R 2 , wherein is selected from an aryl group; and R 2 is selected from an alkyl group. Into a 10 ml reaction flask, add O.Olg
Figure imgf000014_0001
0.04ml polyethylene glycol PEG-400, after the raw material is dissolved, add 0.86ml phosphate buffer ClOOmM, pH=6.0), the substrate is uniformly dispersed in the buffer; add 1mg NAD + , 4.12mg ammonium formate, 2mg coenzyme formic acid Dehydrogenase and 4 mg of dicarbonyl reductase, system pH=6.0, and incubated at 30±3 °C for 15-24 h; stop the reaction with 1 ml of ethyl acetate, filter with 0.25 g of diatomaceous earth, and extract 1 ml of ethyl acetate twice , standing still, the organic phase is dried, filtered, concentrated to give a crude product.
Figure imgf000014_0002
, yield 90%, ee value 93.9%, de value 90.7%, the nuclear magnetic data of the obtained product are as follows: 400Hz, CDC13: 57.29 (m, 5H), 4.54 (s, 2H), 4.22 (m, lH), 4.07 ( m, 1H), 3.45~3.40 (m, 4H), 2.4 l(d, 2H), 1.65 (t, 2H), 1.43 (S, 9H). The mass spectrum data of the obtained product was as follows: MW = 310 ± 1. Example 4: A biscarbonyl reductase having a sequence homology of greater than 90% in Example 1 (SEQ ID NO: 13); the gene is a mutant of SEQ NO. To facilitate expression and identification of the dicarbonyl reductase gene, compatible restriction sites were designed at the 5' and 3' ends of the oligonucleotide primers. Its primer pair is as follows: Upstream primer SEQ ID N0.14: 5 '-GGAATTCCATATGACCGAACTGAAACAAATCACC-3 '; Downstream bow|Material SEQ ID NO. 15: 5 '-CCGCTCGAGACCTTTGTAGTTGTAAAAGCCGTCAC-3 '. Amplified gene sequence (SEQ
Figure imgf000015_0001
其 氨 基 酸 序 列 为 （ SEQ ID NO.16 ： MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDIN
Figure imgf000015_0001
Its amino acid sequence is (SEQ ID NO. 16: MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDIN
DEVLEAAKARFEKLAETYA EVPDARDGKAREALGRLTLTSDLKAAVADADLVIEA
Figure imgf000015_0002
DEVLEAAKARFEKLAETYA EVPDARDGKAREALGRLTLTSDLKAAVADADLVIEA
Figure imgf000015_0002
ENYIDKGKLGLASGEGFYKYN) 双羰基还原酶基因全合成后， 连接到 pUC57载体上， 用 Nde l和 Xho l分别将目 的基因和 pET-22b (+) (pET-22b (+) 也可以为 pET-3a (+)， pET-3d (+)， pET-l la (+)， pET-12a (+)， pET-14b (+)， pET-15b (+)， pET-16b (+)， pET-17b (+)， pET-19b ( + )， pET-20b ( + )， pET-21a ( + )， pET-23a ( + )， pET-23b ( + )， pET-24a ( + )， pET-25b ( + )， pET-26b ( + )， pET-27b ( + )， pET-28a ( + )， pET-29a ( + )， pET-30a ( + )， pET-31b ( + )， pET-32a ( + )， pET-35b ( + )， pET-38b ( + )， pET-39b ( + )， pET-40b ( + )， pET-41a ( + )， pET-41b ( + )， pET-42a ( + )， pET-43a ( + )， pET-43b ( + )， pET-44a ( + )， pET-49b ( + )， pQE2, pQE9, pQE30, pQE31 , pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B， pRSET-C， pGEX-5X- 1， pGEX-6p- 1， pGEX-6p-2， pBV220， pBV221， pBV222， pTrc99A, pTwinl , pEZZ18， pKK232-18, pUC-18, pUC-19) 质粒同时进行酶切， T4ENYIDKGKLGLASGEGFYKYN) After the total synthesis