WO2018126467A1 - PROCÉDÉ DE PRÉPARATION D'ACIDE 3α-HYDROXY-7-OXO-5β-CHOLANOÏQUE ET ENZYME 2 POUR SA PRÉPARATION - Google Patents

PROCÉDÉ DE PRÉPARATION D'ACIDE 3α-HYDROXY-7-OXO-5β-CHOLANOÏQUE ET ENZYME 2 POUR SA PRÉPARATION Download PDF

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Publication number
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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Prior art keywords
hydroxy
steroid dehydrogenase
acid
cholanoic
oxo
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PCT/CN2017/070603
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English (en)
Chinese (zh)
Inventor
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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Application filed by 深圳市邦泰绿色生物合成研究院 filed Critical 深圳市邦泰绿色生物合成研究院
Priority to PCT/CN2017/070603 priority Critical patent/WO2018126467A1/fr
Priority to CN201780001727.3A priority patent/CN107980060B/zh
Publication of WO2018126467A1 publication Critical patent/WO2018126467A1/fr

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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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  • Chemical & Material Sciences (AREA)
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  • Life Sciences & Earth Sciences (AREA)
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  • Preparation Of Compounds By Using Micro-Organisms (AREA)
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Abstract

L'invention un procédé qui utilise une catalyse enzymatique biologique pour préparer l'acide 3α-hydroxy-7-oxo-5β-cholanoïque et la 7α-stéroïde déshydrogénase pour sa préparation. Le procédé utilise de l'acide chénodésoxycholique en tant que substrat ; en présence de NAD, de lactate déshydrogénase, de pyruvate de sodium et d'une solution tampon, la 7α-stéroïde déshydrogénase est utilisée pour catalyser l'acide chénodésoxycholique pour préparer l'acide 3α-hydroxy-7-oxo-5β-cholanoïque, la 7α-stéroïde déshydrogénase étant dérivée de cyanothécène sp. ATCC 29155.
PCT/CN2017/070603 2017-01-09 2017-01-09 PROCÉDÉ DE PRÉPARATION D'ACIDE 3α-HYDROXY-7-OXO-5β-CHOLANOÏQUE ET ENZYME 2 POUR SA PRÉPARATION WO2018126467A1 (fr)

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Application Number Priority Date Filing Date Title
PCT/CN2017/070603 WO2018126467A1 (fr) 2017-01-09 2017-01-09 PROCÉDÉ DE PRÉPARATION D'ACIDE 3α-HYDROXY-7-OXO-5β-CHOLANOÏQUE ET ENZYME 2 POUR SA PRÉPARATION
CN201780001727.3A CN107980060B (zh) 2017-01-09 2017-01-09 一种3α-羟基-7氧代-5β-胆烷酸的制备方法及其制备用酶2

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CN114107237B (zh) * 2021-09-07 2024-06-04 伊犁川宁生物技术股份有限公司 一种发酵生产鹅去氧胆酸氧化酶的方法

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