WO2019114157A1 - 一种酶法转化生产9α-羟基雄甾-4-烯-3,17-二酮的方法 - Google Patents

一种酶法转化生产9α-羟基雄甾-4-烯-3,17-二酮的方法 Download PDF

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WO2019114157A1
WO2019114157A1 PCT/CN2018/081871 CN2018081871W WO2019114157A1 WO 2019114157 A1 WO2019114157 A1 WO 2019114157A1 CN 2018081871 W CN2018081871 W CN 2018081871W WO 2019114157 A1 WO2019114157 A1 WO 2019114157A1
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hydroxylase
subunit
kshc
kshb
pet
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饶志明
沙宗焱
张显
杨套伟
徐美娟
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江南大学
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    • 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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
    • 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
    • C12P33/06—Hydroxylating
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
    • C12Y114/13142—3-Ketosteroid 9alpha-monooxygenase (1.14.13.142)

Definitions

  • the invention relates to a method for enzymatically converting 9 ⁇ -hydroxyandrost-4-ene-3,17-dione, belonging to the fields of genetic engineering and enzyme engineering.
  • Steroidal compounds are an important class of natural organic compounds widely found in living tissues. Because steroidal drugs have a variety of physiological functions and exert unique therapeutic effects, they are widely used in clinical practice and are second only to antibiotics. The second largest category of drugs, with an annual growth rate of more than 15%. Terpenoids usually have a unique set of physiological functions, mainly due to differences in substituents, double bond positions or stereoconfigurations on the parent nucleus of the steroid. Terpenoids have similar structures and are complex in structure. They are ubiquitous in animal and plant tissues and certain microorganisms. Commonly used are cholesterol, bile acids, sex hormones, adrenocortical hormones, progesterone and androsterone in animal tissues. Potato pre-saponin and sitosterol in plants, and ergosterol in yeast cells.
  • 9 ⁇ -hydroxyandrost-4-ene-3,17-dione (9 ⁇ -OH-AD) is chemically structurally present in the presence of a 9-hydroxyl group, which can form a C9,11-double bond system by means of conventional steroid chemical synthesis. It is convenient to introduce a halogen atom at the C9 position to form a functional hydroxyl group which is indispensable for glucocorticoids.
  • 9 ⁇ -OH-AD is an important steroidal drug intermediate, and its 9 ⁇ position is used as a hydroxylated site.
  • a halogen substituent such as F or Cl
  • corticosteroids such as dexamethasone, betamethasone, mometasone furoate and beclomethasone
  • the realization of the process can fundamentally solve the low conversion rate of C11 ⁇ -hydroxylation present in industrial production.
  • the problem of many products has extremely high commercial value.
  • 3-ketosteroid-9-alpha-hydroxylase is a key enzyme in the metabolism of steroidal microorganisms, which is widely found in microorganisms, such as Rhodococcus, Nocardia. Nocardia, Arthrobacter, Mycobacteriu. 3-indolone
  • the 9 ⁇ -hydroxylase system is a two-component enzyme consisting of KshA (3-indolone 9 ⁇ -hydroxylase oxidase) and KshB (3-indolone 9 ⁇ -hydroxylase reductase).
  • KshB is a reducing component of the 3-indolone 9 ⁇ -hydroxylase system, which is responsible for the transfer of the reducing power from NADH to KshA, returning it from the oxidized state back to the reduced state and continuously producing hydroxylation at the corresponding position in the steroid
  • KshB is a multifunctional enzyme that not only functions in the 3-indolone 9 ⁇ -hydroxylase system, but also plays an important role in other parts of the organism that require reducing power.
  • FDH Formate dehydrogenase catalyzes the decomposition of formic acid to form CO 2 , while reducing NAD + to form NADH, which can be combined with the 3-indolone 9 ⁇ -hydroxylase system.