of the dicarbonyl reductase gene, it is ligated to the pUC57 vector, and the target gene and pET-22b (+) (pET-22b (+) can also be pET-3a (+) with Nde l and Xho l respectively. , pET-3d (+), pET-l la (+), pET-12a (+), pET-14b (+), pET-15b (+), pET-16b (+), pET-17b (+) , pET-19b ( + ), pET-20b ( + ), pET-21a ( + ), pET-23a ( + ), pET-23b ( + ), pET-24a ( + ), pET-25b ( + ), pET-26b ( + ), pET-27b ( + ), pET-28a ( + ), pET-29a ( + ), pET-30a ( + ), pET-31b ( + ), pET-32a ( + ), pET -35b ( + ), pET-38b ( + ), pET-39b ( + ), pET-40b ( + ), pET-41a ( + ), pET-41b ( + ), pET-42a ( + ), pET- 43a ( + ), pET-43b ( + ), pET-44a ( + ), pET-49b ( + ), pQE2, pQE9, pQE30, pQE31 , pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X- 1, pGEX-6p- 1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwinl, pEZZ18, pKK232-18, pUC-18, pUC-19) plasmid cleavage, T4
DNA连接酶进行连接反应， 将连接产物转化到大肠杆菌 DH5(x菌株的感受态中， 涂布 于含有 50μ§/ιη1含氨苄青霉素的固体 LB培养基中， 37°C培养过夜。 挑取上述培养基 上的单菌落接种于含有 50μ§/ιη1含氨苄青霉素的 LB液体培养基中， 37°C振荡培养过 夜， 经过质粒提取， PCR鉴定和双酶切鉴定后， 将正确的克隆载体 pET22b ( + ) -DKR 转化到大肠杆菌 BL21 (DE3 )中， 涂布于含有 50μ§/ιη1含氨苄青霉素的 LB培养基中， 37°C培养过夜。 挑取上述培养基中上的单菌落接种于 5ml含 50μ§/ιη1含氨苄青霉素的 LB液体培养基中， 经过菌落 PCR鉴定正确的表达载体进行诱导表达， 将上述菌液转 接于 5ml含 50μ§/ιη1含氨苄青霉素的 LB液体培养基中， 37°C振荡培养至 OD6QQ=0.6时， 加入 IPTG至终浓度分别为 0.02mM， 0.05mM， O. lmM, 0.2mM, 0.4mM， 0.6mM， 0.8mM 禾口 1.0mM， 在 18°C下进行诱导表达， 并设立不加 IPTG诱导剂的阴性对照。 诱导 16h 后， 取出菌液， 8000rpm/min离心 lOmin收集菌体。菌体用超声破碎仪破碎细胞， 4°C， 12000rpm/min离心 20min获得上清液和沉淀，上清液用垂直电泳仪进行 SDS-PAGE检 测。结果发现在终浓度为 0.02mM IPTG浓度时诱导表达双羰基还原酶的量最多。为提 高双羰基还原酶的表达量， 将含有重组质粒的大肠杆菌 BL21 (DE3 ) 在 IPTG终浓度 为 0.02mM， 分别于 37°C诱导 8h， 30°C诱导 8h以及 25°C诱导 16h条件下表达， 并同 时设立不加 IPTG诱导剂的阴性对照。 诱导表达结束后， 分别将三个温度的诱导菌体 于 8000rpm/min离心 lOmin收集菌体。菌体用超声破碎仪破碎细胞， 4°C , 12000rpm/min 离心 20min获得上清液和沉淀， 上清液用垂直电泳仪进行 SDS-PAGE检测。 结果发现 在 25°C诱导 16h时表达双羰基还原酶的量最多。 选用已经在市场上商业化的原料或者容易制备的酮类化合物 The ligation reaction was carried out by DNA ligase, and the ligation product was transformed into E. coli DH5 (competent state of x strain, and plated in solid LB medium containing 50 μ § /ιη1 containing ampicillin, and cultured overnight at 37 ° C. A single colony on the medium was inoculated into LB liquid medium containing 50 μ § /ιη1 containing ampicillin, and cultured overnight at 37 ° C with shaking. After plasmid extraction, PCR identification and double enzyme digestion, the correct cloning vector pET22b ( + ) -DKR was transformed into E. coli BL21 (DE3), plated in LB medium containing 50 μ § /ιη1 containing ampicillin, and cultured overnight at 37 ° C. Single colonies in the above medium were picked and inoculated into 5 ml. In the LB liquid medium containing 50 μ § /ιη1 containing ampicillin, the correct expression vector was identified by colony PCR to induce expression, and the above bacterial solution was transferred to 5 ml of LB liquid medium containing 50 μ § /ιη1 containing