WO2018126467A1 - PREPARATION METHOD FOR 3α-HYDROXY-7-OXO-5β-CHOLANOIC ACID AND ENZYME 2 FOR PREPARATION THEREOF - Google Patents

PREPARATION METHOD FOR 3α-HYDROXY-7-OXO-5β-CHOLANOIC ACID AND ENZYME 2 FOR PREPARATION THEREOF Download PDF

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WO2018126467A1
WO2018126467A1 PCT/CN2017/070603 CN2017070603W WO2018126467A1 WO 2018126467 A1 WO2018126467 A1 WO 2018126467A1 CN 2017070603 W CN2017070603 W CN 2017070603W WO 2018126467 A1 WO2018126467 A1 WO 2018126467A1
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hydroxy
steroid dehydrogenase
acid
cholanoic
oxo
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PCT/CN2017/070603
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French (fr)
Chinese (zh)
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傅荣昭
刘立辉
曹磊
刘滔滔
彭亭
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深圳市邦泰绿色生物合成研究院
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Priority to PCT/CN2017/070603 priority Critical patent/WO2018126467A1/en
Priority to CN201780001727.3A priority patent/CN107980060B/en
Publication of WO2018126467A1 publication Critical patent/WO2018126467A1/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
    • C12P33/00—Preparation of steroids
    • C12P33/02—Dehydrogenating; Dehydroxylating
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01159—7-Alpha-hydroxysteroid dehydrogenase (1.1.1.159)

Definitions

  • the present invention relates to the field of molecular biology and biotechnology, and more particularly to a method for preparing 3d-hydroxy-7 oxo-5 ⁇ -cholanoic acid by using a biological enzyme catalytic technique and a 7 ⁇ -steroid dehydrogenase for the preparation thereof.
  • 3d-hydroxy-7 oxo-5 ⁇ -cholanoic acid also known as 7-ketolithic acid, is an important intermediate for the preparation of ursodeoxycholic acid.
  • Ursodeoxycholic acid is the main active ingredient of the valuable Chinese medicine bear bile. It has the effects of increasing bile acid secretion, changing bile composition, lowering cholesterol and cholesterol in bile, and is mainly used for the treatment of gallstone disease.
  • bear bile is a very scarce resource, because the traditional way of obtaining it depends mainly on the method of artificially breeding live bears. At present, this traditional method with long cycle, low yield and inhumanity is gradually replaced by synthetic methods, and the known method of synthesizing ursodeoxycholic acid, 3d-hydroxy-7 oxo-5 ⁇ -cholane Acids are extremely important intermediates.
  • Chinese invention patent application CN105368828A discloses a method for preparing 3ot-hydroxy-7 ox-5 ⁇ -cholanoic acid by whole cell catalysis, but this method requires cell fermentation culture, and the reaction period is long and the operation is cumbersome. The product is complicated and so on.
  • An object of the present invention is to provide a novel preparation method of 30C-hydroxy-7 oxo-5 ⁇ -cholanoic acid to solve the residual organic solvent existing in the prior preparation method mentioned in the above background art,
  • the present invention provides the biological enzyme to which the new preparation method is applicable, such as harsh conditions, long reaction time, cumbersome operation, high cost, and environmental pollution.
  • the biological enzyme and optimized on the basis of this sequence, obtains a mutant enzyme with increased activity and substrate inhibition, thereby eliciting a new method for preparing 3 ⁇ -hydroxy-7 ox-5 ⁇ -cholanoic acid , characterized by: using chenodeoxycholic acid as a substrate, in the presence of NAD, lactate dehydrogenase, sodium pyruvate and a buffer solution, catalyzing the synthesis of chenodeoxycholic acid with 7 ⁇ -steroid dehydrogenase 3d- Hydroxy-7 oxo-5 ⁇ cholanoic acid, the 7oc-steroid dehydrogenase is derived from Cyanobacterium Cymwthece sp. ATCC 29155, and the nucleotide sequence of the lactate dehydrogenase is SEQ ID
  • the concentration of the substrate is 50 to 100 mg/mL
  • the concentration of the NAD is 0.01 to 0.25 mg/mL
  • the concentration of the sodium pyruvate is 10 to 30 mg. /mL.
  • the specific forms of the two enzymes used in the above methods include liquid enzymes, solid enzymes, and various immobilized enzymes, either in the form of unpurified crude enzymes or in partially or completely purified form. .
  • the catalytic process is controlled at a temperature of 25 to 35 ° C and a pH of 7.5 to 8.5.
  • the buffer solution is a 50-100 mM potassium phosphate buffer.
  • the above preparation method further comprises the following purification step: after the end of the reaction of the catalytic process, the pH is adjusted to 1.0 to 2.0, stirred for 20 to 30 minutes, and after being cooled, filtered and dried to obtain 3oc- Finished product of hydroxy-7 oxo-5 ⁇ -cholanoic acid.
  • the above preparation method further comprises the following purification step: the obtained 3 ⁇ -hydroxy-7 oxo-5 ⁇ -cholanoic acid is obtained by using 8-15 times absolute ethanol 50-60. The mixture was stirred and refluxed for 0.5-lh under a water bath condition, filtered, and the filtrate was concentrated under vacuum to a volume of 1/4 to 1/5, and then stirred for 4 hours with 4-5 times of pure water, filtered, and the filter cake was vacuum dried overnight. A 3 ⁇ -hydroxy-7ox-5 ⁇ -cholanoic acid product is obtained.
  • the 7 ⁇ -steroid dehydrogenase used in the above preparation method is a protein of the following (a) or (b):
  • the 7ot-steroid dehydrogenase has at least one mutation selected from at least one of the following positions compared to the amino acid sequence set forth in SEQ ID NO: 2: position 42, 44 Bit, 97th, 99th, 117th, 159th, 192th, and 195th.
  • the 7ot-steroid dehydrogenase has at least one of the following mutations: D42N, D44A, G97
  • the present invention also provides a 7oc-steroid dehydrogenase derived from Bluerod
  • Cyanothece sp. ATCC 29155 used to catalyze the preparation of chenodeoxycholic acid 3 ⁇ -hydroxy-7 oxo-5 ⁇ cholanoic acid
  • the 7ot-steroid dehydrogenase is a protein of (a) or (b) as follows:
  • the 7ot-steroid dehydrogenase has at least one mutation selected from at least one of the following positions compared to the amino acid sequence set forth in SEQ ID NO: 2: position 42 , 44th , 97th , 99th
  • the 7 ⁇ -steroid dehydrogenase has at least one of the following mutations: D42N, D44A, G97D, G99A, L117E, Y159F, I192K, and D195N.
  • the method provided by the invention has the advantages of simple operation, mild and easy control of reaction conditions, short reaction time, and no use.
  • the organic solvent, non-toxic, non-polluting and low-cost advantages have been proved by practice that the reaction time of the method provided by the invention only needs 4 to 12 hours, and the conversion rate of the substrate is as high as 99.8% or more. The content is above 96.8%.
  • the present invention screens a 7ot-steroid dehydrogenase gene suitable for extracellular biocatalysis to prepare 3ot-hydroxy-7 ox-5 ⁇ -cholanoic acid, and optimizes on the basis of the sequence. Mutant enzymes with increased activity and removal of substrate inhibition, these mutant enzymes exhibit high selectivity such that the method does not form by-products, and the high catalytic activity and high specificity of these mutant enzymes make 3 ⁇ -hydroxy-7 Oxy-5 ⁇ -cholanoic acid method Embodiments of the invention
  • the chenodeoxycholic acid was suspended in 50-100 mM potassium phosphate buffer (pH 8.0), the pH was adjusted to 8.0 with 10 M NaOH, and sodium pyruvate was added at a final concentration of 10 to 30 mg/mL and 10 M was used.
  • NaOH adjusts the pH to 8.0, adds 7 ⁇ -steroid dehydrogenase and lactate dehydrogenase, and finally adds NAD at a final concentration of 0.01 ⁇ 0.25mg/ml, the final concentration of the substrate is 50 ⁇ 100mg/mL, and the reaction is at temperature 25. ⁇ 35 ° C, 200 ⁇ 400 rpm and pH 7.5 ⁇ 8.5, the reaction time is 41! ⁇ 12h.
  • buffer solution take sodium dihydrogen phosphate 0.78g, dissolve 1L water, adjust the pH value with phosphoric acid to 3
  • methanol 30:37:40, with 0.45
  • the filter membrane is filtered and used.
  • the column temperature was 40
  • the specific forms of the two enzymes used in the above methods include liquid enzymes, solid enzymes, and various immobilized enzymes, either in the form of unpurified crude enzymes or in partially or completely purified form. .
  • the 7 ⁇ -steroid dehydrogenase gene AHSDH4 and the W eh/to sp lactate dehydrogenase gene LDH use the primer pair 5'CGCCATATGATGTTCAACAGCGATAC, respectively CTT3' and 5'CCGGAATTCTTAGTCCAGTTCTTGAACGC3'
  • GTA3' and 5'CCGCTCGAGTTAATATTCCACCGCAATGC3' were obtained by PCR amplification technology
  • the PCR product was digested and inserted into the fe l and EcoR I sites of the expression vector pET22b (+) and the EcoR I site and Xho I site to obtain the co-expression recombinant plasmid pET22b-AHSDH4-LDH.
  • the nucleotide sequence of the cloned parent 7ot-steroid dehydrogenase is determined by DNA sequencing as shown in SEQ ID NO: 1, and the amino acid sequence thereof is shown in SEQ ID NO: 2; determining the cloned parental lactate
  • the nucleotide sequence of the hydrogenase is shown in SEQ ID NO: 3, and the amino acid sequence thereof is shown in SEQ ID NO: 4.
  • the PCR system is: TaKaRa EX Taq HS 0.25ul; ⁇ Taq Buffer
  • the PCR procedure is: First 98. C2min; then 98. C10s, 55-56 ° C 30s, 72. C7min, 30 cycles
  • Example 3 The parental and mutant co-expression recombinant plasmids prepared in Example 1 and Example 2 were separately transferred into Escherichia coli Ro setta (de3), and the obtained recombinant Escherichia coli was inoculated into a small volume of LB medium (containing 10 (Vg/mL of Amp), after 30 ⁇ 37 °C overnight culture, transfer to a volume of LB medium (containing 10 (Vg/mL of Amp), in 30 ⁇ with an inoculation amount of 1 ⁇ 5 ⁇ 3 ⁇ 4
  • the OD 600 was further cultured at 37 ° C to reach 0.6 to 1.0, and isopropyl- ⁇ -D-thiogalactoside (IPTG) was added at a final concentration of 0.1 mM to 1 M, and the expression was induced at 20 to 37 ° C for 10 to 20 hours.
  • IPTG isopropyl- ⁇ -D-thiogalactoside
  • the cells are collected by centrifugation.
  • the fermenting cells are suspended in a volume of 50-100 mM potassium phosphate buffer (pH 8.0) and ultrasonically disrupted, and centrifuged to contain lactate dehydrogenase and 7 ⁇ -steroid dehydrogenase parent or A crude enzyme solution of a 7 ⁇ -steroid dehydrogenase mutant, which can be used for the determination of enzyme activity and biocatalytic preparation of 3d-hydroxy-7 oxo-5 ⁇ -cholanoic acid.
  • Lactate dehydrogenase enzyme activity assay method using sodium pyruvate as a substrate, in a 3mL reaction system, add 100uL of 50mM sodium pyruvate, lOOuL of diluted enzyme solution, NADH final concentration of 0.2mM, pH8.
  • the reaction at 0 and 25 ° C was constant, and the decrease in absorbance was measured at 340 nm.
  • the crude enzyme solution prepared in Example 3 is used, and the input amount of the enzyme solution accounts for the entire reaction system by the weight of the enzyme solution.
  • the volumetric meter, the final concentration of the control substrate chenodeoxycholic acid was 100 mg/mL, and the other specific parameters are shown in Table 3. Reaction 41! After ⁇ 12h, the substrate conversion rate was above 99.8%, the finished product content was above 96.8%, and the yield was 90 ⁇ 95%.
  • reaction solution was dropwise added with a hydrochloric acid solution to a pH of 1.2, and stirring was continued for 30 minutes, and then filtered, cooled, washed three times, and then dried in vacuo to give 60 g of 3oc-hydroxy-7-oxo-5?-cholane acid.
  • the product is stirred and refluxed with 1080 ml of absolute ethanol under a water bath at 60 ° C for 1 h.
  • the filtrate is filtered and concentrated under vacuum to a volume of 250 ml, and then added with 1 L of pure water for 1 h, filtered, and the filter cake is vacuum dried overnight to obtain 3 ⁇ -hydroxy- 7 oxo-5 ⁇ -cholanoic acid concentrate 54g

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Abstract

A method which uses biological enzyme catalysis to prepare 3α-hydroxy-7-oxo-5β-cholanoic acid and 7α-steroid dehydrogenase for the preparation thereof. The method uses chenodeoxycholic acid as a substrate; in the presence of NAD, lactate dehydrogenase, sodium pyruvate and a buffer solution, the 7α-steroid dehydrogenase is used to catalyze the chenodeoxycholic acid to prepare the 3α-hydroxy-7-oxo-5β-cholanoic acid, wherein the 7α-steroid dehydrogenase is derived from Cyanothece sp. ATCC 29155.

Description

一种 3(x-羟基 -7氧代 -5β-胆垸酸的制备方法及其制备用酶 2 技术领域  Method for preparing 3(x-hydroxy-7 oxo-5β-cholesteric acid and enzyme for preparation thereof 2
[0001] 本发明涉及分子生物学与生物技术领域， 特别涉及一种利用生物酶催化技术制 备 3d-羟基 -7氧代 -5β-胆烷酸的方法及其制备用 7α-类固醇脱氢酶。  [0001] The present invention relates to the field of molecular biology and biotechnology, and more particularly to a method for preparing 3d-hydroxy-7 oxo-5β-cholanoic acid by using a biological enzyme catalytic technique and a 7α-steroid dehydrogenase for the preparation thereof.