  • FDH is the best NADH regenerative enzyme, which has unique advantages in the synthesis of chiral compounds: (1) FDH-catalyzed reaction is irreversible, the recovery rate of main reaction product can reach more than 99%; (2) substrate The formate ion is relatively cheap, the product CO 2 easily escapes from the system, simplifying the separation and purification step; (3) the broad tolerance of FDH, the reaction produces only one by-product -CO 2 , and CO 2 to any enzyme The activity has no effect and the reaction is complete, and CO 2 as a gas easily escapes from the reaction system.
  • the present invention successfully establishes a method for converting androsten-4-ene-3,17-dione (AD) into 9-OH-AD by using a crude enzyme solution based on different catalytic functions of KSH and FDH.
  • the coupling of the hydroxylation system and the coenzyme regeneration system was used.
  • a first object of the present invention is to provide a composition for converting androst-4-ene-3,17-dione to form 9 ⁇ -hydroxyandrost-4-ene-3,17-dione, said composition
  • the 3-anthrone 9 ⁇ -hydroxylase mixed enzyme solution consisting of a 3-anthracene 9 ⁇ -hydroxylase reducing subunit KshB and a 3-indolone 9 ⁇ -hydroxylase oxidizing subunit KshC is included.
  • the composition further comprises coenzyme NAD + , NADH, formate dehydrogenase or any combination of the three.
  • amino acid sequence of the 3-indolone 9 ⁇ -hydroxylase reducing subunit KshB is SEQ ID NO.
  • amino acid sequence of the 3-indolone 9 ⁇ -hydroxylase oxidative subunit KshC is SEQ ID NO.
  • the composition is a 3-fluorenone 9 ⁇ consisting of a 3-indolone 9 ⁇ -hydroxylase reducing subunit KshB and a 3-indolone 9 ⁇ -hydroxylase oxidizing subunit KshC.
  • - Hydroxylase mixed enzyme solution, NADH and formate dehydrogenase is a 3-fluorenone 9 ⁇ consisting of a 3-indolone 9 ⁇ -hydroxylase reducing subunit KshB and a 3-indolone 9 ⁇ -hydroxylase oxidizing subunit KshC.
  • the 3-anthroquinone 9 ⁇ -hydroxylase oxidative subunit KshC, 3-anthone 9 ⁇ -hydroxylase reducing subunit KshB and formate dehydrogenase are added in an enzyme-based ratio. 1 to 10: 1 to 10: 1 to 10.
  • the NADH is added in an amount of 0.01 to 1 mol/L.
  • a second object of the invention is a process for the preparation of said composition, said method being specifically:
  • step (1) and step (2) were separately transformed into E. coli to obtain recombinant Escherichia coli BL21/pET-28a(+)-kshB and BL21/pET-Duet1(+)-kshC;
  • a third object of the present invention is to provide a method for improving the efficiency of conversion of androst-4-ene-3,17-dione to 9 ⁇ -hydroxyandrost-4-ene-3,17-dione, the method Bioconversion is carried out using the above composition as a catalyst.
  • a fourth object of the invention is to provide the use of the composition in the field of medicine.
  • the invention utilizes molecular technique to clone the oxidative subunit gene kshA of 3-nonanone 9 ⁇ -hydroxylase from Mycobacterium sp. Strain VKM Ac-1817D, the reducing subunit gene kshB, and the unknown active subunit gene kshC to construct recombinant expression.
  • the vector pET-28a(+)-kshA, pET-28a(+)-kshB, pET-Duet1(+)-kshC was chemically transformed into E.coli BL21, and the genetically engineered strain BL21/pET was successfully constructed.
  • the KSH assay system included: 105 ⁇ M NADH, 200 ⁇ M substrate AD (dissolved in 100% isopropanol), 50 ⁇ M Tris-HCL (200 ⁇ L, pH 7.0), and KSH enzyme. Therefore, its activity is defined as the amount of enzyme required to oxidize the steroidal substrate AD within 1 min, expressed as the amount of enzyme required to oxidize 1 nmol of NADH in 1 min, and the specific activity unit is nmol min -1 mg -1 (U/mg).
