WO2021139689A1 - 一种生物酶用于制备奥利司他中间体的用途及制备方法 - Google Patents

一种生物酶用于制备奥利司他中间体的用途及制备方法 Download PDF

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WO2021139689A1
WO2021139689A1 PCT/CN2021/070495 CN2021070495W WO2021139689A1 WO 2021139689 A1 WO2021139689 A1 WO 2021139689A1 CN 2021070495 W CN2021070495 W CN 2021070495W WO 2021139689 A1 WO2021139689 A1 WO 2021139689A1
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ketoreductase
enzyme
amino acid
glucose dehydrogenase
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French (fr)
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徐天帅
龚大勇
肖玉梅
王章洪
黄治川
黄山
张磊
高鑫
沈军伟
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植恩生物技术股份有限公司
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Priority to US17/790,787 priority Critical patent/US20240158819A1/en
Priority to JP2022541240A priority patent/JP7498278B2/ja
Priority to EP21738086.4A priority patent/EP4086343A1/en
Priority to KR1020227027087A priority patent/KR20220125300A/ko
Priority to BR112022013466A priority patent/BR112022013466A2/pt
Publication of WO2021139689A1 publication Critical patent/WO2021139689A1/zh

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    • 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)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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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
    • 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
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • 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)
    • C12Y101/01047—Glucose 1-dehydrogenase (1.1.1.47)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
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    • 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)
    • 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)
    • C12Y101/01002—Alcohol dehydrogenase (NADP+) (1.1.1.2), i.e. aldehyde reductase

Definitions

  • the present invention belongs to the technical field of biopharmaceuticals and biochemical industry. Specifically, it relates to a method for biosynthesis of (R)- ⁇ -hydroxytetradecanoate compounds.
  • Obesity treatment drugs are mainly divided into two categories, namely, central nervous system action weight loss drugs and non-central nervous system action weight loss drugs.
  • the weight loss effect of the central nervous system weight loss drugs is obvious, but the side effects are relatively large, and the clinical application is greatly restricted.
  • fenfluramine was delisted in 1997 because it caused pulmonary hypertension and hypertrophic heart valve disease.
  • Sibutramine was announced by the State Food and Drug Administration in 2010 to stop production, sales and use because it may increase serious cardiovascular risks.
  • Locaserin brand name Belviq
  • Orlistat is the first weight loss drug with non-central nervous function, and it is currently the only OTC weight loss drug in the world. Since it was launched in 1998, it has become the first choice for weight loss treatment due to its precise curative effect and high safety. Orlistat acts directly on the gastrointestinal lipase and has a significant effect on inhibiting fat absorption. Its target is highly specific, has no effect on other gastrointestinal enzymes, and does not require systemic absorption to exert its efficacy. Its systemic drug exposure in the body The amount is very low, the main side effect is gastrointestinal reactions, and the safety is excellent. Orlistat has been sold globally for more than 20 years and has been approved for marketing in more than 145 countries. Its excellent efficacy and safety have been verified by a large number of clinical applications.
  • Orlistat has a large market demand, and it is extremely important to find an efficient synthesis method for orlistat and its intermediates.
  • (R)- ⁇ -hydroxytetradecanoate is an important intermediate in the synthesis of orlistat, and the optical purity of this intermediate is extremely critical. How to efficiently obtain optically pure (R)- ⁇ -hydroxytetradecanoate?
  • one of the objectives of the present invention is to provide a new use of a biological enzyme in the preparation of orlistat intermediates, which can effectively act on the substrate ⁇ -carbonyl myristate to produce pure The higher orlistat intermediate.
  • the biological enzyme is ketoreductase, and the amino acid sequence of the biological enzyme is shown in SEQ ID NO:1 and its amino acid sequence with a homology of greater than 90% is derived from Singulisphaera acidiphila.
  • the code in the NCBI database is WP_015245403.1 , Belongs to the short chain dehydrogenase family; and/or the amino acid sequence shown in SEQ ID NO: 2 and its homology greater than 90%, derived from Sphingomonas echinoides, the code in the NCBI database is WP_010403640.1, which belongs to the short chain Dehydrogenase family.
  • the amino acid sequence shown in SEQ ID NO: 4 and its identity ⁇ 80%, derived from Rhodotorula toruloides, is a truncated protein encoding EGU12837.1 protein in the NCBI database, and belongs to the short-chain dehydrogenase family.
  • the biological enzyme is a fusion enzyme of ketoreductase and glucose dehydrogenase, and the amino acid sequence of the fusion enzyme is as shown in SEQ ID NO: 8 and/or as shown in SEQ ID NO: 9.
  • the inventors of the present invention surprisingly found that the natural or genetic/protein engineered biological enzyme has a good catalytic effect on the substrate and obtains the final product with high optical purity.
  • identity/homology ⁇ 90% with SEQ ID NO:1 and/or SEQ ID NO:2, identity/homology ⁇ 80% with SEQ ID NO: 4, and SEQ ID NO: 8 and/ Or the biological enzyme of SEQ ID NO: 9 can also be used to prepare orlistat intermediates.
  • ketoreductase and glucose dehydrogenase are connected through a linker
  • amino acid sequence of the linker is shown in SEQ ID NO: 5.
  • the fusion enzyme of the ketoreductase and glucose dehydrogenase is ketoreductase-linker-glucose dehydrogenase, or glucose dehydrogenase-linker-ketoreductase; optionally, the glucose dehydrogenase is The amino acid sequence is shown in SEQ ID NO: 3.
  • any one of the aforementioned biological enzymes is enzyme powder and/or enzyme solution and/or immobilized enzyme.
  • R in structural formula I is any one of methyl, ethyl, n-propyl or isopropyl
  • R in structural formula II is any one of methyl, ethyl, n-propyl or isopropyl.
  • the second objective of the present invention is to provide a fusion enzyme, which is a fusion enzyme of ketoreductase and glucose dehydrogenase, and the amino acid sequence of the fusion enzyme is as SEQ ID NO: 8 and/or as SEQ ID NO: 9 shown.
  • ketoreductase and glucose dehydrogenase in the fusion enzyme are connected by a linker; optionally, the amino acid sequence of the linker is shown in SEQ ID NO: 5.
  • the fusion enzyme is ketoreductase-linker-glucose dehydrogenase or glucose dehydrogenase-linker-ketoreductase;
  • amino acid sequence of the ketoreductase is as shown in SEQ ID NO: 1 and/or as shown in SEQ ID NO: 2, and/or as shown in SEQ ID NO: 4 and its identity/homology ⁇ 80 % Amino acid sequence;
  • amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO: 3.
  • the third objective of the present invention is to provide a nucleotide sequence encoding any one of the aforementioned fusion enzymes and a construction method thereof.
  • the nucleotide sequence of the fusion enzyme is shown in SEQ ID NO: 6 or SEQ ID NO: 7.
  • the method for constructing the nucleotide sequence includes: inserting the linker sequence shown in SEQ ID NO: 5 at the 3'end of the glucose dehydrogenase gene fragment shown in SEQ ID NO: 3, followed by The ketoreductase gene fragment shown in SEQ ID NO: 4 constitutes a recombinant plasmid with the nucleotide sequence shown in SEQ ID NO: 6.
  • nucleotide sequence of the fusion enzyme SEQ ID NO: 6 is connected to the vector pET28a through the restriction sites NdeI and XhoI at both ends to form a double-enzyme fusion expression plasmid pET28a-G3790, which is then transferred into E. coli for screening and inoculation , Cultivate the bacteria; crush the bacteria and centrifuge to obtain the crude enzyme solution, and freeze-dry the crude enzyme solution to obtain the enzyme powder.
  • the method for constructing the nucleotide sequence includes: inserting the linker sequence shown in SEQ ID NO: 5 at the 3'end of the ketoreductase gene fragment shown in SEQ ID NO: 4, followed by The gene fragment of glucose dehydrogenase shown in SEQ ID NO: 3 constitutes the nucleotide sequence shown in SEQ ID NO: 7.
  • nucleotide sequence of the fusion enzyme SEQ ID NO: 7 is connected to the vector pET28a through the restriction sites NdeI and XhoI at both ends to form a double-enzyme fusion expression plasmid pET28a-G3790, which is then transferred to E. coli for screening and inoculation , Cultivate the bacteria; crush the bacteria and centrifuge to obtain the crude enzyme solution, and freeze-dry the crude enzyme solution to obtain the enzyme powder.
  • the fourth object of the present invention is to provide a composition in which the two can form a synergistic relationship between the enzyme and the substrate, and obtain a product as shown in structural formula II.
  • the structural formula of the ⁇ -carbonyltetradecanoate is shown in formula I;
  • the biological enzyme is a ketoreductase or a fusion enzyme of ketoreductase and glucose dehydrogenase, and the amino acid sequence of the ketoreductase is shown in SEQ ID NO :1 and/or as shown in SEQ ID NO: 2, and/or as shown in SEQ ID NO: 4 and its identity/homology ⁇ 80% amino acid sequence;
  • the amino acid sequence of the fusion enzyme is as SEQ ID NO :8 and/or as shown in SEQ ID NO: 9;
  • R in structural formula I refers to a saturated alkyl group containing 1 to 3 carbon atoms.
  • ketoreductase and glucose dehydrogenase in the fusion enzyme are connected by a linker; optionally, the amino acid sequence of the linker is shown in SEQ ID NO: 5.
  • the fusion enzyme is ketoreductase-linker-glucose dehydrogenase or glucose dehydrogenase-linker-ketoreductase.
  • amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO: 3.
  • the biological enzyme is enzyme powder and/or enzyme solution and/or immobilized enzyme.
  • R in structural formula I is any one of methyl, ethyl, n-propyl or isopropyl
  • R in structural formula II is any one of methyl, ethyl, n-propyl or isopropyl.
  • the weight ratio of the biological enzyme to the substrate is 1:1.1-150.
  • the weight ratio of the biological enzyme to the substrate is 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80 , 1:90, 1:100, 1:110, 1:120, 1:130, 1:140, and/or 1:150.
  • the fifth object of the present invention is to provide a reaction system for preparing (R)- ⁇ -hydroxytetradecanoate, which can obtain (R)- ⁇ -hydroxydecanoate without resorting to high temperature, high pressure and severe conditions. Tetraalkanoate.
  • the reaction system is composed of the substrate shown in I, biological enzyme, glucose, glucose dehydrogenase, NADP + and buffer; the biological enzyme described here is that the biological enzyme is ketoreductase, and the ketoreductase
  • the amino acid sequence of is as shown in SEQ ID NO: 1 and/or as shown in SEQ ID NO: 2, and/or as shown in SEQ ID NO: 4 and the amino acid sequence with identity/homology ⁇ 80%; or,
  • the reaction system consists of the substrate shown in I, biological enzyme, glucose NADP + and buffer; here, the biological enzyme is a fusion enzyme of ketoreductase and glucose dehydrogenase, and the amino acid sequence of the ketoreductase is as SEQ ID NO:1 and/or as shown in SEQ ID NO:2, and/or as shown in SEQ ID NO:4 and its identity/homology ⁇ 80% amino acid sequence; said ketoreductase and glucose dehydrogenation
  • the amino acid sequence of the enzyme fusion enzyme is as shown in SEQ
  • R in structural formula I refers to a saturated alkyl group containing 1 to 3 carbon atoms.
  • the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO: 3. Further, the ketoreductase and glucose dehydrogenase in the fusion enzyme are connected by a linker; optionally, the amino acid sequence of the linker is shown in SEQ ID NO: 5.
  • the fusion enzyme is ketoreductase-linker-glucose dehydrogenase or glucose dehydrogenase-linker-ketoreductase.
  • amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO: 3.
  • the biological enzyme is enzyme powder and/or enzyme solution and/or immobilized enzyme.
  • R in structural formula I is any one of methyl, ethyl, n-propyl or isopropyl
  • R in structural formula II is any one of methyl, ethyl, n-propyl or isopropyl.
  • the pH of the reaction system is 6.0-8.0.
  • the buffer solution in the reaction system is PBS buffer or Tris-HCl buffer.
  • the concentration of the substrate in the reaction system is 20 g/L-150 g/L.
  • reaction system is characterized in that the concentration of NADP + is 0.1-0.5 g/L.
  • the molar ratio of the compound represented by the structural formula I to the glucose is 1:(1.2-4).
  • the concentration of the buffer is 0.01-0.5 mol/L.
  • reaction time of the reaction system does not exceed 15 hours to obtain a reaction liquid.
  • the sixth object of the present invention is to provide a preparation method of orlistat intermediate, which is suitable for industrialized large-scale production.
  • R refers to a saturated alkyl group containing 1 to 3 carbon atoms, preferably, methyl, ethyl, n-propyl or isopropyl;
  • the biosynthesis method includes reacting compound I in ketoreductase, glucose, glucose dehydrogenase and NADP+, or reacting in the fusion enzyme of ketoreductase and glucose dehydrogenase, glucose and NADP+ to obtain compound II .
  • the biosynthesis method includes the following steps: taking compound I into a buffer solution, and then adding ketoreductase or a fusion enzyme of ketoreductase and glucose dehydrogenase, glucose, glucose Dehydrogenase (glucose dehydrogenase does not need to be added when adding the fusion enzyme of ketoreductase and glucose dehydrogenase) and NADP+ to obtain a mixed solution.
  • the mixed solution is stirred and reacted at 20-40°C, and NaOH aqueous solution is used throughout the process Adjust the pH, monitor the conversion rate of the reaction by liquid chromatography, until the conversion rate reaches more than 99%, the reaction is over, add extraction solvent to extract, combine the organic phases and concentrate under reduced pressure, cooling and crystallization, the product is precipitated to obtain a white solid compound II, optional It is further recrystallized with n-hexane to obtain a product of higher purity.
  • the concentration of the aqueous NaOH solution may be 2M.
  • the buffer solution has a pH of 6.0-8.0; preferably, the buffer solution is PBS (ie, phosphate) buffer or Tris-HCl buffer; more preferably, the The concentration of the buffer is 0.01-0.5mol/L PBS phosphate buffer or 0.01-0.5mol/L Tris-HCl buffer.
  • the pH range is controlled at 7.0-7.5 during the reaction process.
  • the amino acid sequence of the ketoreductase is shown in SEQ ID NO: 1 and SEQ ID NO: 2 and SEQ ID NO: 4 of the present application; the ketoreductase and glucose dehydrogenase
  • the amino acid sequence of the fusion enzyme is as shown in SEQ ID NO: 8 and/or as shown in SEQ ID NO: 9; the form of the ketoreductase or the fusion enzyme of ketoreductase and glucose dehydrogenase can be enzyme powder, enzyme solution , Or immobilized enzyme; the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO: 3 of the present application, and the form of the glucose dehydrogenase may be enzyme powder, enzyme solution, or immobilized enzyme.
  • the NADP+ refers to nicotinamide adenine dinucleotide phosphate, which is the oxidized form of reduced coenzyme II (NADPH); the concentration of NADP+ in the mixed solution is 0.1-0.5g/ L.
  • the concentration of Compound I in the mixed solution is 20 g/L-150 g/L.
  • the molar ratio of compound I to glucose in the mixed solution is 1:(1.2-4).
  • the temperature of the reaction is maintained at 35°C.
  • the extraction solvent is extracted twice; the extraction solvent is absolute ethanol or ethyl acetate.
  • the reaction system is stirred and reacted at 20-40° C. to obtain the reaction solution of the orlistat intermediate.
  • the intermediate of the intermediate is (R)- ⁇ -hydroxytetradecanoate, and the structural formula is shown in II.
  • an aqueous NaOH solution is used as a pH adjuster.
  • reaction time does not exceed 15 hours.
  • the orlistat intermediate is extracted with a solvent in the resulting reaction liquid.
  • the solvent is absolute ethanol or ethyl acetate.
  • the obtained extract is concentrated under reduced pressure, and the temperature is lowered to crystallize, and the white crystalline solid is compound II.
  • Orlistat prepared by the orlistat intermediate prepared by the orlistat intermediate.
  • the biological enzyme used in the present invention can tolerate a substrate concentration of up to 150 g/L, and the enzyme activity is not inhibited by the substrate or product.
  • the method for preparing orlistat intermediates of the present invention is a biological enzymatic method.
  • the method has mild conditions, simple equipment requirements, and simple operation.
  • the method produces less three wastes, has no heavy metal pollution, is environmentally friendly, and is conducive to industrial production. .
  • the conversion rate of the enzyme-catalyzed process of the present invention is as high as 99% or more, the chiral ee value can reach 99% or more, the reaction can be completed within 15 hours, the product concentration is high, and the almost complete conversion of the substrate can simplify the post-treatment of the reaction solution. Steps, only a simple extraction and crystallization step is required to obtain a high-purity product, which greatly reduces the production cost.
  • Figure 1 is an HPLC chart of the conversion rate measured in Example 5.
  • Figure 2 is an HPLC chart of the purity of the product in Example 5.
  • Figure 3 is an HPLC chart of the chiral purity of the product in Example 5.
  • Figure 4 is a plasmid map of the fusion enzyme G3790 expression plasmid.
  • Figure 5 is a plasmid map of the fusion enzyme 3790G expression plasmid.
  • Figure 6 shows the G3790 fusion enzyme protein band.
  • Figure 7 shows the 3790G fusion enzyme protein band.
  • ⁇ -Carbonyl tetradecanoate is used as a substrate in an environment containing NADP + coenzyme, glucose and glucose dehydrogenase, under the catalysis of certain enzymes, to obtain (R)- ⁇ -hydroxyl group with extremely high chiral purity Myristate.
  • the catalytic enzyme is generally a family of short-chain dehydrogenases, such as different types of ketoreductases in the embodiments of the present invention; it can also be a fusion enzyme of different enzymes, such as reductase and glucose dehydrogenase in some embodiments of the present invention. Fusion fusion enzyme.
  • glucose is dehydrogenated under the action of glucose dehydrogenase to obtain H + , and then NADP + coenzyme carries H + and participates in the reduction reaction of ⁇ -carbonyl tetradecanoate to obtain ⁇ -hydroxy tetradecanoate.
  • the ketoreductase JR3789 of the present invention is derived from Singulisphaera acidiphila, and the code in the NCBI database is WP_015245403.1, which belongs to the family of short-chain dehydrogenases.
  • the amino acid sequence of the ketoreductase is shown in SEQ ID NO: 1, and the size is 249. Amino acids.
  • the ketoreductase JR37150 of the present invention is derived from Sphingomonas echinoides, and the code in the NCBI database is WP_010403640.1, belonging to the family of short-chain dehydrogenases.
  • the amino acid sequence of the ketoreductase is shown in SEQ ID NO: 2, with a size of 259 Amino acids.
  • the glucose dehydrogenase GDH of the present invention is derived from Bacillus subtilis QB928, the code in the NCBI database is AFQ56330.1, and belongs to the short-chain dehydrogenase family.
  • the amino acid sequence of the dehydrogenase is shown in SEQ ID NO: 3, and the size is 263 amino acids.
  • the ketoreductase JR3790 of the present invention is derived from Rhodotorula toruloides, is a truncated protein (position 70-317) encoding the EGU12837.1 protein in the NCBI database, and belongs to the short-chain dehydrogenase family.
  • the amino acid sequence of the ketoreductase is as SEQ ID NO: 4, the size is 248 amino acids.
  • the biological enzymes (SEQ ID NO: 1, SEQ ID NO: 2) and glucose dehydrogenase (SEQ ID NO: 3) were synthesized by Nanjing GenScript Biotechnology Co., Ltd. and commercialized.
  • the crude product was heated and dissolved by adding 2 times the volume of n-hexane, cooled and crystallized, collected and dried at room temperature to obtain 0.89 g of a white crystalline product with a measured purity of 99.99%, an ee value of 99.91%, and a total yield of 89%.
  • the crude product was heated and dissolved by adding 2 times the volume of n-hexane, cooled and crystallized, filtered, and dried at room temperature to obtain 6.53 g of a white crystal product with a measured purity of 99.99%, an ee value of 99.86%, and a total yield of 87%.
  • the purity data of the detected product is shown in Table 2, the HPLC profile is shown in Fig. 2, the chiral purity data of the product is shown in Table 3, and the HPLC profile is shown in Fig. 3.
  • IPTG isopropyl- ⁇ -D-thiogalactoside
  • FIG. 6 shows the protein electrophoresis diagram of the G3790 fusion enzyme, where lane M is the protein Marker (GenScript), lane 1 is the total protein after whole cell disruption, and lane 2 is the supernatant of the whole cell disruption centrifugation.
  • GenScript protein Marker
  • lane 1 is the total protein after whole cell disruption
  • lane 2 is the supernatant of the whole cell disruption centrifugation.
  • the theory of the fusion enzyme The molecular weight should be 67kDa, and its amino acid sequence is shown in SEQ ID NO: 8.
  • Figure 7 shows the protein electrophoresis diagram of the 3790G fusion enzyme, in which lane M is the protein Marker (GenScript), lane 1 is the total protein after whole cell disruption, and lane 2 is the supernatant of the whole cell disruption centrifugation.
  • the theoretical molecular weights of the two fusion enzymes are both 67kDa, and their amino acid sequences are shown in SEQ ID NO: 9.
  • the two fusion enzymes are both soluble proteins, and the molecular weight is close to the corresponding theoretical molecular weight.
  • the crude enzyme solution was pre-frozen overnight and freeze-dried for 24h-36h to obtain fusion enzyme G3790 enzyme powder and fusion enzyme 3790G enzyme powder.
  • the crude product was heated and dissolved by adding 2 times the volume of n-hexane, cooled and crystallized, filtered, and dried at room temperature to obtain 6.45 kg of white crystal product, with a measured purity of 99.70%, an ee value of 99.88%, and a total yield of 86%.

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Abstract

提供了一种(R)-β-羟基十四烷酸酯类化合物的生物合成方法。该方法是将β-羰基十四烷酸酯在酮还原酶与葡萄糖脱氢酶或它们的融合酶、葡萄糖、NADP+和缓冲液中反应得到(R)-β-羟基十四烷酸酯类化合物。该方法为酶催化生物合成方法，操作简单，使用设备为常规设备，工艺环保，制得的产品纯度高、产率高、ee值高。

Description

一种生物酶用于制备奥利司他中间体的用途及制备方法 技术领域
本发明属于本发明属于生物制药和生物化工技术领域，具体地，涉及一种(R)-β-羟基十四烷酸酯类化合物的生物合成方法。
背景技术
肥胖症治疗药物主要分为两大类，即中枢神经作用减肥药和非中枢神经作用减肥药。中枢神经作用减肥药的减肥效果明显，但是副作用较大，临床应用受到较大限制。其中芬氟拉明由于会引起肺动脉高压和肥厚性心脏瓣膜病变于1997年退市。西布曲明由于可能增加严重心血管风险于2010年被国家食品药品监督管理局宣布停止生产、销售和使用。绿卡色林(locaserin，商品名Belviq)2012年6月由FDA批准上市，尚存在增加心瓣膜病和不良心血管事件的风险，临床应用的安全性还有待进一步验证。
奥利司他(Orlistat)是第一个非中枢神经作用的减肥药物，且是目前全球唯一一种OTC减肥药物。自1998年上市以来，因疗效确切、安全性高，已成为减肥治疗的首选药物。奥利司他直接作用于胃肠道脂肪酶，抑制脂肪吸收效果显著，其靶点高度专一，对其他胃肠道酶没有影响，且无需通过全身吸收而发挥药效，其体内系统药物暴露量很低，主要副作用为胃肠道反应，安全性优异。奥利司他全球销售已超过20年，目前已在超过145个国家获得批准上市，其优异的疗效和安全性已被大量的临床应用验证。
奥利司他的市场需求量大，而找寻高效的奥利司他及其中间物的合成方法是极其重要的。而(R)-β-羟基十四烷酸酯是合成药物奥利司他的重要中间体，该中间体的光学纯度极为关键。如何高效的获得光学纯的(R)-β-羟基十四烷酸酯呢？
如中国专利CN101538285B报道了一种利用制备的(R)-金属催化剂[(R)-Ru(MeOBIPHEP)Cl2]2·NEt3催化不对称氢化反应，但该化学催化反应需要在强酸且60bar高压条件下反应才能得到ee值>98.5％的产品。此类方法的不足之处在于：1)需要使用昂贵的(R)-配体与贵金属催化剂；2)催化剂需要现 做现用，工艺稳定性难以保证；3)需要高压氢化设备；4)需要耐酸设备；5)反应控制得不好会影响ee值。
发明内容
针对现有技术的上述缺点，本发明人用生物酶法进行系统的改进，解决奥利司他关键中间体的规模化生产。
有鉴于此，本发明的目的之一在于提供一种生物酶在制备奥利司他中间体中的新用途，该生物酶能够有效地作用底物β-羰基十四烷酸酯，制备出纯度较高的奥利司他中间体。
为实现上述目的，本发明的技术方案为：
生物酶在制备奥利司他中间体(终物)的用途，所述生物酶作用的底物为β-羰基十四烷酸酯(原料)，结构式如I所示；所述奥利司他中间体为(R)-β-羟基十四烷酸酯，结构式如II所示；结构式I中的R指含有1-3个碳原子的饱和烷基，结构式II中R指含有1-3个碳原子的饱和烷基。
Figure PCTCN2021070495-appb-000001
所述生物酶为酮还原酶，所述生物酶的氨基酸序列如SEQ ID NO:1所示及其同源性大于90％的氨基酸序列，来源于Singulisphaera acidiphila，NCBI数据库中的编码为WP_015245403.1，属于短链脱氢酶家族；和/或如SEQ ID N0:2所示及其同源性大于90％的氨基酸序列，来源于Sphingomonas echinoides，NCBI数据库中的编码为WP_010403640.1，属于短链脱氢酶家族。和/或SEQ ID NO:4所示及其同一性≥80％的氨基酸序列，来源于Rhodotorula toruloides，为NCBI数据库中的编码EGU12837.1蛋白的截短蛋白，属于短链脱氢酶家族。或所述生物酶为酮还原酶与葡萄糖脱氢酶的融合酶，所述融合酶的氨基酸序列如SEQ ID NO:8和/或如SEQ ID N0:9所示。本发明人惊奇地发现，天然的或者经过基因/蛋白工程改造后的所述生物酶对所述底物有很好的催化作用，并得到光学纯度 高的所述终物。
进一步，与SEQ ID NO:1和/或SEQ ID N0:2同一性/同源性≥90％，与SEQ ID NO:4同一性/同源性≥80％，与SEQ ID NO:8和/或SEQ ID N0:9的生物酶也可应用于制备奥利司他中间体。
进一步，所述酮还原酶与葡萄糖脱氢酶的融合酶中，酮还原酶与葡萄糖脱氢酶之间通过linker进行连接；
进一步，所述的linker的氨基酸序列如SEQ ID NO:5所示。
进一步，所述酮还原酶与葡萄糖脱氢酶的融合酶为酮还原酶-linker-葡萄糖脱氢酶，或葡萄糖脱氢酶-linker-酮还原酶；任选地，所述葡萄糖脱氢酶的氨基酸序列如SEQ ID NO:3所示。
进一步，前面任一所述生物酶为酶粉和/或酶液和/或固定化酶。
进一步，结构式I中的R为甲基、乙基、正丙基或异丙基中任一种，结构式II中的R为甲基、乙基、正丙基或异丙基中任一种。
本发明目的之二在于提供一种融合酶，所述融合酶为酮还原酶与葡萄糖脱氢酶的融合酶，所述融合酶的氨基酸序列如SEQ ID NO:8和/或如SEQ ID N0:9所示。
进一步，所述融合酶中酮还原酶与葡萄糖脱氢酶之间通过linker进行连接；任选地，所述的linker的氨基酸序列如SEQ ID NO:5所示。
进一步，所述融合酶为酮还原酶-linker-葡萄糖脱氢酶，或葡萄糖脱氢酶-linker-酮还原酶；
任选地，所述酮还原酶的氨基酸序列如SEQ ID NO:1和/或如SEQ ID N0:2所示，和/或SEQ ID NO:4所示及其同一性/同源性≥80％的氨基酸序列；
任选地，所述葡萄糖脱氢酶的氨基酸序列如SEQ ID NO:3所示。
本发明目的之三在于提供一种编码前任一所述融合酶的核苷酸序列及其构建方法。
所述融合酶的核苷酸序列如SEQ ID NO：6或SEQ ID NO：7所示。
进一步，所述核苷酸序列的构建方法，包括：在SEQ ID NO:3所示的葡萄 糖脱氢酶的基因片段的3’端插入SEQ ID NO：5所示的linker序列，后面再接上SEQ ID NO:4所示的酮还原酶的基因片段，构成如SEQ ID NO：6所示的核苷酸序列重组质粒。
进一步，所述融合酶SEQ ID NO：6核苷酸序列在通过两端酶切位点NdeI和XhoI与载体pET28a连接，构成双酶融合表达质粒pET28a-G3790，后经转入大肠杆菌筛选、接种、培养得菌体；菌体破碎、离心得粗酶液，粗酶液冷冻干燥得酶粉。
进一步，所述的核苷酸序列的构建方法，包括：在SEQ ID NO:4所示的酮还原酶的基因片段的3’端插入SEQ ID NO：5所示的linker序列，后面再接上SEQ ID NO:3所示的葡萄糖脱氢酶的基因片段，构成如SEQ ID NO：7所示的核苷酸序列。
进一步，所述融合酶SEQ ID NO：7核苷酸序列在通过两端酶切位点NdeI和XhoI与载体pET28a连接，构成双酶融合表达质粒pET28a-G3790，后经转入大肠杆菌筛选、接种、培养得菌体；菌体破碎、离心得粗酶液，粗酶液冷冻干燥得酶粉。
本发明的目的之四在于提供一种组合物，该组合物中两者能形成酶和底物的协同关系，并得到如结构式II所示的产物。
为实现上述目的，本发明的技术方案为：
含有上面任一所述的生物酶和底物的组合物，所述底物为所述β-羰基十四烷酸酯。所述β-羰基十四烷酸酯结构式如式I所示；所述生物酶为酮还原酶或酮还原酶与葡萄糖脱氢酶的融合酶，所述酮还原酶的氨基酸序列如SEQ ID NO:1和/或如SEQ ID N0:2所示，和/或SEQ ID NO:4所示及其同一性/同源性≥80％的氨基酸序列；所述融合酶的氨基酸序列如SEQ ID NO:8和/或如SEQ ID N0:9所示；
Figure PCTCN2021070495-appb-000002
结构式I中的R指含有1-3个碳原子的饱和烷基。
进一步，所述融合酶中酮还原酶与葡萄糖脱氢酶之间通过linker进行连接；任选地，所述的linker的氨基酸序列如SEQ ID NO:5所示。
进一步，所述融合酶为酮还原酶-linker-葡萄糖脱氢酶，或葡萄糖脱氢酶-linker-酮还原酶。
进一步，所述葡萄糖脱氢酶的氨基酸序列如SEQ ID NO:3所示。
进一步，所述生物酶为酶粉和/或酶液和/或固定化酶。
进一步，结构式I中的R为甲基、乙基、正丙基或异丙基中任一种，结构式II中的R为甲基、乙基、正丙基或异丙基中任一种。
进一步，所述的组合物中，所述生物酶与所述底物的重量比为1:1.1-150。
优选地，所述的组合物中，所述生物酶与所述底物的重量比为1:20、1：30、1：40、1：50、1：60、1：70、1：80、1：90、1：100、1：110、1：120、1：130、1：140和/或1：150。
本发明的目的之五在于提供一种用于制备(R)-β-羟基十四烷酸酯的反应体系，该反应体系无需借助高温高压严苛条件即可得到(R)-β-羟基十四烷酸酯。
为实现上述目的，本发明的技术方案为：
所述反应体系由I所示的底物、生物酶、葡萄糖，葡萄糖脱氢酶、NADP +和缓冲液组成；这里所述的生物酶为所述生物酶为酮还原酶，所述酮还原酶的氨基酸序列如SEQ ID NO:1和/或如SEQ ID N0:2所示，和/或SEQ ID NO:4所示及其同一性/同源性≥80％的氨基酸序列；或者，所述反应体系由I所示的底物、生物酶、葡萄糖NADP +和缓冲液组成；这里，所述生物酶为酮还原酶与葡萄糖脱氢酶的融合酶，所述酮还原酶的氨基酸序列如SEQ ID NO:1和/或如SEQ ID N0:2所示，和/或SEQ ID NO:4所示及其同一性/同源性≥80％的氨基酸序列；所述酮还原酶与葡萄糖脱氢酶的融合酶的氨基酸序列如SEQ ID NO:8和/或如SEQ ID N0:9所示；
Figure PCTCN2021070495-appb-000003
结构式I中的R指含有1-3个碳原子的饱和烷基。
进一步，所述葡萄糖脱氢酶的氨基酸序列如SEQ ID NO:3所示。进一步，所述融合酶中酮还原酶与葡萄糖脱氢酶之间通过linker进行连接；任选地，所述的linker的氨基酸序列如SEQ ID NO:5所示。
进一步，所述融合酶为酮还原酶-linker-葡萄糖脱氢酶，或葡萄糖脱氢酶-linker-酮还原酶。
进一步，所述葡萄糖脱氢酶的氨基酸序列如SEQ ID NO:3所示。
进一步，所述生物酶为酶粉和/或酶液和/或固定化酶。
进一步，结构式I中的R为甲基、乙基、正丙基或异丙基中任一种，结构式II中的R为甲基、乙基、正丙基或异丙基中任一种。
进一步，所述的反应体系的pH值为6.0-8.0。
进一步，所述的反应体系中所述缓冲溶液为PBS缓冲液或Tris-HCl缓冲液。
进一步，所述的反应体系中所述底物的浓度为20g/L-150g/L。
进一步，所述的反应体系，其特征在于，所述NADP +的浓度为0.1-0.5g/L。
进一步，所述的反应体系，结构式如I所示的化合物与所述葡萄糖的摩尔比为1:(1.2-4)。
进一步，反应体系中，所述缓冲液的浓度为0.01-0.5mol/L。
进一步，所述反应体系的反应时间不超过15小时，得反应液。
本发明的目的之六在于提供奥利司他中间体的制备方法，该方法适用于工业化大规模生产。
(R)-β-羟基十四烷酸酯的生物合成方法，反应式如下所示：
Figure PCTCN2021070495-appb-000004
化合物I和化合物II中R指含有1-3个碳原子的饱和烷基，优选地，为甲基、乙基、正丙基或异丙基；
所述的生物合成方法包括，将化合物I在酮还原酶、葡萄糖、葡萄糖脱氢酶以及NADP+中反应，或者在酮还原酶与葡萄糖脱氢酶的融合酶、葡萄糖以及NADP+中反应，得到化合物II。
在本发明的实施方案中，所述的生物合成方法包括如下步骤：取化合物I置于缓冲溶液中，再向其中加入酮还原酶或酮还原酶与葡萄糖脱氢酶的融合酶，葡萄糖，葡萄糖脱氢酶(在加入酮还原酶与葡萄糖脱氢酶的融合酶时不需要加入葡萄糖脱氢酶)以及NADP+，得到混合溶液，所述混合溶液在20-40℃下搅拌反应，全程用NaOH水溶液调节pH，利用液相色谱监测反应的转化率，至转化率达99％以上反应结束，加入萃取溶剂萃取，合并有机相并减压浓缩，降温结晶，产物析出得到白色固体即化合物II，任选地，进一步用正己烷重结晶得到更高纯度的产品。
在本发明的一些实施方案中，所述NaOH水溶液的浓度可以是2M。
在本发明的一些实施方案中，所述缓冲溶液为pH值为6.0-8.0；优选地，所述缓冲溶液为PBS(即磷酸盐)缓冲液或Tris-HCl缓冲液；更优选地，所述缓冲液的浓度为0.01-0.5mol/L的PBS磷酸缓冲液或0.01-0.5mol/L的Tris-HCl缓冲液。
在本发明的实施方案中，在所述反应过程中pH范围控制在7.0-7.5。
在本发明的实施方案中，所述酮还原酶的氨基酸序列如本申请的SEQ ID NO:1和SEQ ID NO:2和SEQ ID NO:4所示；所述酮还原酶与葡萄糖脱氢酶的融合酶的氨基酸序列如SEQ ID NO:8和/或如SEQ ID N0:9所示；所述酮还原酶或酮还原酶与葡萄糖脱氢酶的融合酶的形式可以是酶粉、酶液、或固定化酶；所述葡萄糖脱氢酶的氨基酸序列如本申请的SEQ ID NO:3所示，所述葡萄糖脱氢酶的形式可以是酶粉、酶液、或固定化酶。
在本发明的实施方案中，所述NADP+是指烟酰胺腺嘌呤二核苷酸磷酸，即还原型辅酶II(NADPH)的氧化形式；在所述混合溶液中NADP+的浓度为 0.1-0.5g/L。
在本发明的实施方案中，化合物I在所述混合溶液中的浓度为20g/L-150g/L。
在本发明的实施方案中，在所述混合溶液中化合物I与葡萄糖的摩尔比为1:(1.2-4)。
在本发明的一些实施方案中，优选地，所述反应的温度维持在35℃。
在本发明的一些实施方案中，所述萃取溶剂萃取两次；所述萃取溶剂为无水乙醇或乙酸乙酯。
对上述内容的技术方案进行概括，具体为：
基于所述的反应体系制备奥利司他中间体的方法，将所述反应体系在20-40℃条件下搅拌反应，得所述奥利司他中间体的反应液，所述奥利司他中间体的中间体为(R)-β-羟基十四烷酸酯，结构式如II所示。
进一步，所述的方法中，用NaOH水溶液作为pH值调节剂。
进一步，所述的方法中，所述反应时间不超过15小时。
进一步，所述的方法中，在所得反应液中用溶剂萃取所述奥利司他中间体。
进一步，所述的方法中，所述溶剂为无水乙醇或乙酸乙酯。
进一步，所述的方法中，所得萃取物进行减压浓缩，并降温结晶，白色结晶固体即化合物II。
所述的奥利司他中间体制备的奥利司他。
本发明的有益效果在于：
本发明中使用的所述生物酶可以耐受高达150g/L的底物浓度，酶活性不受底物或产物的抑制作用。
本发明制备奥利司他中间体方法为生物酶法，该方法条件温和、设备要求简单，且操作简单，且该方法产生的三废量较少，没有重金属污染，对环境友好，有利于工业化生产。
本发明中的酶催化工艺转化率高达99％以上，手性ee值可达到99％以上，15个小时以内可以反应完全，产物浓度高，而且底物近乎完全转化可以简化对 反应液后处理的步骤，只需要通过简单的萃取结晶步骤即可得到高纯度的产品，大幅降低了生产成本。
附图说明
图1为实施例5中测得转化率的HPLC图谱。
图2为实施例5中产品纯度HPLC图谱。
图3为实施例5中产品手性纯度HPLC图谱。
图4为融合酶G3790表达质粒的质粒图谱。
图5为融合酶3790G表达质粒的质粒图谱。
图6为G3790融合酶蛋白条带。
图7为3790G融合酶蛋白条带。
具体实施方式
所举实施例是为了更好地对本发明进行说明，但并不是本发明的内容仅局限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整，仍属于本发明的保护范围。
本发明涉及β-羰基十四烷酸酯在生物酶的催化下还原成(R)-β-羟基十四烷酸酯，反应式如下：
Figure PCTCN2021070495-appb-000005
β-羰基十四烷酸酯作为底物在含有NADP +辅酶、葡萄糖及葡萄糖脱氢酶的环境中，在某种酶的催化作用下，得到手性纯度极高的(R)-β-羟基十四烷酸酯。其中催化酶一般为短链脱氢酶家族，如本发明实施例中不同种类的酮还原酶；还可以是不同酶的融合酶，比如本发明某些实施例例中还原酶和葡萄糖脱氢酶融合的融合酶。其中，葡萄糖在葡萄糖脱氢酶的作用下脱氢得H +，然后NADP + 辅酶携带H +，参与β-羰基十四烷酸酯的还原反应得到β-羟基十四烷酸酯。
本发明的酮还原酶JR3789来源于Singulisphaera acidiphila，NCBI数据库中的编码为WP_015245403.1，属于短链脱氢酶家族，该酮还原酶的氨基酸序列如SEQ ID NO:1所示，大小为249个氨基酸。
氨基酸序列SEQ ID NO:1:
Figure PCTCN2021070495-appb-000006
本发明的酮还原酶JR37150来源于Sphingomonas echinoides，NCBI数据库中的编码为WP_010403640.1，属于短链脱氢酶家族，该酮还原酶的氨基酸序列如SEQ ID NO:2所示，大小为259个氨基酸。
氨基酸序列SEQ ID.NO:2:
Figure PCTCN2021070495-appb-000007
本发明的葡萄糖脱氢酶GDH来源于Bacillus subtilis QB928，NCBI数据库中的编码为AFQ56330.1，属于短链脱氢酶家族，该脱氢酶的氨基酸序列如SEQ ID NO:3所示，大小为263个氨基酸。
氨基酸序列SEQ ID NO:3:
Figure PCTCN2021070495-appb-000008
本发明的酮还原酶JR3790来源于Rhodotorula toruloides，为NCBI数据库中的编码EGU12837.1蛋白的截短蛋白(70-317位)，属于短链脱氢酶家族， 该酮还原酶的氨基酸序列如SEQ ID NO:4所示，大小为248个氨基酸。
氨基酸序列SEQ ID NO:4：
Figure PCTCN2021070495-appb-000009
linker序列SEQ ID NO：5：
Figure PCTCN2021070495-appb-000010
HPLC检测条件：OD-H色谱柱；流动相为正己烷∶异丙醇＝98∶2；流速1.0mL/min。检测仪器规格为：DAD1C,Sig＝210,4Ref＝360,100。
所述生物酶(SEQ ID NO:1、SEQ ID NO:2)以及葡萄糖脱氢酶(SEQ ID NO:3)均由南京金斯瑞生物科技有限公司合成，且商品化。
实施例1
称取β-羰基十四烷酸甲酯1g、葡萄糖1.5g置于100mL的三口烧瓶中，再向其中加入50mL的浓度50mM的pH值为7.0的PBS缓冲液；将三口烧瓶放入恒温水浴锅，搅拌转速调至800rpm，温度35℃，然后分别加入10mg NADP +、25mg葡萄糖脱氢酶GDH酶粉(SEQ ID NO:3所示)，以及100mg酮还原酶JR3789酶粉(SEQ ID NO:1所示)，得到混合反应液，用2M浓度的NaOH溶液调节pH维持在7.0-7.5之间，温度维持在35℃，采用HPLC监测反应进程，9h反应结束，并测得转化率＞99％。
反应结束后先升温到60℃保温15min，然后降温到20-25℃，加入80mL乙酸乙酯萃取搅拌20min，过滤，滤液分层取有机相。水相再用50mL乙酸乙酯萃取一次，分层，取有机相。合并有机相，减压浓缩后，缓慢降温，析出产物，收得粗产品，纯度98.10％，光学纯度98.45％。粗产品加入2倍体积正己烷加热溶解，冷却结晶，收集晶体，室温晾干，收得白色结晶产品0.89g，测得纯度99.99％，ee值99.91％，总收率89％。
实施例2
称取β-羰基十四烷酸乙酯1g、葡萄糖1.5g置于100mL的三口烧瓶中，再向其中加入50mL的浓度50mM的pH值为7.0的PBS缓冲液；将三口烧瓶放入恒温水浴锅，搅拌转速调至900rpm，温度35℃，然后分别加入10mg NADP+、35mg葡萄糖脱氢酶GDH酶粉(SEQ ID NO:3所示)，以及150mg酮还原酶JR3789酶粉(SEQ ID NO:1所示)，得到混合反应液，用2M浓度的NaOH溶液调节pH维持在7.0-7.5之间，温度维持在35℃，采用HPLC监测反应进程，10h反应结束，并测得转化率＞99％。
反应结束后先升温到60℃保温15min，然后降温到20-25℃，加入80mL乙酸乙酯萃取搅拌20min，过滤，滤液分层取有机相。水相再用50mL乙酸乙酯萃取一次，分层，取有机相。合并有机相，减压浓缩后，缓慢降温，析出产物，收得白色产品0.84g，纯度98.42％，ee值98.15％，总收率84％。
实施例3
称取β-羰基十四烷酸甲酯5g、葡萄糖7.5g置于100mL的三口烧瓶中，再向其中加入50mL的浓度50mM的pH值为7.0的PBS缓冲液；将三口烧瓶放入恒温水浴锅，搅拌转速调至800rpm，温度35℃，然后分别加入50mg NADP+、125mg葡萄糖脱氢酶GDH酶粉(SEQ ID NO:3所示)，以及500mg酮还原酶JR3789酶粉(SEQ ID NO:1所示)，得到混合反应液，用2M浓度的NaOH溶液调节pH维持在7.0-7.5之间，温度维持在35℃，采用HPLC监测反应进程，13h反应结束，并测得转化率＞99％。
反应结束后先升温到60℃保温15min，然后降温到20-25℃，加入80mL乙酸乙酯萃取搅拌20min，过滤，滤液分层取有机相。水相再用50mL乙酸乙酯萃取一次，分层，取有机相。合并有机相，减压浓缩后，缓慢降温，析出产物，收得白色产品4.65g，纯度98.12％，ee值98.45％，总收率93％。
实施例4
称取β-羰基十四烷酸甲酯5g、葡萄糖7.5g置于100mL的三口烧瓶中，再向其中加入50mL的浓度50mM的pH值为7.0的PBS缓冲液；将三口烧瓶放入恒 温水浴锅，搅拌转速调至800rpm，温度35℃，然后分别加入50mg NADP+、300mg葡萄糖脱氢酶GDH酶粉(SEQ ID NO:3所示)，以及800mg酮还原酶JR37150酶粉(SEQ ID NO:2所示)，得到混合反应液，用2M浓度的NaOH溶液调节pH维持在7.0-7.5之间，温度维持在35℃，采用HPLC监测反应进程，35h反应结束，并测得转化率＞99％。
反应结束后先升温到60℃保温15min，然后降温到20-25℃，加入100mL乙酸乙酯萃取搅拌20min，过滤，滤液分层取有机相。水相再用60mL乙酸乙酯萃取一次，分层，取有机相。合并有机相，减压浓缩后，缓慢降温，析出产物，收得白色产品4.55g，纯度98.62％，光学纯度99.66％，总收率91％。
实施例5
称取β-羰基十四烷酸甲酯7.5g、葡萄糖11.25g置于100mL的三口烧瓶中，再向其中加入50mL的浓度50mM的pH值为7.0的PBS缓冲液；将三口烧瓶放入恒温水浴锅，搅拌转速调至800rpm，温度35℃，然后分别加入150mg NADP+、200mg葡萄糖脱氢酶GDH酶粉(SEQ ID NO:3所示)，以及750mg酮还原酶JR3789酶粉(SEQ ID NO:1所示)，得到混合反应液，用2M浓度的NaOH溶液调节pH维持在7.0-7.5之间，温度维持在35℃，采用HPLC监测反应进程，15h反应结束，并测得转化率＞99％。检测结果如下表1，HPLC图谱如图1。
反应结束后先升温到60℃保温15min，然后降温到20-25℃，加入100mL乙酸乙酯萃取搅拌20min，过滤，滤液分层取有机相。水相再用80mL乙酸乙酯萃取一次，分层，取有机相。合并有机相，减压浓缩后，缓慢降温，析出产物，收得粗产品，纯度99.60％，ee值98.68％。粗产品加入2倍体积正己烷加热溶解，冷却结晶，过滤，室温晾干，收得白色晶体产品6.53g，测得纯度99.99％，ee值99.86％，总收率87％。检测到的产品的纯度数据如表2，HPLC图谱如图2，产品的手性纯度数据如表3，HPLC图谱如图3。
表1测得的转化率的数据表
RT(min) Type Width(min) Area Height Area％
12.431 BB 1.86 3001.36 142.52 99.06
15.684 BBA 0.79 28.40 1.48 0.94
    Sum 3029.76    
表2测得的产品纯度的数据表
RT(min) Type Width(min) Area Height Area％
12.410 BB 1.36 3062.49 145.21 100.00
    Sum 3062.49    
表3测得的产品手性纯度数据表
RT(min) Type Width(min) Area Height Area％
6.189 BBA 0.52 2944.36 298.07 99.93
7.639 BB 0.30 2.00 0.23 0.07
    Sum 2946.336    
实施例6
(1)构建融合酶G3790表达质粒
在葡萄糖脱氢酶GDH(SEQ ID NO:3)基因片段的3’端插入linker(SEQ ID NO：5)的基因序列，后面再接上酮还原酶JR3790(SEQ ID NO:4)的基因片段，构成融合酶G3790序列(SEQ ID NO：6)(由南京金斯瑞生物科技有限公司合成)，在通过两端酶切位点NdeI和XhoI与载体pET28a连接，构成双酶融合表达质粒pET28a-G3790，所述重组质粒的质粒图谱如图4所示。
(2)构建融合酶3790G表达质粒
在酮还原酶JR3790(SEQ ID NO:4)的基因片段的3’端插入linker(SEQ ID NO：5)的基因序列，后面再接上葡萄糖脱氢酶GDH(SEQ ID NO:3)基因片段，构成融合酶序列(SEQ ID NO：7)(由南京金斯瑞生物科技有限公司合成)，在通过两端酶切位点NdeI和XhoI与载体pET28a连接，构成双酶融合表达质粒pET28a-3790G，所述重组质粒的质粒图谱如图5所示。
(3)制备融合酶G3790和融合酶3790G
将构建的融合酶表达质粒pET28a-G3790和pET28a-3790G转入大肠杆菌感受态E.coli BL21(DE3)菌株，筛选得到阳性克隆转化子，挑选含有重组质粒的单克隆接种到含有5ml的LB培养基(100μg/ml卡那霉素)试管中，37℃，200rpm 过夜培养后，将菌液以2％的接种量转接到含有1L的LB培养基的三角摇瓶中，37℃，200rpm培至OD600达到0.6左右，加入异丙基-β-D-硫代半乳糖苷(IPTG)诱导剂(终浓度0.3mM)，在25℃下继续培养12h，离心收集菌体。将菌体用PBS(pH＝7.0)稀释后重悬并进行超声破碎，离心得到上清液，即粗酶液。
将表达获得的粗酶液分别进行SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)蛋白条带鉴定。图6所示为G3790融合酶的蛋白电泳图，其中泳道M为蛋白Marker(GenScript)，泳道1为全细胞破碎后总蛋白，泳道2位全细胞破碎离心的上清液，其中融合酶的理论分子量应为67kDa，其氨基酸序列如SEQ ID NO：8所示。图7所示为3790G融合酶的蛋白电泳图，其中泳道M为蛋白Marker(GenScript)，泳道1为全细胞破碎后总蛋白，泳道2位全细胞破碎离心的上清液。其中两个融合酶的理论分子量均为67kDa，其氨基酸序列如SEQ ID NO：9所示。所述两个融合酶均为可溶蛋白，且分子量与相应的理论分子量接近。
将粗酶液分别预冻过夜，冷冻干燥24h-36h，即得到融合酶G3790酶粉和融合酶3790G酶粉。
(4)制备奥利司他中间体
称取β-羰基十四烷酸甲酯7.5kg、葡萄糖9.375kg置于100L的玻璃反应釜中，再向其中加入25L的浓度50mM的pH值为7.0的PBS缓冲液；高低温循环一体机控制反应釜温度维持35℃，机械搅拌转速调至180rpm，然后分别加入18g NADP+、800g的3790G酶粉，得到混合反应液，用2M浓度的NaOH溶液调节pH维持在7.0-7.5之间，采用HPLC监测反应进程，13h反应结束，并测得转化率＞99％。
反应结束后先升温到60℃保温30min，然后降温到20-25℃，抽滤收集滤饼，将滤饼加入15L乙酸乙酯萃取搅拌20min，抽滤收集有机相。滤饼再用2L乙酸乙酯萃取洗一次，取有机相。合并有机相，减压浓缩后，缓慢降温，析出产物，收得粗产品，纯度98.5％，ee值98.6％。粗产品加入2倍体积正己烷加热溶解，冷却结晶，过滤，室温晾干，收得白色晶体产品6.45kg，测得纯度99.70％，ee值99.88％，总收率86％。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。

Claims (28)

  1. 一种生物酶在制备奥利司他中间体中的用途，其特征在于，所述生物酶作用的底物为β-羰基十四烷酸酯，结构式如I所示,所述奥利司他中间体为(R)-β-羟基十四烷酸酯，结构式如II所示,结构式I中的R指含有1-3个碳原子的饱和烷基，结构式II中R指含有1-3个碳原子的饱和烷基；
    Figure PCTCN2021070495-appb-100001
    所述生物酶为酮还原酶或酮还原酶与葡萄糖脱氢酶的融合酶，所述酮还原酶的氨基酸序列如SEQ ID NO:1和/或如SEQ ID N0:2所示，和/或SEQ ID NO:4所示及其同一性/同源性≥80％的氨基酸序列；所述酮还原酶与葡萄糖脱氢酶的融合酶的氨基酸序列如SEQ ID NO:8和/或如SEQ ID N0:9所示。
  2. 根据权利要求1所述的用途，其特征在于，所述生物酶为酮还原酶与葡萄糖脱氢酶的融合酶，并且所述融合酶中酮还原酶与葡萄糖脱氢酶之间通过linker进行连接；任选地，所述的linker的氨基酸序列如SEQ ID NO:5所示。
  3. 根据权利要求2所述的用途，其特征在于，所述生物酶为酮还原酶与葡萄糖脱氢酶的融合酶，为酮还原酶-linker-葡萄糖脱氢酶，或葡萄糖脱氢酶-linker-酮还原酶。
  4. 根据权利要求1-3中任一项所述的用途，其特征在于，所述葡萄糖脱氢酶的氨基酸序列如SEQ ID NO:3所示。
  5. 根据权利要求1-3中任一项所述的用途，其特征在于，所述生物酶为酶粉和/或酶液和/或固定化酶。
  6. 根据权利要求1-3中任一项所述的用途，其特征在于，结构式I中的R为甲基、乙基、正丙基或异丙基中任一种，结构式II中的R为甲基、乙基、正丙基或异丙基中任一种。
  7. 一种融合酶，其特征在于，所述融合酶为酮还原酶与葡萄糖脱氢酶的融合酶；所述融合酶的氨基酸序列如SEQ ID NO:8和/或如SEQ ID N0:9所示。
  8. 根据权利要求7所述的融合酶，其特征在于，所述融合酶中酮还原酶与葡萄糖脱氢酶之间通过linker进行连接；任选地，所述的linker的氨基酸序列如SEQ ID NO:5所示。
  9. 根据权利要求7或8所述的融合酶，其特征在于，所述融合酶为酮还原酶-linker-葡萄糖脱氢酶，或葡萄糖脱氢酶-linker-酮还原酶；
    任选地，所述酮还原酶的氨基酸序列如SEQ ID NO:1和/或如SEQ ID N0:2所示，和/或SEQ ID NO:4所示及其同一性/同源性≥80％的氨基酸序列；
    任选地，所述葡萄糖脱氢酶的氨基酸序列如SEQ ID NO:3所示。
  10. 一种用于编码权利要求7-9中任一项所述融合酶的核苷酸序列，其特征在于，所述融合酶的核苷酸序列如SEQ ID NO：6或SEQ ID NO：7所示。
  11. 根据权利要求10所述的核苷酸序列的构建方法，包括：在SEQ ID NO:3所示的葡萄糖脱氢酶的基因片段的3’端插入SEQ ID NO：5所示的linker序列，后面再接上SEQ ID NO:4所示的酮还原酶的基因片段，构成如SEQ ID NO：6所示的核苷酸序列重组质粒。
  12. 根据权利要求10所述的核苷酸序列的构建方法，包括：在SEQ ID NO:4所示的酮还原酶的基因片段的3’端插入SEQ ID NO：5所示的linker序列，后面再接上SEQ ID NO:3所示的葡萄糖脱氢酶的基因片段，构成如SEQ ID NO：7所示的核苷酸序列。
  13. 一种含有生物酶和底物的组合物，其特征在于，所述底物为所述β-羰基十四烷酸酯，其结构式如式I所示；所述生物酶为酮还原酶或酮还原酶与葡萄糖脱氢酶的融合酶，所述酮还原酶的氨基酸序列如SEQ ID NO:1和/或如SEQ ID N0:2所示，和/或SEQ ID NO:4所示及其同一性/同源性≥80％的氨基酸序列；所述酮还原酶与葡萄糖脱氢酶的融合酶的氨基酸序列如 SEQ ID NO:8和/或如SEQ ID N0:9所示；
    Figure PCTCN2021070495-appb-100002
    结构式I中的R指含有1-3个碳原子的饱和烷基。
  14. 根据权利要求13所述的组合物，其特征在于，所述生物酶与所述底物的重量比为1:1.1-150。
  15. 根据权利要求14所述的组合物，其特征在于，所述生物酶与所述底物的重量比为1:20、1：30、1：40、1：50、1：60、1：70、1：80、1：90、1：100、1：110、1：120、1：130、1：140和/或1：150。
  16. 一种反应体系，其特征在于，所述反应体系由I所示的底物、生物酶、葡萄糖，葡萄糖脱氢酶、NADP +和缓冲液组成；这里所述的生物酶为所述生物酶为酮还原酶，所述酮还原酶的氨基酸序列如SEQ ID NO:1和/或如SEQ ID N0:2所示，和/或SEQ ID NO:4所示及其同一性/同源性≥80％的氨基酸序列；或者，所述反应体系由I所示的底物、生物酶、葡萄糖NADP +和缓冲液组成；这里，所述生物酶为酮还原酶与葡萄糖脱氢酶的融合酶，所述酮还原酶的氨基酸序列如SEQ ID NO:1和/或如SEQ ID N0:2所示，和/或SEQ ID NO:4所示及其同一性/同源性≥80％的氨基酸序列；所述酮还原酶与葡萄糖脱氢酶的融合酶的氨基酸序列如SEQ ID NO:8和/或如SEQ ID N0:9所示；
    Figure PCTCN2021070495-appb-100003
    结构式I中的R指含有1-3个碳原子的饱和烷基。
  17. 根据权利要求16所述的反应体系，其特征在于，所述反应体系的pH值为6.0-8.0；任选地，所述缓冲溶液为PBS缓冲液或Tris-HCl缓冲液。
  18. 根据权利要求16所述的反应体系，其特征在于，所述底物的浓度 为20g/L-150g/L。
  19. 根据权利要求16所述的反应体系，其特征在于，所述NADP +的浓度为0.1-0.5g/L。
  20. 根据权利要求16所述的反应体系，其特征在于，化合物I与所述葡萄糖的摩尔比为1:1.2-4。
  21. 根据权利要求16所述的反应体系，其特征在于，所述缓冲液的浓度为0.01-0.5mol/L。
  22. 根据权利要求16所述的反应体系，其特征在于，所述反应体系的反应时间不超过15小时，得反应液。
  23. 基于权利要求16至22中任一项所述的反应体系制备奥利司他中间体的方法，其特征在于，将所述反应体系在20-40℃条件下搅拌反应，得所述奥利司他中间体的反应液，所述奥利司他中间体的中间体为(R)-β-羟基十四烷酸酯，结构式如II所示；
    Figure PCTCN2021070495-appb-100004
    结构式II中的R指含有1-3个碳原子的饱和烷基。
  24. 根据权利要求23所述的方法，其特征在于，用NaOH水溶液作为pH值调节剂。
  25. 根据权利要求23所述的方法，其特征在于，所述反应时间不超过15小时。
  26. 根据权利要求23所述的方法，其特征在于，在所得反应液中用溶剂萃取所述奥利司他中间体。
  27. 根据权利要求23所述的方法，其特征在于，所述溶剂为无水乙醇或乙酸乙酯。
  28. 根据权利要求27所述的方法，所得萃取物进行减压浓缩，并降温结晶，白色结晶固体即化合物II。
PCT/CN2021/070495 2020-01-07 2021-01-06 一种生物酶用于制备奥利司他中间体的用途及制备方法 WO2021139689A1 (zh)

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