背景技术  Background technique
[0002] 3d-羟基 -7氧代 -5β-胆烷酸又名 7-酮石胆酸， 是制备熊去氧胆酸的重要中间体。  [0002] 3d-hydroxy-7 oxo-5β-cholanoic acid, also known as 7-ketolithic acid, is an important intermediate for the preparation of ursodeoxycholic acid.
而熊去氧胆酸是名贵中药熊胆的主要有效成分， 具有增加胆汁酸分泌、 并使胆 汁成分改变、 降低胆汁中胆固醇及胆固醇脂等功效， 主要用于治疗胆石疾病。 众所周知， 熊胆是一种非常稀缺的资源， 原因在于其获取的传统途径主要是依 赖于人工养殖活熊取胆的方法。 目前， 这种周期长、 收率低且不人道的传统途 径逐渐被人工合成方法所取代， 而现知的人工合成熊去氧胆酸的方法中， 3d-羟 基 -7氧代 -5β-胆烷酸都是极为重要的中间体。  Ursodeoxycholic acid is the main active ingredient of the valuable Chinese medicine bear bile. It has the effects of increasing bile acid secretion, changing bile composition, lowering cholesterol and cholesterol in bile, and is mainly used for the treatment of gallstone disease. As we all know, bear bile is a very scarce resource, because the traditional way of obtaining it depends mainly on the method of artificially breeding live bears. At present, this traditional method with long cycle, low yield and inhumanity is gradually replaced by synthetic methods, and the known method of synthesizing ursodeoxycholic acid, 3d-hydroxy-7 oxo-5β-cholane Acids are extremely important intermediates.
[0003] 目前 3ot-羟基 -7氧代 -5β-胆烷酸的工业化生产多采用化学法， 但是存在操作条件 苛刻、 选择性低、 污染环境、 使用大量有机溶剂、 存在有机溶剂残留、 有毒有 害等缺点。 为了解决化学法存在的诸多缺点， 人们另辟蹊径， 寻找更好的生产 途径。 中国发明专利 CN1912192B公幵了一种采用电化学合成制备 3d-羟基 -7氧代 -5β-胆烷酸的方法， 但是此种方法依然需要使用有机溶剂， 并且成本较高。 中国 发明专利申请 CN105368828A公幵了一种采用全细胞催化制备 3ot-羟基 -7氧代 -5β- 胆烷酸的方法， 但是此种方法需进行细胞发酵培养， 存在反应吋间长、 操作繁 琐、 产物复杂等缺点。  [0003] At present, industrial production of 3ot-hydroxy-7 ox-5β-cholanoic acid is mostly carried out by chemical methods, but it has harsh operating conditions, low selectivity, environmental pollution, use of a large amount of organic solvents, residual organic solvents, toxic and harmful And so on. In order to solve the many shortcomings of chemical law, people have taken a different approach and sought better ways of production. Chinese invention patent CN1912192B discloses a method for preparing 3d-hydroxy-7 oxo-5β-cholanoic acid by electrochemical synthesis, but this method still requires the use of an organic solvent and is costly. Chinese invention patent application CN105368828A discloses a method for preparing 3ot-hydroxy-7 ox-5β-cholanoic acid by whole cell catalysis, but this method requires cell fermentation culture, and the reaction period is long and the operation is cumbersome. The product is complicated and so on.
技术问题  technical problem
[0004] 本发明的目的在于提供一种 30C-羟基 -7氧代 -5β-胆烷酸的新的制备方法， 以解决 上述背景技术中提到的现有制备方法所存在的有机溶剂残留、 条件苛刻、 反应 吋间长、 操作繁琐、 成本较高、 污染环境等缺点， 本发明同吋提供了该新制备 方法适用的生物酶。  [0004] An object of the present invention is to provide a novel preparation method of 30C-hydroxy-7 oxo-5β-cholanoic acid to solve the residual organic solvent existing in the prior preparation method mentioned in the above background art, The present invention provides the biological enzyme to which the new preparation method is applicable, such as harsh conditions, long reaction time, cumbersome operation, high cost, and environmental pollution.
问题的解决方案 技术解决方案 Problem solution Technical solution
[0005] 为实现上述目的， 发明人经过长期大量的实验摸索， 在经历上百次的失败尝试 之后， 终于筛选出适用于细胞外生物催化制备 3d-羟基 -7氧代 -5β-胆烷酸的生物酶 ， 并在此序列基础上进行优化， 获得了活性提高和去除底物抑制的突变体酶， 从而幵发出一种新的制备 3α-羟基 -7氧代 -5β-胆烷酸的方法， 其特征在于： 以鹅去 氧胆酸为底物， 在 NAD、 乳酸脱氢酶、 丙酮酸钠以及缓冲溶液存在的条件下， 用 7α-类固醇脱氢酶催化鹅去氧胆酸制备 3d-羟基 -7氧代 -5β胆烷酸， 所述 7oc-类固 醇脱氢酶来源于蓝杆藻 Cymwthece sp. ATCC 29155， 所述乳酸脱氢酶的核苷酸 序列如 SEQ ID  [0005] In order to achieve the above object, the inventors have explored for a long time, a large number of experiments, and finally selected for extracellular biocatalysis to prepare 3d-hydroxy-7 oxo-5β-cholanoic acid after hundreds of failed attempts. The biological enzyme, and optimized on the basis of this sequence, obtains a mutant enzyme with increased activity and substrate inhibition, thereby eliciting a new method for preparing 3α-hydroxy-7 ox-5β-cholanoic acid , characterized by: using chenodeoxycholic acid as a substrate, in the presence of NAD, lactate dehydrogenase, sodium pyruvate and a buffer solution, catalyzing the synthesis of chenodeoxycholic acid with 7α-steroid dehydrogenase 3d- Hydroxy-7 oxo-5β cholanoic acid, the 7oc-steroid dehydrogenase is derived from Cyanobacterium Cymwthece sp. ATCC 29155, and the nucleotide sequence of the lactate dehydrogenase is SEQ ID
NO : 3所示， 在整个催化反应体系中， 所述底物的浓度为 50〜100mg/mL， 所述 NAD的浓度为 0.01〜0.25mg/mL， 所述丙酮酸钠的浓度为 10〜30mg/mL。  As indicated by NO: 3, in the entire catalytic reaction system, the concentration of the substrate is 50 to 100 mg/mL, the concentration of the NAD is 0.01 to 0.25 mg/mL, and the concentration of the sodium pyruvate is 10 to 30 mg. /mL.
[0006] 上述方法中所使用的两种酶的具体存在形式包括液态酶、 固态酶以及各种固定 化酶， 可以是未经纯化的粗酶形式， 也可以是经部分纯化或完全纯化的形式。  [0006] The specific forms of the two enzymes used in the above methods include liquid enzymes, solid enzymes, and various immobilized enzymes, either in the form of unpurified crude enzymes or in partially or completely purified form. .
[0007] 优选地， 控制所述催化过程在温度为 25〜35°C， pH值为 7.5〜8.5的条件下进行  [0007] Preferably, the catalytic process is controlled at a temperature of 25 to 35 ° C and a pH of 7.5 to 8.5.
[0008] 优选地， 所述缓冲溶液为 50〜100mM磷酸钾缓冲液。 Preferably, the buffer solution is a 50-100 mM potassium phosphate buffer.
[0009] 优选地， 上述制备方法还包括如下提纯步骤： 待所述催化过程反应结束后， 调 节 pH值为 1.0〜2.0， 搅拌 20〜30min， 待冷却后再经过滤水洗干燥后即得 3oc-羟基 -7氧代 -5β-胆烷酸成品。  [0009] Preferably, the above preparation method further comprises the following purification step: after the end of the reaction of the catalytic process, the pH is adjusted to 1.0 to 2.0, stirred for 20 to 30 minutes, and after being cooled, filtered and dried to obtain 3oc- Finished product of hydroxy-7 oxo-5β-cholanoic acid.
[0010] 更优选地， 上述制备方法还包括如下精制步骤： 将获得的 3α-羟基 -7氧代 -5β-胆 烷酸成品用 8- 15倍无水乙醇 50-60。C水浴条件下搅拌回流 0.5-lh， 过滤， 取滤液进 行真空减压浓缩至 1/4- 1/5体积， 再加入 4-5倍纯水搅拌 lh， 过滤， 将滤饼真空干 燥过夜， 即得 3α-羟基 -7氧代 -5β-胆烷酸精制品。  More preferably, the above preparation method further comprises the following purification step: the obtained 3α-hydroxy-7 oxo-5β-cholanoic acid is obtained by using 8-15 times absolute ethanol 50-60. The mixture was stirred and refluxed for 0.5-lh under a water bath condition, filtered, and the filtrate was concentrated under vacuum to a volume of 1/4 to 1/5, and then stirred for 4 hours with 4-5 times of pure water, filtered, and the filter cake was vacuum dried overnight. A 3α-hydroxy-7ox-5β-cholanoic acid product is obtained.
[0011] 优选地， 上述制备方法中所使用的 7α-类固醇脱氢酶为如下 （a) 或 （b) 的蛋 白质：  [0011] Preferably, the 7α-steroid dehydrogenase used in the above preparation method is a protein of the following (a) or (b):
[0012] (a) 其氨基酸序列如 SEQ ID NO: 2所示的蛋白质，  (a) a protein having the amino acid sequence of SEQ ID NO: 2,
[0013] (b) 在 （a) 限定的氨基酸序列中经过取代、 缺失或添加一个或几个氨基酸并 且在 NAD存在下以鹅去氧胆酸为底物具有比氨基酸序列如 SEQ ID NO: 2所示的 亲本高的 7α-类固醇脱氢酶催化活性的由 （a) 衍生的蛋白质。 (b) substituting, deleting or adding one or several amino acids in the (a) defined amino acid sequence and using chenodeoxycholic acid as a substrate in the presence of NAD has an amino acid sequence such as SEQ ID NO: 2. Shown The parental high 7α-steroid dehydrogenase catalytically active protein derived from (a).
[0014] 更优选地， 所述 7ot-类固醇脱氢酶与如 SEQ ID NO: 2所示的氨基酸序列相比在 选自至少一个下述位点处具有至少一个突变： 第 42位、 第 44位、 第 97位、 第 99 位、 第 117位、 第 159位、 第 192位以及第 195位。 More preferably, the 7ot-steroid dehydrogenase has at least one mutation selected from at least one of the following positions compared to the amino acid sequence set forth in SEQ ID NO: 2: position 42, 44 Bit, 97th, 99th, 117th, 159th, 192th, and 195th.
[0015] 更优选地， 所述 7ot-类固醇脱氢酶具有至少一个下述突变： D42N、 D44A、 G97More preferably, the 7ot-steroid dehydrogenase has at least one of the following mutations: D42N, D44A, G97
D、 G99A、 L117E、 Y159F、 I192K以及 D195N。 D, G99A, L117E, Y159F, I192K and D195N.
[0016] 本发明还提供了一种 7oc-类固醇脱氢酶， 所述 7α-类固醇脱氢酶来源于蓝杆藻[0016] The present invention also provides a 7oc-steroid dehydrogenase derived from Bluerod
Cyanothece sp. ATCC 29155， 用于催化鹅去氧胆酸制备 3α-羟基 -7氧代 -5β胆烷酸Cyanothece sp. ATCC 29155, used to catalyze the preparation of chenodeoxycholic acid 3α-hydroxy-7 oxo-5β cholanoic acid
， 所述 7ot-类固醇脱氢酶为如下 （a) 或 （b) 的蛋白质： The 7ot-steroid dehydrogenase is a protein of (a) or (b) as follows:
[0017] (a) 其氨基酸序列如 SEQ ID NO: 2所示的蛋白质， (a) a protein having the amino acid sequence of SEQ ID NO: 2,
[0018] (b) 在 （a) 限定的氨基酸序列中经过取代、 缺失或添加一个或几个氨基酸并 且在 NAD存在下以鹅去氧胆酸为底物具有比氨基酸序列如 SEQ ID NO: 2所示的 亲本高的 7α-类固醇脱氢酶催化活性的由 （a) 衍生的蛋白质。  (b) substituting, deleting or adding one or several amino acids in the (a) defined amino acid sequence and using chenodeoxycholic acid as a substrate in the presence of NAD has an amino acid sequence such as SEQ ID NO: 2. The parental high 7α-steroid dehydrogenase shown catalytically active protein derived from (a).
[0019] 优选地， 所述 7ot-类固醇脱氢酶与如 SEQ ID NO: 2所示的氨基酸序列相比在选 自至少一个下述位点处具有至少一个突变： 第 42位、 第 44位、 第 97位、 第 99位 Preferably, the 7ot-steroid dehydrogenase has at least one mutation selected from at least one of the following positions compared to the amino acid sequence set forth in SEQ ID NO: 2: position 42 , 44th , 97th , 99th
、 第 117位、 第 159位、 第 192位以及第 195位。 , No. 117, No. 159, No. 192 and No. 195.
[0020] 优选地， 所述 7α-类固醇脱氢酶具有至少一个下述突变： D42N、 D44A、 G97D 、 G99A、 L117E、 Y159F、 I192K以及 D195N。 Preferably, the 7α-steroid dehydrogenase has at least one of the following mutations: D42N, D44A, G97D, G99A, L117E, Y159F, I192K, and D195N.
发明的有益效果  Advantageous effects of the invention
有益效果  Beneficial effect
[0021] 1、 与现有的 3α-羟基 -7氧代 -5β-胆烷酸的制备方法相比， 本发明提供的方法具 有操作简单、 反应条件温和易控、 反应吋间短、 不使用有机溶剂、 无毒无污染 且成本低廉的优点， 经实践证明， 本发明提供的方法的反应吋长仅需 4〜12小吋 ， 其对底物的转化率高达 99.8%以上， 所获得的产品的含量在 96.8%以上。  [0021] 1. Compared with the existing preparation method of 3α-hydroxy-7 ox-5β-cholanoic acid, the method provided by the invention has the advantages of simple operation, mild and easy control of reaction conditions, short reaction time, and no use. The organic solvent, non-toxic, non-polluting and low-cost advantages have been proved by practice that the reaction time of the method provided by the invention only needs 4 to 12 hours, and the conversion rate of the substrate is as high as 99.8% or more. The content is above 96.8%.
[0022] 2、 本发明筛选出了适用于细胞外生物催化制备 3ot-羟基 -7氧代 -5β-胆烷酸的 7ot- 类固醇脱氢酶基因， 并在此序列基础上进行优化， 获得了活性提高和去除底物 抑制的突变体酶， 这些突变体酶表现出高的选择性使得该方法不会形成副产物 ， 这些突变体酶的高催化活性和高专一性使得 3α-羟基 -7氧代 -5β-胆烷酸酶法大规 本发明的实施方式 [0022] 2. The present invention screens a 7ot-steroid dehydrogenase gene suitable for extracellular biocatalysis to prepare 3ot-hydroxy-7 ox-5β-cholanoic acid, and optimizes on the basis of the sequence. Mutant enzymes with increased activity and removal of substrate inhibition, these mutant enzymes exhibit high selectivity such that the method does not form by-products, and the high catalytic activity and high specificity of these mutant enzymes make 3α-hydroxy-7 Oxy-5β-cholanoic acid method Embodiments of the invention
[0023] 下面结合具体实施例对本发明做进一步的详细说明， 以下实施例是对本发明的 解释， 本发明并不局限于以下实施例， 实施例中未注明具体条件者， 按常规条 件或者制造商建议的条件进行。  [0023] The present invention will be further described in detail below with reference to specific embodiments. The following examples are illustrative of the present invention. The present invention is not limited to the following examples, and the specific conditions are not specified in the examples, according to conventional conditions or manufacturing. The conditions suggested by the business are carried out.
[0024] 本发明提供的 3α-羟基 -7氧代 -5β-胆烷酸的制备方法的具体实施过程如下： [0024] The specific implementation process of the method for preparing 3α-hydroxy-7 oxo-5β-cholanoic acid provided by the present invention is as follows:
[0025] 将鹅去氧胆酸悬浮于 50〜100mM磷酸钾缓冲液 （pH8.0) 中， 用 10M的 NaOH调 节 pH到 8.0， 再加入终浓度为 10〜30mg/mL的丙酮酸钠并用 10M的 NaOH调节 pH 到 8.0， 加入 7α-类固醇脱氢酶和乳酸脱氢酶， 最后加入终浓度为 0.01〜0.25mg/m L的 NAD， 底物终浓度为 50〜100mg/mL， 反应在温度 25〜35°C、 200〜400rpm和 pH7.5〜8.5进行， 反应吋间为 41！〜 12h。 每隔一定吋间取反应液用流动相稀释 50 〜100倍， 微孔过滤后进样进行液相分析。 液相检测使用 Phenomenx Gemini
Figure imgf000005_0001
110Α 250x4.6mm为分析柱， 流动相为乙腈：缓冲溶液 （取磷酸二氢 钠 0.78g,溶解 1L水中， 用磷酸调 pH值为 3， 即可） ：甲醇 =30:37:40， 用 0.45醒滤 膜过滤后备用。 柱温为 40°C， 示差检测器 （RID) ， 流速为 0.8mIJmin。 待催化 过程反应结束后， 在快速搅拌的情况下加入盐酸至 pH为 1.0〜2.0， 继续搅拌 20〜 30min , 待冷却后过滤、 经水洗三次后真空干燥后即得 3d-羟基 -7氧代 -5β-胆烷酸 成品。 成品再用 8- 15倍无水乙醇 50-60°C水浴条件下搅拌回流 0.5-lh， 过滤， 取滤 液进行真空减压浓缩至 1/4- 1/5体积， 再加入 4-5倍纯水搅拌 lh， 过滤， 将滤饼真 空干燥过夜， 即得 3α-羟基 -7氧代 -5β-胆烷酸精制品。 [0025] The chenodeoxycholic acid was suspended in 50-100 mM potassium phosphate buffer (pH 8.0), the pH was adjusted to 8.0 with 10 M NaOH, and sodium pyruvate was added at a final concentration of 10 to 30 mg/mL and 10 M was used. NaOH adjusts the pH to 8.0, adds 7α-steroid dehydrogenase and lactate dehydrogenase, and finally adds NAD at a final concentration of 0.01~0.25mg/ml, the final concentration of the substrate is 50~100mg/mL, and the reaction is at temperature 25. ~35 ° C, 200 ~ 400 rpm and pH 7.5 ~ 8.5, the reaction time is 41! ~ 12h. The reaction solution was diluted 50 to 100 times with the mobile phase at regular intervals, and the liquid phase was analyzed by microfiltration. Liquid phase testing using Phenomenx Gemini
Figure imgf000005_0001
110Α 250x4.6mm is the analytical column, the mobile phase is acetonitrile: buffer solution (take sodium dihydrogen phosphate 0.78g, dissolve 1L water, adjust the pH value with phosphoric acid to 3): methanol = 30:37:40, with 0.45 The filter membrane is filtered and used. The column temperature was 40 ° C, the differential detector (RID), and the flow rate was 0.8 mIJmin. After the reaction in the catalytic process is finished, hydrochloric acid is added to the pH of 1.0~2.0 with rapid stirring, and stirring is continued for 20 to 30 minutes. After cooling, filtering, washing three times, and vacuum drying, 3d-hydroxy-7 oxo is obtained. Finished product of 5β-cholanoic acid. The finished product is further refluxed with 8- 15 times absolute ethanol in a water bath at 50-60 ° C for 0.5-lh, filtered, and the filtrate is concentrated under vacuum to a volume of 1/4 - 1/5, and then added 4-5 times pure. The mixture was stirred for 1 h with water, filtered, and the filter cake was dried under vacuum overnight to obtain a 3α-hydroxy-7 oxo-5β-cholanoic acid product.
[0026] 上述方法中所使用的两种酶的具体存在形式包括液态酶、 固态酶以及各种固定 化酶， 可以是未经纯化的粗酶形式， 也可以是经部分纯化或完全纯化的形式。  The specific forms of the two enzymes used in the above methods include liquid enzymes, solid enzymes, and various immobilized enzymes, either in the form of unpurified crude enzymes or in partially or completely purified form. .
[0027] 实施例 1  Embodiment 1
[0028] 含有亲本基因的共表达重组质粒 pET22b-AHSDH4-LDH的制备  Preparation of co-expression recombinant plasmid pET22b-AHSDH4-LDH containing parental gene
[0029] 将来源于蓝杆藻 ( Cyanothece sp. ATCC  [0029] Will be derived from Cyanothece sp. ATCC
29155) 的 7α-类固醇脱氢酶基因 AHSDH4和来源于魏斯氏菌 （ W ^e/to sp) 的 乳酸脱氢酶基因 LDH分别利用引物对 5'CGCCATATGATGTTCAACAGCGATAC CTT3'和 5'CCGGAATTCTTAGTCCAGTTCTTGAACGC3' 29155) The 7α-steroid dehydrogenase gene AHSDH4 and the W eh/to sp lactate dehydrogenase gene LDH use the primer pair 5'CGCCATATGATGTTCAACAGCGATAC, respectively CTT3' and 5'CCGGAATTCTTAGTCCAGTTCTTGAACGC3'
GTA3'和 5'CCGCTCGAGTTAATATTCCACCGCAATGC3'通过 PCR扩增技术获得GTA3' and 5'CCGCTCGAGTTAATATTCCACCGCAATGC3' were obtained by PCR amplification technology
PCR产物后经过酶切处理， 同吋***到表达载体 pET22b (+) 的 fe l和 EcoR I 位点以及 EcoR I位点和 Xho I位点， 得到共表达重组质粒 pET22b-AHSDH4-LDH 。 经 DNA测序， 确定该被克隆的亲本 7ot-类固醇脱氢酶的核苷酸序列如 SEQ ID NO : 1所示， 其氨基酸序列如 SEQ ID NO: 2所示； 确定该被克隆的亲本乳酸脱 氢酶的核苷酸序列如 SEQ ID NO: 3所示， 其氨基酸序列如 SEQ ID NO : 4所示。 The PCR product was digested and inserted into the fe l and EcoR I sites of the expression vector pET22b (+) and the EcoR I site and Xho I site to obtain the co-expression recombinant plasmid pET22b-AHSDH4-LDH. The nucleotide sequence of the cloned parent 7ot-steroid dehydrogenase is determined by DNA sequencing as shown in SEQ ID NO: 1, and the amino acid sequence thereof is shown in SEQ ID NO: 2; determining the cloned parental lactate The nucleotide sequence of the hydrogenase is shown in SEQ ID NO: 3, and the amino acid sequence thereof is shown in SEQ ID NO: 4.
[0030] 实施例 2 [0030] Example 2
[0031] 含有 7α-类固醇脱氢酶突变体的共表达重组质粒的制备  Preparation of co-expression recombinant plasmid containing 7α-steroid dehydrogenase mutant
[0032] 通过反向 PCR技术对 7ot-类固醇脱氢酶亲本进行定点突变， 在突变位置通过设 计反向引物， 利用上下游突变引物扩增目的片段， 并在引物上引入相应突变， 以重组质粒 pET22b-AHSDH4-LDH作为模板进行反向 PCR， PCR产物经 Dpn I酶 消化模板处理后转化到大肠杆菌 ROSetta(de3)， 经过 Amp的筛选后挑取菌落送测 序。 突变位点及引物设计如表 1所示。 [0032] Site-directed mutagenesis of 7ot-steroid dehydrogenase parent by reverse PCR, design of reverse primer at the mutation position, amplification of the target fragment by upstream and downstream mutant primers, and introduction of corresponding mutations on the primer to recombinant plasmid pET22b-AHSDH4-LDH was used as a template for reverse PCR. The PCR product was transformed into E. coli R OSe tt a (de3) by Dpn I digestion template, and the colonies were picked for sequencing after Amp screening. The mutation sites and primer design are shown in Table 1.
[0033] PCR体系为： TaKaRa EX Taq HS 0.25ul; ΙΟχΕχ Taq Buffer  [0033] The PCR system is: TaKaRa EX Taq HS 0.25ul; ΙΟχΕχ Taq Buffer
5ul; 模板质粒 lul; dNTP (2.5mM each) 4ul; 上游引物 lul;下游引物 lul; 无菌 水 up to 50 ul。  5 ul; template plasmid lul; dNTP (2.5 mM each) 4 ul; upstream primer lul; downstream primer lul; sterile water up to 50 ul.
[0034] PCR程序为： 首先 98。C2min; 然后 98。C10s， 55-56°C30s , 72。C7min， 30个循环 [0034] The PCR procedure is: First 98. C2min; then 98. C10s, 55-56 ° C 30s, 72. C7min, 30 cycles
； 最后 72°C10min。 ; Last 72 ° C 10 min.
[0035] 表 1 Table 1
[表 1] [Table 1]
Figure imgf000007_0001
Figure imgf000007_0001
[0036] 实施例 3  Embodiment 3
[0037] 酶液的制备 [0037] Preparation of enzyme solution
[0038] 将实施例 1和实施例 2制备的亲本和突变体共表达重组质粒分别转入大肠杆菌 Ro setta(de3), 再将获得的重组大肠杆菌接种在小体积的 LB培养基 （含有 10(Vg/mL 的 Amp) ， 30〜37°C过夜培养后， 以 1〜5<¾的接种量转接到一定体积的 LB培养 基中 （含有 10(Vg/mL的 Amp) ， 在 30〜37°C继续培养 OD 600达到 0.6〜1.0加入终 浓度为 0.1mM〜lmM的异丙基 -β-D-硫代半乳糖苷 (IPTG)， 在 20〜37°C诱导表达 1 0〜20h后离心收集菌体。 发酵菌体悬浮于一定体积的 50〜100mM的磷酸钾缓冲 液 （pH8.0) 中并超声波破胞， 离心即得含有乳酸脱氢酶与 7α-类固醇脱氢酶亲本 或者与 7α-类固醇脱氢酶突变体的粗酶液， 可用于酶活力的测定以及 3d-羟基 -7氧 代 -5β-胆烷酸的生物催化制备。 [0038] The parental and mutant co-expression recombinant plasmids prepared in Example 1 and Example 2 were separately transferred into Escherichia coli Ro setta (de3), and the obtained recombinant Escherichia coli was inoculated into a small volume of LB medium (containing 10 (Vg/mL of Amp), after 30~37 °C overnight culture, transfer to a volume of LB medium (containing 10 (Vg/mL of Amp), in 30~ with an inoculation amount of 1~5<3⁄4 The OD 600 was further cultured at 37 ° C to reach 0.6 to 1.0, and isopropyl-β-D-thiogalactoside (IPTG) was added at a final concentration of 0.1 mM to 1 M, and the expression was induced at 20 to 37 ° C for 10 to 20 hours. The cells are collected by centrifugation. The fermenting cells are suspended in a volume of 50-100 mM potassium phosphate buffer (pH 8.0) and ultrasonically disrupted, and centrifuged to contain lactate dehydrogenase and 7α-steroid dehydrogenase parent or A crude enzyme solution of a 7α -steroid dehydrogenase mutant, which can be used for the determination of enzyme activity and biocatalytic preparation of 3d-hydroxy-7 oxo-5β-cholanoic acid.
[0039] 实施例 4  Example 4
[0040] 酶活力的测定  [0040] Determination of enzyme activity
[0041] 7ot-类固醇脱氢酶的酶活测定方法： 以鹅去氧胆酸为底物， 在一个 3mL的反应 体系中加入 lOuL的 150mM鹅去氧胆酸， lOOuL的稀释酶液， NAD+终浓度为 0.2m[0041] 7ot-steroid dehydrogenase enzyme activity assay method: using chenodeoxycholic acid as a substrate, in a 3mL reaction Add lOuL of 150mM chenodeoxycholic acid, lOOuL diluted enzyme solution, NAD+ final concentration of 0.2m
M， 在 pH8.0和 25°C反应一定吋间， 在 340nm处测定吸光值增加。 M, reacted at pH 8.0 and 25 ° C for a certain period of time, and measured the increase in absorbance at 340 nm.
[0042] 乳酸脱氢酶的酶活测定方法： 以丙酮酸钠为底物， 在一个 3mL的反应体系中加 入 lOOuL的 50mM丙酮酸钠， lOOuL的稀释酶液， NADH终浓度为 0.2mM， 在 pH8.[0042] Lactate dehydrogenase enzyme activity assay method: using sodium pyruvate as a substrate, in a 3mL reaction system, add 100uL of 50mM sodium pyruvate, lOOuL of diluted enzyme solution, NADH final concentration of 0.2mM, pH8.
0和 25°C反应一定吋间， 在 340nm处测定吸光值减少。 The reaction at 0 and 25 ° C was constant, and the decrease in absorbance was measured at 340 nm.
[0043] 酶活力的测定结果如表 2所示， 其中 LDH为乳酸脱氢酶， 7ot-HSDH为 7ot-类固醇 脱氢酶。 The results of the measurement of the enzyme activity are shown in Table 2, wherein LDH is lactate dehydrogenase and 7ot-HSDH is 7ot-steroid dehydrogenase.
[0044] 表 2 Table 2
[] [表 2] [] [Table 2]
Figure imgf000008_0001
Figure imgf000008_0001
[0045] 实施例 5  [0045] Example 5
[0046] 3ot-羟基 -7氧代 -5β-胆烷酸的制备  Preparation of 3ot-hydroxy-7-oxo-5β-cholanoic acid
[0047] 参照前述 3α-羟基 -7氧代 -5β-胆烷酸的制备方法的具体实施过程， 使用实施例 3 制备的粗酶液， 酶液的投入量以酶液的重量占整个反应体系的体积计， 控制底 物鹅去氧胆酸的终浓度为 100mg/mL， 其余各具体参数如表 3所示。 反应 41！〜 12h 后测得， 底物转化率在 99.8%以上， 成品含量在 96.8%以上， 收率为 90〜95%。  Referring to the specific implementation process of the preparation method of the above 3α-hydroxy-7 oxo-5β-cholanoic acid, the crude enzyme solution prepared in Example 3 is used, and the input amount of the enzyme solution accounts for the entire reaction system by the weight of the enzyme solution. The volumetric meter, the final concentration of the control substrate chenodeoxycholic acid was 100 mg/mL, and the other specific parameters are shown in Table 3. Reaction 41! After ~12h, the substrate conversion rate was above 99.8%, the finished product content was above 96.8%, and the yield was 90~95%.
[0048] 表 3  Table 3
[] [表 3] [] [table 3]
Figure imgf000009_0001
Figure imgf000009_0001
[0049] 实施例 6  Example 6
[0050] 3ot-羟基 -7氧代 -5β-胆烷酸的制备  Preparation of 3ot-hydroxy-7-oxo-5β-cholanoic acid
[0051] 总体系 1L， 取 50g含量为 99%的鹅去氧胆酸， 悬浮于 lOOmM的磷酸钾缓冲液 （p H8.0) ，用 lO M NaOH的调节 pH到 8.0后加入终浓度 20g/L的丙酮酸钠， 并依次加 入 0.09g7ot-类固醇脱氢酶冻干粉 （Y159F突变体酶） 和 0.07g乳酸脱氢酶冻干粉， 最后加入终浓度为 O.lg/L的 NAD， 底物终浓度为 50g/L。 在 25°C 250rpm和 pH8.0 左右进行反应 4h， 转化率达 99.8%。 反应结束后， 反应液滴加盐酸溶液至 pH为 1. 2, 继续搅拌 30min后待冷却过滤、 经水洗三次后真空干燥得到 3oc-羟基 -7氧代 -5β -胆烷酸成品 60g。 成品用 1080ml无水乙醇 60°C水浴条件下搅拌回流 lh， 过滤取滤 液进行真空减压浓缩至 250ml体积， 再加入 1L纯水搅拌 lh， 过滤， 将滤饼真空干 燥过夜即得 3α-羟基 -7氧代 -5β-胆烷酸精制品 54g  [0051] 1L of the total system, 50g of 99% of chenodeoxycholic acid, suspended in 100 mM potassium phosphate buffer (p H8.0), adjusted to pH 8.0 with 10 M NaOH and added to the final concentration of 20g / L sodium pyruvate, and sequentially added 0.09g 7ot-steroid dehydrogenase lyophilized powder (Y159F mutant enzyme) and 0.07g lactate dehydrogenase lyophilized powder, and finally added NAD at a final concentration of O.lg/L, bottom The final concentration was 50 g/L. The reaction was carried out at 25 ° C, 250 rpm and pH 8.0 for 4 h, and the conversion rate was 99.8%. After the completion of the reaction, the reaction solution was dropwise added with a hydrochloric acid solution to a pH of 1.2, and stirring was continued for 30 minutes, and then filtered, cooled, washed three times, and then dried in vacuo to give 60 g of 3oc-hydroxy-7-oxo-5?-cholane acid. The product is stirred and refluxed with 1080 ml of absolute ethanol under a water bath at 60 ° C for 1 h. The filtrate is filtered and concentrated under vacuum to a volume of 250 ml, and then added with 1 L of pure water for 1 h, filtered, and the filter cake is vacuum dried overnight to obtain 3α-hydroxy- 7 oxo-5β-cholanoic acid concentrate 54g

Claims

权利要求书 Claim
[权利要求 1] 一种 3d-羟基 -7氧代 -5β-胆烷酸的制备方法， 其特征在于： 以鹅去氧胆 酸为底物， 在 NAD、 乳酸脱氢酶、 丙酮酸钠以及缓冲溶液存在的条 件下， 用 7α-类固醇脱氢酶催化鹅去氧胆酸制备 3α-羟基 -7氧代 -5β胆烷 酸， 所述 7α-类固醇脱氢酶来源于蓝杆藻 Cyanothece sp.ATCC 29155 ， 所述乳酸脱氢酶的核苷酸序列如 SEQ ID NO: 3所示， 在整个催化 反应体系中， 所述底物的浓度为 50〜100mg/mL， 所述 NAD的浓度为 0.01〜0.25mg/mL， 所述丙酮酸钠的浓度为 10〜30mg/mL。  [Claim 1] A method for producing 3d-hydroxy-7ox-5β-cholanoic acid, characterized by: using chenodeoxycholic acid as a substrate, in NAD, lactate dehydrogenase, sodium pyruvate and In the presence of a buffer solution, 3α-hydroxy-7ox-5-cholanoic acid is prepared by catalyzing chenodeoxycholic acid with 7α-steroid dehydrogenase, which is derived from Cyanothece sp. ATCC 29155, the nucleotide sequence of the lactate dehydrogenase is as shown in SEQ ID NO: 3, the concentration of the substrate is 50 to 100 mg/mL, and the concentration of the NAD is 0.01 in the entire catalytic reaction system. ~0.25 mg/mL, the concentration of the sodium pyruvate is 10 to 30 mg/mL.
[权利要求 2] 根据权利要求 1所述的 3d-羟基 -7氧代 -5β-胆烷酸的制备方法， 其特征 在于： 控制所述催化过程在温度为 25〜35°C， pH值为 7.5〜8.5的条件 下进行。  [Claim 2] The method for preparing 3d-hydroxy-7 ox-5β-cholanoic acid according to claim 1, wherein: the catalytic process is controlled at a temperature of 25 to 35 ° C, and the pH is Performed under conditions of 7.5 to 8.5.
[权利要求 3] 根据权利要求 1所述的 3d-羟基 -7氧代 -5β-胆烷酸的制备方法， 其特征 在于： 所述缓冲溶液为 50〜100mM磷酸钾缓冲液。  [Claim 3] The method for producing 3d-hydroxy-7-oxo-5β-cholanoic acid according to claim 1, wherein the buffer solution is a 50-100 mM potassium phosphate buffer.
[权利要求 4] 根据权利要求 1所述的 3d-羟基 -7氧代 -5β-胆烷酸的制备方法， 其特征 在于， 所述制备方法还包括如下提纯步骤： 待所述催化过程反应结束 后， 调节 pH值为 1.0〜2.0， 搅拌 20〜30min， 待冷却后再经过滤水洗 干燥后即得 3α-羟基 -7氧代 -5β-胆烷酸成品。  [Claim 4] The method for producing 3d-hydroxy-7ox-5β-cholanoic acid according to claim 1, wherein the preparation method further comprises the following purification step: After that, the pH value is adjusted to 1.0 to 2.0, stirred for 20 to 30 minutes, and after cooling, it is filtered and washed with water to obtain a finished product of 3α-hydroxy-7 oxo-5β-cholanoic acid.
[权利要求 5] 根据权利要求 1至 4任一项所述的 3d-羟基 -7氧代 -5β-胆烷酸的制备方法 [Claim 5] The method for preparing 3d-hydroxy-7-oxo-5β-cholanoic acid according to any one of claims 1 to 4
， 其特征在于所述 7α-类固醇脱氢酶为如下 （a) 或 （b) 的蛋白质：, characterized in that the 7α-steroid dehydrogenase is a protein of the following (a) or (b):
(a) 其氨基酸序列如 SEQ ID NO: 2所示的蛋白质， (a) a protein having the amino acid sequence of SEQ ID NO: 2,
(b) 在 （a) 限定的氨基酸序列中经过取代、 缺失或添加一个或几个 氨基酸并且在 NAD存在下以鹅去氧胆酸为底物具有比氨基酸序列如 S EQ ID NO: 2所示的亲本高的 7α-类固醇脱氢酶催化活性的由 （a) 衍 生的蛋白质。  (b) Substituting, deleting or adding one or several amino acids in the (a) defined amino acid sequence and using chenodeoxycholic acid as a substrate in the presence of NAD has an amino acid sequence as shown by S EQ ID NO: 2 The parental high 7α-steroid dehydrogenase catalytically active protein derived from (a).
[权利要求 6] 根据权利要求 5所述的 3d-羟基 -7氧代 -5β-胆烷酸的制备方法， 其特征 在于所述 7ot-类固醇脱氢酶与如 SEQ ID NO: 2所示的氨基酸序列相比 在选自至少一个下述位点处具有至少一个突变： 第 42位、 第 44位、 第 97位、 第 99位、 第 117位、 第 159位、 第 192位以及第 195位。 [Claim 6] The method for producing 3d-hydroxy-7ox-5-cholic acid according to claim 5, characterized in that the 7ot-steroid dehydrogenase is as shown in SEQ ID NO: The amino acid sequence has at least one mutation selected from at least one of the following positions: 42nd, 44th, 97th, 99th, 117th, 159th, 192th, and 195th .
[权利要求 7] 根据权利要求 6所述的 3α-羟基 -7氧代 -5β-胆烷酸的制备方法， 其特征 在于所述 7ot-类固醇脱氢酶具有至少一个下述突变： D42N、 D44A、 G 97D、 G99A、 L117E、 Y159F、 I192K以及 D195N。 [Claim 7] The method for producing 3?-hydroxy-7oxo-5?-cholanoic acid according to claim 6, wherein the 7ot-steroid dehydrogenase has at least one of the following mutations: D42N, D44A , G 97D, G99A, L117E, Y159F, I192K and D195N.
[权利要求 8] —种 7α-类固醇脱氢酶， 其特征在于： 所述 7α-类固醇脱氢酶来源于蓝 杆藻 Cyanothece sp. ATCC  [Claim 8] A 7α-steroid dehydrogenase, characterized in that the 7α-steroid dehydrogenase is derived from Cyanothece sp. ATCC
29155， 用于催化鹅去氧胆酸制备 3α-羟基 -7氧代 -5β胆烷酸， 所述 7oc- 类固醇脱氢酶为如下 ) 或 （b) 的蛋白质：  29155, for catalyzing the preparation of chenodeoxycholic acid 3α-hydroxy-7 oxo-5β cholanoic acid, the 7oc-steroid dehydrogenase is as follows: or (b) protein:
(a) 其氨基酸序列如 SEQ ID NO: 2所示的蛋白质，  (a) a protein having the amino acid sequence of SEQ ID NO: 2,
(b) 在 （a) 限定的氨基酸序列中经过取代、 缺失或添加一个或几个 氨基酸并且在 NAD存在下以鹅去氧胆酸为底物具有比氨基酸序列如 S EQ ID NO: 2所示的亲本高的 7α-类固醇脱氢酶催化活性的由 （a) 衍 生的蛋白质。  (b) Substituting, deleting or adding one or several amino acids in the (a) defined amino acid sequence and using chenodeoxycholic acid as a substrate in the presence of NAD has an amino acid sequence as shown by S EQ ID NO: 2 The parental high 7α-steroid dehydrogenase catalytically active protein derived from (a).
[权利要求 9] 根据权利要求 8所述的 7α-类固醇脱氢酶， 其特征在于所述 7α-类固醇 脱氢酶与如 SEQ ID NO: 2所示的氨基酸序列相比在选自至少一个下 述位点处具有至少一个突变： 第 42位、 第 44位、 第 97位、 第 99位、 第 117位、 第 159位、 第 192位以及第 195位。  [Claim 9] The 7α-steroid dehydrogenase according to claim 8, wherein the 7α-steroid dehydrogenase is selected from at least one of amino acid sequences as shown in SEQ ID NO: 2. There are at least one mutation at the position: 42nd, 44th, 97th, 99th, 117th, 159th, 192th, and 195th.
[权利要求 10] 根据权利要求 9所述的 7α-类固醇脱氢酶， 其特征在于所述 7α-类固醇 脱氢酶具有至少一个下述突变： D42N、 D44A、 G97D、 G99A、 L117 E、 Y159F、 I192K以及 D195N。  [Claim 10] The 7α-steroid dehydrogenase according to claim 9, wherein the 7α-steroid dehydrogenase has at least one of the following mutations: D42N, D44A, G97D, G99A, L117 E, Y159F, I192K and D195N.
PCT/CN2017/070603 2017-01-09 2017-01-09 PREPARATION METHOD FOR 3α-HYDROXY-7-OXO-5β-CHOLANOIC ACID AND ENZYME 2 FOR PREPARATION THEREOF WO2018126467A1 (en)

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