  • FDH enzyme activity determination method prepare 17mg ⁇ mL -1 sodium formate solution (pH 7.5), 3mg ⁇ mL -1 NAD + solution, take 1.48mL sodium formate solution in quartz cuvette, add 80 ⁇ L NAD + The solution and 10 ⁇ L of the crude enzyme solution were measured for changes in absorbance per minute at 340 nm, and the enzyme activity defined by 1 min catalyzes the amount of enzyme required to convert 1 ⁇ mol of NAD + to NADH.
  • LB medium peptone 10g / L, yeast extract 5g / L, NaCl 10g / L (solid medium added 2% agar powder)
  • PCR primers P1 and P2 for the 3-anthracene 9 ⁇ -hydroxylase oxidative subunit KshA were designed according to the kshA gene sequence (SEQ ID NO.1) in the genome-wide nucleic acid sequence of Mycobacterium sp. Strain VKM Ac-1817D in NCBI (SEQ ID NO.1) ID NO. 6 and SEQ ID NO. 7).
  • PCR primers P3 and P4 for the 3-anthrone 9 ⁇ -hydroxylase reducing subunit KshB were designed according to the kshB gene sequence (SEQ ID NO. 2) in the whole genome nucleic acid sequence of Mycobacterium sp. Strain VKM Ac-1817D in NCBI (SEQ ID NO. 2) ID NO. 8 and SEQ ID NO. 9).
  • PCR primers P5 and P6 of 3-anthrone 9 ⁇ -hydroxylase KshC were designed according to the kshC gene sequence (SEQ ID NO. 3) in the whole genome nucleic acid sequence of Mycobacterium sp. Strain VKM Ac-1817D in NCBI (SEQ ID NO. 10 and SEQ ID NO. 11).
  • Example 2 3-anthrone cloning of the 9 ⁇ -hydroxylase gene kshA, kshB, kshC
  • the synthesized DNA was used as a template, and the primers provided above were used for PCR amplification.
  • the amplification conditions were: 94 °C pre-denaturation, 5 min, one cycle; 94 °C denaturation, lmin, 58 °C re-ignition, lmin, 72 °C extension, 90s , 34 cycles; end extension at 72 ° C for 10 min.
  • PCR amplification system 1 ⁇ L of template, 0.4 ⁇ L of upstream and downstream primers, 4 ⁇ L of dNTP Mix, 5 ⁇ L of 10 x Ex Taq Buffer, 37 ⁇ L of sterilized double distilled water, and 1 ⁇ L of Ex Taq DNA polymerase.
  • the PCR product was purified and recovered by a gel recovery kit, and the concentration of the recovered product was examined by electrophoresis.
  • the recovered product was stored in a 1.5 ml centrifuge tube and stored in a refrigerator at 20 ° C until use.
  • the recovered product was ligated to pMD18-T Vector, the ligation product was transformed into E.coil JM109, and the transformed product was coated with ampicillin-resistant LB plate, cultured at 37 ° C overnight, and the colony was picked up into 10 ml of liquid LB medium, and shaken at 37 ° C. After overnight culture, the plasmid was extracted and named as pMD18 T-kshA, pMD18 T-kshB, pMD18 T-kshC. After successful ligation, glycerol was added to a final concentration of 15% to 20% (w/v), -70 °C. Refrigerated storage.
  • Example 3 Construction of recombinant expression vector pET-28a(+)-kshA, pET-28a(+)-kshB, pET-Duet1(+)-kshC
  • the plasmids pMD18 T-kshA, pMD18 T-kshB, pMD18 T-kshC and pET-28a(+), pET-Duet1(+) stored in E. coli jM109 were extracted and double digested with BamH I/Hind III, respectively.
  • the ligation system 7 ⁇ L of the target gene digestion product, 1 ⁇ L of pET-28a (+) or pET-Duet1 (+) digestion product, T4 DNA ligase buffer 111L, T4 DNA ligase 1 ⁇ L , overnight at 16 ° C.
  • the ligated recombinant plasmid pET-28a(+)-kshA, pET-28a(+)-kshB, pET-Duet1(+)-kshC was transformed into competent E.coil jM109 with LB kanamycin resistance and Ampicillin resistant medium, picking positive colonies. After overnight culture at 37 °C shaker, the plasmid was extracted and named pET-28a(+)-kshA, pET-28a(+)-kshB, pET-Duet1(+)-kshC. After correct digestion, glycerol was added to the final concentration. 15%-20% (w/v), -70 °C refrigerator preservation reserve.
  • Example 4 Recombinant plasmid pET-28a(+)-kshA, pET-28a(+)-kshB, pET-Duet1(+)-kshC transformed E.coli BL21
  • E.coli BL21 Competent Preparation (1) Pick a newly activated single colony from the LB plate, inoculate it in 10 mL of LB liquid medium for about 12 h, and inoculate the bacterial suspension with 1% inoculum. 50 mL of LB liquid medium was cultured for 2-3 h until the OD 600 reached about 0.5. (2) The bacterial liquid was placed in an ice water bath for 15 minutes to rapidly cool the bacterial liquid. (3) Dispensing the bacterial solution in several 1.5 mL centrifuge tubes, centrifuging at 8000 rpm for 1 min, then discarding the supernatant for use. The cells were washed 2-3 times with a .1M CaCl 2 solution. (4) The washed bacterial solution was suspended in 80 ⁇ L of CaCl 2 and 40 ⁇ L of 50% glycerol, and stored at -40 ° C for a long period of time.
  • the recombinant strain BL21/pET-28a(+)-kshA, BL21/pET-28a(+)-kshB constructed in Example 4 and the starting strain BL21/pET-28a(+) were inoculated separately into 10 ml of kanamycin-containing In LB medium, BL21/pET-Duet1(+)-kshC was inoculated in 10 ml of ampicillin-containing LB medium and shaken overnight at 37 ° C. The next day, 1% of the inoculum was transferred to 50 ml of LB medium at 37 ° C. After 2 hours of culture, when the OD 600 was 0.8, IPTG was added to a final concentration of 0.8 mM.
  • the KSH assay system was 105 ⁇ M NADH, 200 ⁇ M substrate AD (dissolved in 100% isopropanol), 50 ⁇ M Tris-HCL (200 ⁇ L, pH 7.0), and KSH enzyme (including oxidized subunits and reducing subunits). It was verified that KshC is an oxidized subunit, and the enzyme activity of the same amount of KshB crude enzyme solution + KshC crude enzyme solution is 30.7 U/mL, which is much higher than KshA and the same concentration of the induced culture cells.
  • the KSH enzyme activity of KshB crude enzyme solution was 6.1 U/mL, the former was 5.4 times that of the latter, while the original strain E. coli BL21/pET-28a(+) did not detect the enzyme activity of KSH, thus achieving The activity of 3-nonanone 9 ⁇ -hydroxylase enzyme in E.coli BL21 is from scratch.
  • the KshA or KshC crude enzyme solution obtained in Example 5 and the KshB crude enzyme solution were added to a 10 mL conversion system in a ratio of an enzyme amount of 1:1.5: 0.1 M Tris-Hcl buffer, pH 7.0, 0.32 mMol NADH, 1g ⁇ L-1 substrate AD (dissolved in ethyl acetate) and crude enzyme mixture (KshA+KshB or KshC+KshB), pH adjusted with 20% formic acid and 50% ammonia water, speed 160r ⁇ min-1,30
  • the conversion of °C for 20h, and the need to add substrate AD and coenzyme NADH in time the results show that the yield of 9-OH-AD in KshA+KshB crude enzyme solution conversion system is 0.98g / L, and KshC + KshB crude enzyme solution conversion system The 9-OH-AD yield was 7.8 g/L. It is thus seen that the oxidized subunit KshC of 3-nonan
  • