WO2010025607A1 - 一种采用羰酰还原酶生产(s)-4-氯-3-羟基丁酸乙酯的方法 - Google Patents

一种采用羰酰还原酶生产(s)-4-氯-3-羟基丁酸乙酯的方法 Download PDF

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WO2010025607A1
WO2010025607A1 PCT/CN2009/000408 CN2009000408W WO2010025607A1 WO 2010025607 A1 WO2010025607 A1 WO 2010025607A1 CN 2009000408 W CN2009000408 W CN 2009000408W WO 2010025607 A1 WO2010025607 A1 WO 2010025607A1
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ethyl
ala
chloro
preparation
chloroacetoacetate
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应汉杰
严明
叶齐
许琳
曹厚
熊健
柏建新
陈勇
李振江
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南京工业大学
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    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)

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  • the invention belongs to the field of biotechnology and relates to the application of a carbonyl acyl reductase, in particular to a carbonyl Rreductase CR, which is produced by asymmetric reduction reaction of ethyl 4-chloroacetoacetate as a substrate ( Method for the preparation of ethyl 4-chloro-3-hydroxybutyrate. Background technique
  • S 4-Chloro-3-hydroxybutanoate
  • S)-CHBE is an important organic intermediate that can be used in the synthesis of many active drugs, such as statins.
  • Ethyl 4-chloroacetoacetate (COBE) is used as a latent substrate for the reduction reaction. It is easy to synthesize and inexpensive. Obtaining (S)-CHBE by using it as a substrate for asymmetric reduction is very A cost-effective route to preparation.
  • NADPH-dependent aldehyde-based reductase from Sporobolomyces salmonicolor AKU 4429 to catalyze reduction in a single aqueous phase system [2] and a water/organic solvent two-phase system [3].
  • COBE preparation of chiral CHBE. Since the enzyme used in the enzyme-catalyzed reduction reaction is isolated and purified from the microbial cells, the process is cumbersome and the enzyme is easily inactivated, and the application is less than that of whole cell catalysis.
  • the whole cell rule is divided into two types: (S)-CHBE, which is catalyzed by wild yeast and genetically engineered bacteria.
  • the use of this carbonyl reductase for the asymmetric reduction of COBE to prepare (S)-CHBE has not been reported so far.
  • the technical problem to be solved by the present invention is to provide a method for preparing (S)-CHBE by asymmetrically reducing COBE with a carbonyl acyl reductase.
  • the above preparation method comprises the use of a carbonyl reductase having the amino acid sequence of SEQ ID NO: 2, ethyl 4-chloroacetoacetate as a substrate, and reduced nicotinamide adenine dinucleotide phosphate (reduced coenzyme). II, NADPH) as a cofactor, asymmetric reduction of ethyl 4-chloroacetoacetate to prepare ethyl (S)-4-chloro-3-hydroxybutanoate.
  • Specific reactions include the use of a recombinant gene having the expressed gene sequence as shown in SEQ ID NO: 1, after being crushed, with 200 mmol/L to 2 mol/L of glucose, 1.5 to 300 g/L of ethyl 4-chloroacetoacetate, 50U-5KU glucose dehydrogenase (GDH) and 0.05 ⁇ 0.5 mmol/L oxidized nicotinamide adenine dinucleotide phosphate (oxidized coenzyme II, NADP) at pH 6.0 ⁇ 7.5, 20 ⁇ 30
  • GDH glucose dehydrogenase
  • NADP oxidized coenzyme II
  • the reaction was carried out for 16 to 32 hours under stirring at ° C and 180 to 280 rpm to obtain ethyl (S)-4-chloro-3-hydroxybutyrate.
  • the addition of NADP and GDH generates NADPH, and the reaction cycle is carried out, which reduces the amount of addition to reduce the production cost.
  • ethyl 4-chloroacetoacetate is added in portions.
  • the above preparation method is preferably carried out in a water/organic phase.
  • the above preparation method further comprises adding n-butyl acetate to promote dissolution of ethyl 4-chloroacetoacetate, and inhibiting inhibition of enzymes and cells by substrates and products. use.
  • the present inventors Based on the idea of modern bioinformatics, the present inventors combined with molecular biology techniques to clone the gene of carboxyl reductase from Pichia Stipitis CBS 6054 by means of genetic engineering, and found it in the aqueous phase after expression in Escherichia coli.
  • the COBE can be efficiently catalyzed as (S)-CHBE with an ee value of 100%.
  • the inhibition of the cells and enzymes by the substrate and the product is eliminated, and the transformation effect is remarkably improved.
  • the present invention firstly applies a carboxyl acyl reductase having an amino acid sequence of SEQ ID NO: 2 to the asymmetric reduction of COBE to prepare (S)-CHBE, and has achieved good effects, and the enzyme activity is as high as 32 U/mg.
  • the carbonyl reductase isolated from Yasharahara from Candida utilis ' ag «o/ a£? has an enzyme activity of only 13.7 U/mg [6] .
  • the amino acid sequence such as the carbonyl acyl reductase shown in SEQ ID NO: 2 has a high yield (more than 95%) for the substrate COBE, and the optical activity of the product CHBE is high (the ee% is 100%), and the yield is high, which is greatly reduced. Cost of production.
  • Figure 1 is a diagram showing the construction of a carboxyl reductase gene. The best way to implement the invention
  • Example 1 Construction of recombinant Escherichia coli Rosseta ( pET22b-PsCR )
  • Pichia Stipitis CBS 6054 purchased from Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversiry Centre
  • medium YPD yeast extract lOg, peptone 20 g, glucose 20 g, distilled water to 1 L.
  • the Pichia Stipitis CBS 6054 was inoculated into 5 mL of YPD liquid medium and cultured at 30 ° C to logarithmic growth phase, and the genome was extracted using a genomic DNA extraction kit (Beijing Tianwei Bioengineering Co., Ltd. yeast genome extraction kit).
  • the primers used in the construction of the expression vector are ligated to the restriction sites, and the primer sequences are as follows:
  • the upstream primer (CR-sense containing Nde I) is:
  • the downstream primer (CR-anti contains BamH I ) is:
  • Denaturation at 94 °C for 7 min cycle 30 times according to the following parameters: denaturation at 94 °C for 1 min, annealing at 60 °C for 50 s, extension at 72 °C for 1.5 min. Finally, extend at 72 ° C for 10 min. 2.
  • the pET-22b (pET-22b was purchased from Novagen) and the amplified target gene containing two restriction sites were digested with Nde I and BamH I, respectively. Fragment and expression vector, the double-digested expression vector pET-22b was ligated with the target gene by T4 ligase overnight, and 10 ⁇ of the ligated product pET-22b-PsCR was added to 100 ⁇ of Rosetta (DE3) competent cells. , placed on ice for 30 min, heat shock at 42 °C for 90 s. Place on ice for 2 min. Preheated 0.45 mL of medium was added. 220 rpm 37 ° C lh. 200 broth was added to LB plates containing 100 g/mL of ampicillin and chloramphenicol, and cultured overnight at 37 ° C for 12-16 h. The construction map is shown in Figure 1. 3. Determination of enzyme activity
  • the recombinant strain Rosseta ( pET-22b-PsCR ) and the Escherichia coli Rosseta ( DE3 ) were picked up into an antibiotic-containing LB liquid medium and cultured overnight at 37 ° C with shaking. Then, the cells were inoculated into the fresh culture solution according to the 2% inoculation amount, and cultured at 37 ° C until the OD 600 was about 0.6. IPTG was added to a final concentration of 0.8 mmol-L" 1 , 25 °C, 220 rpm, and the expression was induced for 10 hours, and then centrifuged.
  • the slime was resuspended in 100 mM potassium phosphate buffer (pH 7.0), sonicated (power 300W, sonicated 5s, intermittent 5s, 5 min), centrifuged (4°) C, 12000 rpm, 15 min), the enzyme activity in the supernatant was determined.
  • the enzyme reaction system included 100 mM potassium phosphate buffer (pH 6.0), 5 mMNADPH, 20 mM COBE, 30 ° C, and the decrease in absorbance was measured at 340 nm. Enzyme activity is defined as the amount of enzyme required to oxidize 1 ⁇ NADPH per minute as an enzyme unit U. Peptone was determined by the Brandford method.
  • Example 2 Fermentation of recombinant Escherichia coli Rosseta (pET22b-PsCR) The recombinant strain Rosseta (pET-22b-PsCR) was picked up to an antibiotic-containing LB medium and cultured overnight at 37 ° C with shaking. Then, the cells were inoculated into the fresh culture solution according to the 2% inoculation amount, and cultured at 37 ° C until the OD 600 was about 0.6. IPTG was added to the final concentration of O.Smmo L- 1 , 25 ° C, 220 rpm, and the expression was induced for 10 hours. Centrifuge at 8000 rpm for 10 min at 4 ° C, discard the supernatant, and set aside for use.
  • Example 3 Fermentation of recombinant Escherichia coli Rosseta (pET22b-PsCR) The recombinant strain Rosseta (pET-22b-PsCR) was picked up to an antibiotic-containing
  • Example take precipitated with potassium phosphate buffer (lOOmmoH 1, pH 6.5) was washed twice, weighed 0.5g (wet weight) of E. coli bacterial sludge, suspended in 15 mL of potassium phosphate buffer pH 6.5 in 2.
  • the cells were sonicated (power 300 W, ultrasonic 5 s, intermittent 5 s, 5 min), glucose 200 mmol/L, COBE 1.5 g/L, GDH 50 U, NADP 0.05 mmol/L, 20 °C, 180 rpm, 16 h.
  • the yield of the product (S)-CHBE was 1.45 g/L, the yield of the product was 96.7%, and the optical purity ee% was 100%.
  • the detection method of the product was as follows (the detection method of the product in the following examples was the same as in Example 3):
  • PEG-20M capillary column the internal standard is naphthalene.
  • the procedure is: detector FID, temperature 210 ° C, vaporization chamber temperature 210 ° C, column temperature 150 ° C, column head pressure 0.03 MPa, hydrogen 0.05 MPa, air 0.1
  • the yield of the product (S)-CHBE was 24.1 g/L, the yield of the product was: 96.4%, and the optical purity was ee ° / c ⁇ 100%.
  • Example 2 The precipitate embodiment taken in Example 2 using potassium phosphate buffer (lOOmmoH 1, pH 7.0) was washed twice, said E. coli take 2g (wet weight) of bacterial sludge, suspended in 50 mL of pH 7.0 potassium phosphate buffer.
  • Ultrasonic treatment of cells power 300W, ultrasonic 5s, intermittent 5s, 5 minutes total), adding glucose 600 mmol / L, COBE 35g / L (0, 1, 2, 8, 14h each 7g / L), GDH500U, NADP 0.15 mmol / L, 30 °C, 240 rpm, 28h.
  • the yield of the product (S)-CHBE was 33.5 g/L, and the yield of the product was 95.7%, and the optical purity was ee. /c ⁇ 100%.
  • Example 6 The yield of the product (S)-CHBE was 33.5 g/L, and the yield of the product was 95.7%, and the optical purity was ee. /c ⁇ 100%.
  • Example 2 embodiment taken precipitated with potassium phosphate buffer (lOOmmoH 1, pH 6.0) was washed twice, 4G weighed (wet weight) of E. coli bacterial sludge, suspended in 50 mL of pH 6.0 potassium phosphate buffer. Sonicate the cells (power 300W, ultrasonic 5s, intermittent 5s, 5 min), add 1.5 mol/L glucose, 50 mL n-butyl acetate (can promote the dissolution of COBE and inhibit the inhibition of enzymes and cells by substrates and products) ), add COBE 100g / L (0, 2, 4, 6, 10 each 20g / L), GDH3KU, NADP 0.3 mmol / L, 20 ° C, 240 rpm, 28h.
  • the yield of the product (S)-CHBE was 97.1 g/L, and the yield of the product was: 97.1%, optical purity ee. /c ⁇ 100%.
  • Example 2 The precipitate embodiment taken in Example 2 using potassium phosphate buffer (lOOmmoH 1, pH 6.5) was washed twice, said E. coli take 2g (wet weight) of bacterial sludge, suspended in 15 mL of pH 6.5 potassium phosphate buffer. Sonicate the cells (power 300W, ultrasonic 5s, intermittent 5s, 5 min), add 15 mL of n-butyl acetate (can promote the dissolution of COBE and release the inhibition of enzymes and cells by substrate and product), add 1 mol of glucose /L, COBE 50g/L (0, 2, 4, 6, 10 each 10g/L), GDH 2KU, NADP 0.2 mmol/L, 20 °C, 240 rpm, 28h.
  • the yield of the product (S)-CHBE was 47.6 g/L, and the yield of the product was: 95.2%, and the optical purity was ee ° / c ⁇ 100%.
  • Example 8 Example take precipitated with potassium phosphate buffer (lOOmmoH 1, pH 6.5) was washed twice, weighed 10g (wet weight) of E. coli bacterial sludge, suspended in 200 mL of potassium phosphate buffer pH 6.5 in 2. Sonicate the cells (power 300W, 5s for 5s, 5s for 5 minutes), add 200ml of n-butyl acetate (which can promote the dissolution of COBE and inhibit the inhibition of enzymes and cells by substrate and product), add 2mol/glucose/ L, COBE 300g/L (0, 2, 4, 6, 10 each 60g/L), GDH5KU, NADP 0.5mmol/L, 25 °C, 280 rpm, 32h. The yield of the product (S)-CHBE was 288.7 g/L, and the yield of the product was: 96.2%, and the optical purity was ee ° / c ⁇ 100%.
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Description

一种釆用羰酰还原酶生产 (S)-4-氯 -3-羟基丁酸乙酯的方法 技术领域
本发明属于生物技术领域， 涉及一种羰酰还原酶的应用， 尤其涉及一种 釆用羰酰还原酶（ Carbonyl Rreductase CR ) , 以 4-氯乙酰乙酸乙酯为底物不 对称还原反应生产 (S)-4-氯 -3-羟基丁酸乙酯的方法。 背景技术
(S)-4-氯 -3-羟基丁酸乙酯（ Ethyl 4-chloro-3-hydroxybutanoate , (S)-CHBE ) 是一种重要的有机中间体， 可用于很多活性药物的合成， 如他汀类药物—— 羟甲基戊二酰 CoA ( HMG-CoA ) 还原酶抑制剂和 4-羟基吡啶垸酮 ( 4-hydroxypyrrolidone ) 等 [1] 。 以 4-氯 乙 酰 乙 酸 乙 酯 （ Ethyl 4-chloroacetoacetate , COBE )作为还原反应的潜手性底物， 易于合成且价格 低廉，以其为底物进行不对称还原反应获取 (S)-CHBE是非常经济有效的制备 途径。
迄今为止， 关于 COBE不对称还原制备手性 CHBE已进行了很多的研究 报道。 概括起来主要有化学法和生物法。 化学催化不对称还原法所用的催化 剂包括价格昂贵的铑、 钌等金属。 釆用化学法合成手性 CHBE的缺点是产物 的光学纯度不够高， 且催化还原反应需要很高的氢气压， 耗能高， 污染大。 微生物法分为酶催化和全细胞催化法， Shimizu等用来自 Sporobolomyces salmonicolor AKU 4429的 NADPH依赖的醛基还原酶分别在单一水相体系 [2] 和水 /有机溶剂两相体系 [3]催化还原 COBE , 制备手性 CHBE。 由于应用酶催 化还原反应所用的酶要从微生物细胞中分离纯化得到， 过程操作繁瑣且酶容 易失活， 与全细胞催化相比， 应用较少。 全细胞法则分为釆用野生酵母和基 因工程菌催化 COBE为（S)-CHBE两种。 Yasohara等 [4]从 400株酵母菌中筛选 得到了一株 Οζ« · i¾w?/ ae, 在水 /乙酸正丁酯体系中， 在添加葡萄糖、 NADP和葡萄糖脱氢酶以及反应过程中需要控制 pH值的条件下， 产物 (S)-CHBE在有机相的积累浓度可达 90g/L , 产物的光学纯度对映体过量值 ( enantiomeric excess e.e )达到 96%。 由于釆用野生酵母中往往含有多种能 够催化 COBE为不同构型 CHBE的还原酶， 因此釆用野生酵母进行催化获得 的产物的光学活性往往很低， 需要筛选到高立体选择性的优良微生物菌株非 常困难，所以近来的研究着重集中于运用重组大肠杆菌不对称合成具有高立 体选择性的（S)-CHBE。 Yasohara等 [5]从木兰假丝酵母菌 ζ«ί i¾w?/ ae中 分离得到了一个辅酶 NADPH依赖型的羰基还原酶， 将该酶与葡萄糖脱氢酶 基因克隆到大肠杆菌中共表达， 在定时添加适量的辅酶 NADP和葡萄糖以及 分批添加底物的条件下， 催化 COBE不对称还原为 (S)-CHBE, 其得率和光学 纯度分别为 85%和 100% e.e. [6]。
综上所述， 现有催化 COBE为 (S)-CHBE的技术存在底物得率低、 产物 光学活性底、 成本高等问题。
【参考文献】：
[1] Karanewsky DS, Badia MC, Ciosek CP Jr, Robl JF, Sofia MJ, Simpkins
LM, DeLange B, Harrity TW, Biller SA, Gorden EM (1990) Phosphorus-containing inhibitors of HMG-CoA reductase. 1. 4-[2-arylethyl]-hydroxyphosphinyl]-3-hydroxybutanoic acids: a new class of cell-selective inhibitors of cholesterol biosynthesis. J Med Chem 33:2925—2956。
[2] Shimizu S, Kataoka M, Morishita A Katoh M, Morikawa T, Miyoshi T,
Yamada H( 1990a) Microbial asymmetric reduction of ethyl 4-chloro-3-oxobutaoate to optically active ethyl 4-4-chloro-3-hydroxybutanoate. Biotechnol lett 12:593-596。
[3] Shimizu S, Kataoka M, Katoh M, Morikawa T, Miyoshi T, Yamada H( 1990b) Stereoselective reduction of ethyl 4-chloro-3 -oxobutaoate by a microbial aldehyde reductase in an organic solvent-water diphasic system. Appl Environ Microbiol 56: 2374-2377。
[4] Yasohara Y, Kizaki N, Hasegawa J, Takahashi S, Wada M, Kataoka M, Shimizu S (1999) Synthesis of optically activeethyl 4-chloro-3- hydroxybutanoate by microbial reduction. Appl Microbiol Biotechnol 51 :847-851
[5] Wada M, Kataoka M, Kawabata H, Yasohara Y, Kizaki N, Hasegawa J, Shimizu S (1998) Purification and characterization of NADPH-dependent carbonyl reductase involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate, from Candida magnoliae. Biosci Biotechnol Biochem 62:280—285。
[6] Yasohara Y, Kizaki N, Hasegawa J, Wada M, Kataoka M, Shimizu S (2000) Molecular cloning and overexpression of the gene encoding an NADPH-dependent carbonyl reductase, involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate, from Candida magnoliae. Biosci Biotechnol Biochem 64: 1430—1436。 发明内容
本专利中涉及到的还原酶是羰酰还原酶的一种， 其包含 282个氨基酸， 其在 Genbank中的收录号为 XP— 001387287, ( http://www.ncbi.nlm. nih.gov/ entrez/viewer.fcgi?db=protein&id=126273732 ), 其氨基酸序列如 SEQ ID NO: 2所示。 编码该蛋白的基因含有 849bp碱基， 其在 Genbank 中的收录号为 XM— 001387250, ( http：〃 www.ncbi.nlm.nih.gov/entrez/viewer· fcgi?val=XM_00 1387250.1), 其基因序列如 SEQ ID NO: 1所示。 至今未发现该羰酰还原酶 用于 COBE不对称还原制备 (S)-CHBE的报道。
本发明所要解决的技术问题是提供一种釆用羰酰还原酶将 COBE 不对 称还原制备 (S)-CHBE的方法。
为解决上述技术问题， 本发明所釆用的技术方案如下：
一种 (S)-4-氯 -3-羟基丁酸乙酯 （（S)-CHBE ) 的制备方法， 该制备方法包 括釆用氨基酸序列如 SEQ ID NO: 2所示的羰酰还原酶将 4-氯乙酰乙酸乙酯 ( COBE )不对称还原为（S)-4-氯 -3-羟基丁酸乙酯。
上述制备方法包括釆用氨基酸序列如 SEQ ID NO: 2所示的羰酰还原酶， 以 4-氯乙酰乙酸乙酯为底物， 以还原型烟酰胺腺嘌呤二核苷磷酸磷酸（还原 型辅酶 II, NADPH )为辅因子， 不对称还原 4-氯乙酰乙酸乙酯制备 (S)-4-氯 -3-羟基丁酸乙酯。
具体反应包括釆用表达基因序列如 SEQ ID NO: 1所示的重组菌， 经过 破碎后与 200 mmol/L~2 mol/L的葡萄糖、 1.5~300g/L的 4-氯乙酰乙酸乙酯、 50U-5KU的葡萄糖脱氢酶（ glucose dehydrogenase, GDH )和 0.05~0.5 mmol/L 的氧化型烟酰胺腺嘌呤二核苷磷酸（氧化型辅酶 II, NADP ), 在 pH 6.0~7.5、 20~30°C、 180~280 rpm搅拌的条件下反应 16~32h, 得到（S)-4-氯 -3-羟基丁酸 乙酯。 其中， 加入 NADP与 GDH即生成 NADPH, 且使反应循环进行， 降 低了加入量以减少了生产成本。
在上述制备方法中， 优选地， 4-氯乙酰乙酸乙酯分批添加。 上述制备方 法优选是在水 /有机相中进行反应。优选地，上述制备方法还包括加入乙酸正 丁酯促进 4-氯乙酰乙酸乙酯的溶解，并解除底物和产物对酶和细胞的抑制作 用。
本发明人基于现代生物信息学思想， 结合分子生物学技术， 釆用基因工 程的手段从毕赤酵母 Pichia Stipitis CBS 6054克隆羰酰还原酶的基因， 在大 肠杆菌中表达后发现其在水相中能够高效的催化 COBE为 (S)-CHBE, e.e值 为 100%。 同时， 通过在水 /有机相中反应、 分批添加底物 COBE等方式， 解 除了底物和产物对细胞和酶的抑制作用， 显著的提高了转化效果。 通过对羰 酰还原酶的基因进行重组表达， 获得了具有新型催化功能的酶蛋白， 开发了 该条基因的新功能——催化非天然底物 COBE为高立体选择性的 (S)-CHBE。
有益效果： 本发明首次将氨基酸序列如 SEQ ID NO: 2所示的羰酰还原 酶应用于 COBE不对称还原制备 (S)-CHBE中， 取得很好的效果， 其酶活高达 32U/mg , 而 Yasohara从木兰假丝酵母菌 Οϋΐ ' ag«o/ a£? 中分离得到的羰 基还原酶， 其酶活只有 13.7U/mg[6]。 氨基酸序列如 SEQ ID NO: 2所示的羰 酰还原酶对底物 COBE的得率高（大于 95% )、产物 CHBE的光学活性高（e.e% 为 100% ), 且产量高， 大大降低了生产成本。 附图的简要说明
图 1为羰酰还原酶基因的构建图。 实施发明的最佳方式
根据下述实施例， 可以更好地理解本发明。 然而， 本领域的技术人员容 易理解， 实施例所描述的具体的物料配比、 工艺条件及其结果仅用于说明本 发明， 而不应当也不会限制权利要求书中所详细描述的本发明。 实施例 1 : 重组大肠杆菌 Rosseta ( pET22b-PsCR ) 的构建
1、 羰酰还原酶基因的获取
毕赤酵母 Pichia Stipitis CBS 6054 ( 购于 Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversiry Centre ), 培养基 YPD ( g-L"1 ): 酵 母提取物 lOg, 蛋白胨 20g, 葡萄糖 20g, 补蒸馏水至 1L。
将毕赤酵母 Pichia Stipitis CBS 6054接种于 5 mL YPD液体培养基中 30°C培养至对数生长期， 使用基因组 DNA提取试剂盒（北京天为生物工程 有限公司酵母基因组提取试剂盒）提取基因组。
构建表达载体所用的引物加设酶切位点， 引物序列如下： 上游引物 （ CR-sense含 Nde I ) 为：
5' GGAATTCCATATGACCAACAACCCGAGCAT
下游引物 （ CR-anti含 BamH I ) 为：
CGCGGATCCCTATGGCGCACAGTAGCCTCC
所有引物均由上海申能博彩公司合成。
基因的 PCR条件：
94°C变性 7 min,按如下参数循环 30次： 94°C变性 1 min, 60 °C退火 50s, 72°C延伸 1.5 min。 最后 72°C延伸 10min。 2、 基因的表达
用 Nde I及 BamH I分别酶切 pET-22b ( pET-22b购于 Novagen (默克中 国））及所扩增的含有两个酶切位点的目的基因， 分别胶回收已双酶切的目 的片段和表达载体， 将已双酶切的表达载体 pET-22b与目的基因用 T4连接 酶进行连接过夜，将 10 μΐ的连接产物 pET-22b-PsCR加入 100 μΐ的 Rosetta (DE3 ) 感受态细胞中， 冰上放置 30 min, 42°C热激 90s。 冰上放置 2 min。 加入预热的 0.45 mL培养基。 220rpm37°C lh。 将 200 菌液加入分别含有 100 g/mL的氨苄青霉素和氯霉素的 LB平板上， 37°C过夜培养 12— 16h。构 建图谱见图 1。 3、 酶活的测定
挑取重组菌 Rosseta( pET-22b-PsCR )及出发大肠杆菌 Rosseta( DE3 ) 至含抗生素的 LB液体培养基中， 37°C振荡培养过夜。 然后按 2%接种量分别 接种到新鲜培养液中， 37°C培养至 OD600约为 0.6时， 加入 IPTG至终浓度 0.8 mmol-L"1, 25 °C, 220rpm, 诱导表达 10h后， 离心（4°C, 5000 rpm, 15 min), 菌泥用 lOOmM磷酸钾缓冲液 （pH7.0) 重悬， 超声破碎细胞 （功率 300W, 超声 5s, 间歇 5s, 共 5 min), 离心 （4°C, 12000 rpm, 15 min), 测定上清 中的酶活。
酶反应体系包括 lOOmM磷酸钾缓冲液（pH6.0), 5 mMNADPH, 20 mM COBE, 30°C, 340nm处测定吸光值的下降。酶活定义为每分钟内氧化 1 μηιοΐ NADPH所需要的酶量为一个酶活单位 U。 蛋白釆用 Brandford法进行测定。
结果显示， 出发大肠杆菌 Rosseta (DE3 )的比酶活为 0.12U/mg, 而重组 菌 .co/ Rosseta (pET22b-PsCR)的比酶活为 32U/mg, 高于能够催化该底物 COBE为（S)-CHBE的羰基还原酶的最高报道（SI的比酶活为 13.7U/mg[6])。 实施例 2: 重组大肠杆菌 Rosseta ( pET22b-PsCR ) 的发酵 挑取重组菌 Rosseta ( pET-22b- PsCR) 至含抗生素的 LB培养液， 37°C振荡培养过夜。 然后按 2%接种量分别接种到新鲜培养液中， 37°C培养 至 OD600约为 0.6时， 加入 IPTG至终浓度 O.Smmo L-1, 25 °C, 220 rpm, 诱导 表达 10h后， 8000rpm, 4°C离心 lOmin, 弃上清， 沉淀备用。 实施例 3:
取实施例 2的沉淀用磷酸钾缓冲液（ lOOmmoH 1, pH 6.5 ) 洗涤两次， 称取 0.5g (湿重）的大肠杆菌菌泥， 悬浮于 15 mL的 pH 6.5磷酸钾缓冲液中。 超声处理细胞 （功率 300W, 超声 5s, 间歇 5s, 共 5min), 加入葡萄糖 200 mmol/L, COBE 1.5g/L, GDH 50U, NADP 0.05 mmol/L, 20 °C, 180 rpm, 16h。产物 (S)-CHBE的产量为 1.45g/L,产物的得率为： 96.7%, 光学纯度 e.e% 为 100%。
产物的检测方法如下（以下实施例中产物的检测方法与实施例 3相同）: 对于水相反应： 反应结束后， 加入等体积乙酸乙酯， 剧烈振荡 lOmin, 然后放置两小时， 8000 rpm离心 10 min分离有机层和水层。 小心吸取上层 乙酸乙酯过有机膜， 加入内标， 保存测样。
对于水 /有机两相反应： 反应结束后 8000 rpm离心 10 min分离有机层和 水层。 小心吸取上层乙酸乙酯过有机膜， 加入内标， 保存测样。
PEG-20M毛细管柱， 内标物为萘。 程序为： 检测器 FID, 温度 210°C, 汽化室温度 210°C, 柱温 150°C, 柱头压 0.03 MPa, 氢气 0.05 MPa, 空气 0.1
MPa, 尾吹 0.08MPa。 用 HPLC对 (S)- 4-氯 -3-羟基丁酸乙酯的旋光性进行分 析 (手性柱 Chiralcel OB, 4.6x250mm; Daicel Chemical Industries, 日本）， 检 测条件： 流动相为正己垸： 正己垸（9: 1), 波长 214nm, 流量为 0.8 mL/min,
R型和 S型 CHBE的出峰时间分别为： 10.5 min和 11.6 min。 实施例 4:
取实施例 2的沉淀用磷酸钾缓冲液（ lOOmmoH 1, pH 7.5 ) 洗涤两次， 称取 lg (湿重） 的大肠杆菌菌泥， 悬浮于 15mL的 pH7.5磷酸钾缓冲液中。 超声处理细胞 （功率 300W, 超声 5s, 间歇 5s, 共 5 min), 加入葡萄糖 500 mmol/L, COBE 25g/L( 0、 1、 2、 8、 14h各 5g/L ), GDH 200U, NADP 0.1 mmol/L, 25°C, 220 rpm, 24h。产物 (S)-CHBE的产量为 24.1g/L,产物的得率为： 96.4%, 光学纯度 e.e°/c^ 100%。 实施例 5:
取实施例 2的沉淀用磷酸钾缓冲液（ lOOmmoH 1, pH 7.0 ) 洗涤两次， 称取 2g (湿重） 的大肠杆菌菌泥， 悬浮于 50 mL的 pH 7.0磷酸钾缓冲液中。 超声处理细胞 （功率 300W, 超声 5s, 间歇 5s, 共 5 min), 加入葡萄糖 600 mmol/L, COBE35g/L (0、 1、 2、 8、 14h各 7g/L), GDH500U, NADP 0.15 mmol/L, 30 °C, 240 rpm, 28h。 产物 (S)-CHBE的产量为 33.5g/L, 产物的得 率为： 95.7%, 光学纯度 e.e。/c^ 100%。 实施例 6:
取实施例 2的沉淀用磷酸钾缓冲液（ lOOmmoH 1, pH 6.0 ) 洗涤两次， 称取 4g (湿重） 的大肠杆菌菌泥， 悬浮于 50 mL的 pH 6.0磷酸钾缓冲液中。 超声处理细胞（功率 300W,超声 5s,间歇 5s,共 5 min ),加入葡萄糖 1.5 mol/L, 50 mL乙酸正丁酯 （可促进 COBE的溶解并解除底物和产物对酶和细胞的抑 制作用）， 加入 COBE 100g/L (0、 2、 4、 6、 10各 20g/L), GDH3KU, NADP 0.3 mmol/L, 20 °C, 240 rpm, 28h。 产物 (S)-CHBE的产量为 97.1g/L, 产物的 得率为： 97.1%, 光学纯度 e.e。/c^ 100%。 实施例 7:
取实施例 2的沉淀用磷酸钾缓冲液（ lOOmmoH 1, pH 6.5 ) 洗涤两次， 称取 2g (湿重） 的大肠杆菌菌泥， 悬浮于 15 mL的 pH 6.5磷酸钾缓冲液中。 超声处理细胞（功率 300W, 超声 5s, 间歇 5s, 共 5 min), 加入 15 mL乙酸 正丁酯（可促进 COBE的溶解并解除底物和产物对酶和细胞的抑制作用 ), 加 入葡萄糖 1 mol/L, COBE 50g/L (0、 2、 4、 6、 10各 10g/L), GDH 2KU, NADP 0.2 mmol/L, 20 °C, 240 rpm, 28h。 产物 (S)-CHBE的产量为 47.6g/L, 产物的得率为： 95.2%, 光学纯度 e.e°/c^ 100%。
实施例 8: 取实施例 2的沉淀用磷酸钾缓冲液（ lOOmmoH 1, pH 6.5 ) 洗涤两次， 称取 10g (湿重）的大肠杆菌菌泥，悬浮于 200 mL的 pH 6.5磷酸钾缓冲液中。 超声处理细胞 （功率 300W, 超声 5s, 间歇 5s, 共 5 min), 加入 200 mL乙 酸正丁酯 （可促进 COBE的溶解并解除底物和产物对酶和细胞的抑制作用 ), 加入葡萄糖 2mol/L, COBE300g/L (0、 2、 4、 6、 10各 60g/L), GDH5KU, NADP0.5mmol/L, 25 °C, 280 rpm, 32h。 产物 (S)-CHBE的产量为 288.7g/L, 产物的得率为： 96.2%, 光学纯度 e.e°/c^ 100%。
<110> 南京工业大学
<120> 一种釆用羰酰还原酶生产 (S)-4-氯 -3-羟基丁酸乙酯的方法
<130> EIC09310004P
<160> 2
<170> Patentln version 3.3
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<213> 毕赤酵母（ Pichia stipitis CBS 6054 )
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35 40 45
l ie Gly Leu Ala Val Ala Glu Ala Tyr Ala Gin Ala Gly Ala Asp Val 50 55 60
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lie Phe Lys Lys Asn Gly Lys Gly Ser Met lie Phe Thr Ala Ser Met
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245 250 255 " 7 -
082 LZ θ·¾
Figure imgf000015_0001
OLZ 992 092
dsy Jos ^ID jqi jqi jqi ^iv dsv ^9s ^iv 9qd
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Claims

杈 利 要 求
1、 一种 (S)-4-氯 -3-羟基丁酸乙酯的制备方法， 该制备方法包括釆用氨基 酸序列如 SEQ ID NO: 2所示的羰酰还原酶将 4-氯乙酰乙酸乙酯不对称还原 为 (S)-4-氯 -3-羟基丁酸乙酯。
2、 根据权利要求 1 所述的制备方法， 其特征在于， 所述制备方法包括 釆用氨基酸序列如 SEQ ID NO: 2所示的羰酰还原酶， 以 4-氯乙酰乙酸乙酯 为底物，以 NADPH为辅因子，不对称还原 4-氯乙酰乙酸乙酯制备 (S)-4-氯 -3- 羟基丁酸乙酯。
3、 根据权利要求 1 所述的制备方法， 其特征在于， 所述制备方法包括 釆用表达基因序列如 SEQ ID NO: 1 所示的重组菌， 经过破碎后与 200 mmol/L~2 mol/L的葡萄糖、 1.5~300g/L的 4-氯乙酰乙酸乙酯、 50U~5KU的 葡萄糖脱氢酶和 0.05~0.5 mmol/L的 NADP,在 pH 6.0~7.5、20~30°C、 180-280 rpm搅拌的条件下反应 16~32h, 得到 (S)-4-氯 -3-羟基丁酸乙酯。
4、 根据权利要求 1所述的制备方法， 其特征在于， 所述 4-氯乙酰乙酸 乙酯分批添加。
5、 根据权利要求 1 所述的制备方法， 其特征在于， 所述制备方法是在 水 /有机相中进行反应。
6、 根据权利要求 1 所述的制备方法， 其特征在于， 所述制备方法还包 括加入乙酸正丁酯促进 4-氯乙酰乙酸乙酯的溶解，并解除底物和产物对酶和
PCT/CN2009/000408 2008-09-02 2009-04-16 一种采用羰酰还原酶生产(s)-4-氯-3-羟基丁酸乙酯的方法 WO2010025607A1 (zh)

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