ampicillin. When cultured at 37 ° C to OD 6QQ = 0.6, IPTG was added to a final concentration of 0.02 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM and 1.0 mM at 18 ° C. Inducing expression, A negative control without IPTG inducer was established. After induction for 16 hours, the bacterial solution was taken out, and the cells were collected by centrifugation at 8000 rpm/min for 10 min. The cells were disrupted by sonication, centrifuged at 12000 rpm/min for 20 min to obtain the supernatant. The supernatant was precipitated by SDS-PAGE with a vertical electrophoresis apparatus. It was found that the maximum amount of biscarbonyl reductase was induced at a final concentration of 0.02 mM IPTG. In order to increase the expression of biscarbonyl reductase, it would contain recombination. The plasmid Escherichia coli BL21 (DE3) was induced at a final concentration of IPTG of 0.02 mM for 8 h at 37 ° C, 8 h at 30 ° C and 16 h at 25 ° C, and a negative control without IPTG inducer was also established. After the induction of expression, the three cells were centrifuged at 8000 rpm/min for 10 min to collect the cells. The cells were disrupted with a sonicator and centrifuged at 12000 rpm/min for 20 min to obtain the supernatant and precipitate. The supernatant was subjected to SDS-PAGE detection by a vertical electrophoresis apparatus. It was found that the amount of the expression of the dicarbonyl reductase was the highest when induced at 25 ° C for 16 h. The raw materials which have been commercialized on the market or the easily prepared ketones were selected. Compound
为初始原料， 其中 选自芳香基； R2选自烷基。所述的双羟基产物由以下化学通式表 As a starting material, which is selected from an aromatic group; R 2 is selected from an alkyl group. The bishydroxy product is represented by the following chemical formula
OH OH O OH OH O
R2， 其中 选自芳香基； R2选自烷基。 向 10ml反应瓶中， 加入 O.Olg
Figure imgf000016_0001
0.04ml聚乙二醇 PEG-400, 原料溶解后， 加入 0.86ml磷酸盐缓冲液 ClOOmM, pH=6.0)， 底物均匀分散于 缓冲液中； 加入 lmg NAD+， 4.12mg甲酸铵， 2mg辅酶甲酸脱氢酶和 4mg双羰基还原 酶， 体系 pH=6.0， 并于 30±3 °C保温 15-24h; 用 lml乙酸乙酯停止反应， 用 0.25g硅藻 土过滤， lml 乙酸乙酯萃取两次， 静置分液， 有机相经干燥， 过滤， 浓缩得到粗品， R 2 , wherein is selected from an aryl group; and R 2 is selected from an alkyl group. Into a 10 ml reaction flask, add O.Olg
Figure imgf000016_0001
0.04ml polyethylene glycol PEG-400, after the raw material is dissolved, add 0.86ml phosphate buffer ClOOmM, pH=6.0), the substrate is uniformly dispersed in the buffer; add 1mg NAD + , 4.12mg ammonium formate, 2mg coenzyme formic acid Dehydrogenase and 4 mg of dicarbonyl reductase, system pH=6.0, and incubated at 30±3 °C for 15-24 h; stop the reaction with 1 ml of ethyl acetate, filter with 0.25 g of diatomaceous earth, and extract 1 ml of ethyl acetate twice , standing still, the organic phase is dried, filtered, concentrated to give a crude product.
OH 0H 0 I  OH 0H 0 I
50.75 % , 收率 90%， ee值 91.74%， de值 91.6%。 所得产品的核磁数据如下： 400Hz， CDC13: 57.29 (m, 5H),4.54(s,2H), 4.22(m,lH), 4.07 (m, 1H) ,3.45~3.40(m,4H), 2.4 l(d, 2H), 1.65 (t,2H) , 1.43(S,9H)。 所得产品质谱数据如下： MW=310±1。 实施例 5 ： 与实施例 1中序列同源性大于 90%的双羰基还原酶 ( SEQ 1D 0. 17); 该基因为 SEQ NO.l的突变体。 为了便于双羰基还原酶基因的表达以及鉴定， 在寡聚核苷酸引物的 5'和 3'末端设 计了兼容的限制性酶切位点。 其引物对如下： 上游引物 SEQ ID N0.18 : 5 ' -GGAATTCCATATGACCGAACTGAAACAAATCACC-3 '； 下游弓 |物 SEQ ID NO.19： 5 ' -CCGCTCGAGACCTTTGTAGTTGTAAAAGCCGTCAC-3 '。 扩增得基因序列 （SEQ 50.75 %, yield 90%, ee value 91.74%, de value 91.6%. The nuclear magnetic data of the obtained product are as follows: 400 Hz, CDC13: 57.29 (m, 5H), 4.54 (s, 2H), 4.22 (m, lH), 4.07 (m, 1H), 3.45 to 3.40 (m, 4H), 2.4 l (d, 2H), 1.65 (t, 2H), 1.43 (S, 9H). The mass spectrum data of the obtained product was as follows: MW = 310 ± 1. Example 5: A biscarbonyl reductase having a sequence homology of greater than 90% in Example 1 (SEQ ID NO: 17); the gene is a mutant of SEQ NO. To facilitate expression and identification of the dicarbonyl reductase gene, compatible restriction sites were designed at the 5' and 3' ends of the oligonucleotide primers. The primer pairs are as follows: Upstream primer SEQ ID N0.18: 5 '-GGAATTCCATATGACCGAACTGAAACAAATCACC-3 '; Downstream bow | SEQ ID NO. 19: 5 '-CCGCTCGAGACCTTTGTAGTTGTAAAAGCCGTCAC-3 '. Amplified gene sequence (SEQ
Figure imgf000017_0001
其 氨 基 酸 序 列 为 （ SEQ ID NO.20 ： MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDIN
Figure imgf000017_0001
Its amino acid sequence is (SEQ ID NO. 20: MTNSVKTVTVLGTGVLGSQIAFQSAFKGFTVTAYDIN
DEVLEAAKARFEKLAETYA EVPDARDGKAREALGRLTLTSDLKAAVADADLVIEA VPEILDLKRETYQKLGDLAPAKTIFATNSSTLLPSDLKDSTGRPDKFLALHFA RVW KENYIDKGKLGLASGEGFYKYN) 双羰基还原酶基因全合成后， 连接到 pUC57载体上， 用 Nde l和 Xho l分别将目 的基因和 pET-22b (+) (pET-22b (+) 也可以为 pET-3a (+)， pET-3d (+)， pET-l la (+)， pET-12a (+)， pET-14b (+)， pET-15b (+)， pET-16b (+)， pET-17b (+)， pET-19b ( + )， pET-20b ( + )， pET-21a ( + )， pET-23a ( + )， pET-23b ( + )， pET-24a ( + )， pET-25b ( + )， pET-26b ( + )， pET-27b ( + )， pET-28a ( + )， pET-29a ( + )， pET-30a ( + )， pET-31b ( + )， pET-32a ( + )， pET-35b ( + )， pET-38b ( + )， pET-39b ( + )， pET-40b ( + )， pET-41a ( + )， pET-41b ( + )， pET-42a ( + )， pET-43a ( + )， pET-43b ( + )， pET-44a ( + )， pET-49b ( + )， pQE2, pQE9, pQE30, pQE31 , pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-l , pGEX-6p-l , pGEX-6p-2, pBV220, pBV221 , pBV222, pTrc99A, pTwinl , pEZZ18， pKK232-18, pUC-18, pUC-19) 质粒同时进行酶切， T4 DNA连接酶进行连接反应， 将连接产物转化到大肠杆菌 DH5(x菌株的感受态中， 涂布 于含有 50μ§/ιη1含氨苄青霉素的固体 LB培养基中， 37°C培养过夜。 挑取上述培养基 上的单菌落接种于含有 50μ§/ιη1含氨苄青霉素的 LB液体培养基中， 37°C振荡培养过 夜， 经过质粒提取， PCR鉴定和双酶切鉴定后， 将正确的克隆载体 pET22b ( + ) -DKR 转化到大肠杆菌 BL21 (DE3 )中， 涂布于含有 50μ§/ιη1含氨苄青霉素的 LB培养基中， 37°C培养过夜。 挑取上述培养基中上的单菌落接种于 5ml含 50μ§/ιη1含氨苄青霉素的 LB液体培养基中， 经过菌落 PCR鉴定正确的表达载体进行诱导表达， 将上述菌液转 接于 5ml含 50μ§/ιη1含氨苄青霉素的 LB液体培养基中， 37°C振荡培养至 OD6(X)=0.6时， 加入 IPTG至终浓度分别为 0.02mM， 0.05mM， O. lmM, 0.2mM, 0.4mM， 0.6mM， 0.8mM 禾口 1.0mM， 在 18°C下进行诱导表达， 并设立不加 IPTG诱导剂的阴性对照。 诱导 16h 后， 取出菌液， 8000rpm/min离心 lOmin收集菌体。菌体用超声破碎仪破碎细胞， 4°C， 12000rpm/min离心 20min获得上清液和沉淀，上清液用垂直电泳仪进行 SDS-PAGE检 测。结果发现在终浓度为 0.02mM IPTG浓度时诱导表达双羰基还原酶的量最多。为提 高双羰基还原酶的表达量， 将含有重组质粒的大肠杆菌 BL21 (DE3 ) 在 IPTG终浓度 为 0.02mM， 分别于 37°C诱导 8h， 30°C诱导 8h以及 25 °C诱导 16h条件下表达， 并同 时设立不加 IPTG诱导剂的阴性对照。 诱导表达结束后， 分别将三个温度的诱导菌体 于 8000rpm/min离心 lOmin收集菌体。菌体用超声破碎仪破碎细胞， 4°C , 12000rpm/min 离心 20min获得上清液和沉淀， 上清液用垂直电泳仪进行 SDS-PAGE检测。 结果发现 在 25 °C诱导 16h时表达双羰基还原酶的量最多。 ο ο ο 选用已经在市场上商业化的原料或者容易制备的酮类化合物 DEVLEAAKARFEKLAETYA EVPDARDGKAREALGRLTLTSDLKAAVADADLVIEA VPEILDLKRETYQKLGDLAPAKTIFATNSSTLLPSDLKDSTGRPDKFLALHFA RVW KENYIDKGKLGLASGEGFYKYN) After the total synthesis of the dicarbonyl reductase gene, it is ligated to the pUC57 vector, and the target gene and pET-22b (+) (pET-22b (+) can also be pET-3a (+) with Nde l and Xho l respectively. , pET-3d (+), pET-l la (+), pET-12a (+), pET-14b (+), pET-15b (+), pET-16b (+), pET-17b (+) , pET-19b ( + ), pET-20b ( + ), pET-21a ( + ), pET-23a ( + ), pET-23b ( + ), pET-24a ( + ), pET-25b ( + ), pET-26b ( + ), pET-27b ( + ), pET-28a ( + ), pET-29a ( + ), pET-30a ( + ), pET-31b ( + ), pET-32a ( + ), pET -35b ( + ), pET-38b ( + ), pET-39b ( + ), pET-40b ( + ), pET-41a ( + ), pET-41b ( + ), pET-42a ( + ), pET- 43a ( + ), pET-43b ( + ), pET-44a ( + ), pET-49b ( + ), pQE2, pQE9, pQE30, pQE31 , pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-l, pGEX-6p-l, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwinl, pEZZ18, pKK232-18, pUC-18, pUC-19) Cutting, T4 DNA ligase is ligated to convert the ligation product to Enterobacter the DH5 (x competent strain, the coating containing 50μ § / ιη1 solid LB medium containing ampicillin, 37 ° C overnight. Picked single colonies on the above medium was inoculated containing 50μ § / Ign1 was cultured in ampicillin-containing LB liquid medium at 37 °C overnight. After plasmid extraction, PCR identification and double enzyme digestion, the correct cloning vector pET22b ( + ) -DKR was transformed into E. coli BL21 (DE3 ). Among them, it was applied to LB medium containing 50 μ § /ιη1 containing ampicillin, and cultured at 37 ° C overnight. A single colony in the above medium was picked and inoculated into 5 ml of LB liquid medium containing 50 μ § /ιη1 containing ampicillin, and the correct expression vector was identified by colony PCR to induce expression, and the above bacterial solution was transferred to 5 ml containing 50 μ. § /ιη1 ampicillin-containing LB liquid medium, shake culture at 37 °C until OD 6 (X) = 0.6, IPTG was added to a final concentration of 0.02 mM, 0.05 mM, O. lmM, 0.2 mM, 0.4 mM , 0.6 mM, 0.8 mM and 1.0 mM, induced expression at 18 ° C, and a negative control without IPTG inducer was established. After induction for 16 h, the bacterial solution was taken out, and the cells were collected by centrifugation at 8000 rpm/min for 10 min. The cells were disrupted by a sonicator, centrifuged at 12000 rpm/min for 20 min to obtain a supernatant and a precipitate, and the supernatant was subjected to SDS-PAGE detection using a vertical electrophoresis apparatus. As a result, it was found that the amount of expression of the biscarbonyl reductase was most induced at a final concentration of 0.02 mM IPTG. In order to increase the expression level of dicarbonyl reductase, E. coli BL21 (DE3) containing recombinant plasmid was induced at a final concentration of IPTG of 0.02 mM for 8 h at 37 ° C, 8 h at 30 ° C and 16 h at 25 ° C for 16 h. Expression, and a negative control without IPTG inducer was also established. After the induction of expression was completed, the cells at three temperatures were centrifuged at 8000 rpm/min for 10 min to collect the cells. The cells were disrupted by a sonicator, centrifuged at 12000 rpm/min for 20 min to obtain a supernatant and a precipitate, and the supernatant was subjected to SDS-PAGE detection using a vertical electrophoresis apparatus. As a result, it was found that the amount of the expression of the biscarbonylreductase was the highest at 16 h of induction at 25 °C. ο ο ο Use raw materials that have been commercialized on the market or ketone compounds that are easy to prepare
为初始原料， 其中 选自芳香基； R2选自烷基。所述的双羟基产物由以下化学通式表 As a starting material, which is selected from an aromatic group; R 2 is selected from an alkyl group. The bishydroxy product is represented by the following chemical formula
OH OH O  OH OH O
其中 选自芳香基； 选自烷基 c 向 10ml反应瓶中， 加入 O.Olg主原料"^^^^^/"^^， 0.04ml聚乙二醇 PEG-400, 原料溶解后， 加入 0.86ml磷酸盐缓冲液 ClOOmM, pH=6.0)， 底物均匀分散于 缓冲液中； 加入 lmg NAD+， 4.12mg甲酸铵， 2mg辅酶甲酸脱氢酶和 4mg双羰基还原 酶， 体系 pH=6.0， 并于 30±3 °C保温 15-24h; 用 lml乙酸乙酯停止反应， 用 0.25g硅藻 土过滤， lml 乙酸乙酯萃取两次， 静置分液， 有机相经干燥， 过滤， 浓缩得到粗品，
Figure imgf000019_0001
% , 收率 90%， ee值 93.8%， de值 85.9%c 所得产品的核磁数据如下： 400Hz， CDC13: 57.29 (m, 5H),4.54(s,2H), 4.22(m,lH),Which is selected from an aromatic group; is selected from the group consisting of alkyl c to a 10 ml reaction bottle, and added O.Olg main raw material "^^^^^/"^^, 0.04 ml of polyethylene glycol PEG-400, after the raw material is dissolved, 0.86 is added. Ml phosphate buffer ClOOmM, pH=6.0), the substrate is uniformly dispersed in the buffer; add 1mg NAD + , 4.12mg ammonium formate, 2mg coenzyme formate dehydrogenase and 4mg dicarbonyl reductase, system pH=6.0, and Incubate at 30 ± 3 °C for 15-24 h; stop the reaction with 1 ml of ethyl acetate, filter with 0.25 g of celite, extract twice with 1 ml of ethyl acetate, and then stand for separation. The organic phase is dried, filtered and concentrated to give crude ,
Figure imgf000019_0001
%, yield 90%, ee value 93.8%, de value 85.9% c The nuclear magnetic data of the obtained product are as follows: 400 Hz, CDC13: 57.29 (m, 5H), 4.54 (s, 2H), 4.22 (m, lH),
4.07 (m, 1H) ,3.45~3.40(m,4H), 2.4 l(d, 2H), 1.65 (t,2H) , 1.43(S,9H)。 所得产品质谱数据如下： MW=310±1。 从以上的描述中， 可以看出， 本发明上述的实施例实现了如下技术效果： 采用本发 明的双羰基还原酶（Diketoreductase, DKR) 为生物催化剂， 可以一步还原二酮底物， 制备得到单 一光学纯度的 3R， 5S-二羟基化合物， 将其应用到合成他汀类药物中间体， 简化了合成步骤， 降低 了生产污染和生产成本， 适合于大规模工业化生产。 以上所述仅为本发明的优选实施例而已， 并不用于限制本发明， 对于本领域的技 术人员来说， 本发明可以有各种更改和变化。 凡在本发明的精神和原则之内， 所作的 任何修改、 等同替换、 改进等， 均应包含在本发明的保护范围之内。 4.07 (m, 1H), 3.45~3.40 (m, 4H), 2.4 l(d, 2H), 1.65 (t, 2H), 1.43 (S, 9H). The mass spectrum data of the obtained product was as follows: MW = 310 ± 1. From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: By using the dicarbonyloreductase (DKR) of the present invention as a biocatalyst, the diketone substrate can be reduced in one step, and a single preparation is obtained. The optically pure 3R, 5S-dihydroxy compound, which is applied to the synthesis of statin intermediates, simplifies the synthesis step, reduces production pollution and production costs, and is suitable for large-scale industrial production. The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes can be made to the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and scope of the present invention are intended to be included within the scope of the present invention.

Claims

权 利 要 求 书 种双羰基还原酶， 其特征在于， 具有下列之一的氨基酸序列： Claims: A dicarbonyl reductase, characterized in that it has one of the following amino acid sequences:
1 ) SEQ NO. l的氨基酸序列； 1) Amino acid sequence of SEQ NO. 1;
2)将 SEQ NO.l的氨基酸序列经过一个或几个氨基酸的取代、 和 /或缺失、 o o o OH OH O 和 /或添加且具有将 R2立体选择性地还原为 F 2) The amino acid sequence of SEQ NO. 1 is subjected to one or several amino acid substitutions, and/or deletions, ooo OH OH O and/or additions and has the ability to stereoselectively reduce R 2 to F
功能的由 SEQ N0.1衍生的氨基酸序列， 且所述 SEQ N0.1衍生的氨基酸序列 与所述 SEQ NO.l具有 80%以上的同源性， 其中， 选自芳香基、 烷基、 环烷 基、 烷基取代的芳香基、 卤素取代的芳香基、 芳烷杂环基、 环状杂烷基或环状 杂烷化烷基， R2选自烷基、 环烷基、 卤烷基或卤环烷基。 A functional amino acid sequence derived from SEQ NO.1, and the amino acid sequence derived from SEQ NO.1 has more than 80% homology with the SEQ NO.1, wherein, is selected from aryl, alkyl, cyclic Alkyl, alkyl-substituted aryl, halogen-substituted aryl, aralkheterocyclyl, cyclic heteroalkyl or cyclic heteroalkylated alkyl, R 2 is selected from alkyl, cycloalkyl, haloalkyl Or halocycloalkyl.
2. 权利要求 1所述的双羰基还原酶的编码基因。 2. The encoding gene of dicarbonyl reductase according to claim 1.
3. 根据权利要求 2所述的编码基因， 其特征在于， 具有下述之一的脱氧核苷酸序 列： 3. The coding gene according to claim 2, characterized in that it has one of the following deoxynucleotide sequences:
1 ) SEQ N0.2的脱氧核苷酸序列； 1) Deoxynucleotide sequence of SEQ N0.2;
2)与 SEQ N0.2的脱氧核苷酸序列具有 80%同源性且编码的蛋白质具有将 o o o OH OH O 2) It has 80% homology with the deoxynucleotide sequence of SEQ No.2 and the encoded protein has o o o OH OH O
、OR2立体选择性还原为 、QR2功能的脱氧核苷酸序 列' 含有权利要求 1所述的双羰基还原酶的编码基因的重组表达载体。 含有权利要求 1所述的双羰基还原酶的编码基因的转基因细胞系。 含有权利要求 1所述的双羰基还原酶的编码基因的转基因重组菌。 , OR 2 stereoselectively reduced to, QR 2 functional deoxynucleotide sequence' a recombinant expression vector containing the gene encoding the dicarbonyl reductase according to claim 1. A transgenic cell line containing a gene encoding the dicarbonyl reductase of claim 1. A transgenic recombinant bacterium containing a gene encoding the dicarbonyl reductase according to claim 1.
o o o o o o
如权利要求 1 所述的双羰基还原酶在将 R2立体选择性还原为 The dicarbonyl reductase as claimed in claim 1 is capable of stereoselectively reducing R to
OH OH OOH OH O
OR2中的应用。 Application in OR 2 .
8. 根据权利要求 7所述的应用， 其特征在于， 所述双羰基还原酶在制备 3R， 5S- 二羟基 -6-苄氧基-己酸叔丁酯中的应用。 ο ο ο 根据权利要求 8 所述的应用， 其特征在于， 所述 •OR2为
Figure imgf000021_0001
8. Application according to claim 7, characterized in that the dicarbonyl reductase is used in the preparation of 3R, 5S-dihydroxy-6-benzyloxy-hexanoic acid tert-butyl ester. ο ο ο The application according to claim 8, characterized in that, the •OR 2 is
Figure imgf000021_0001
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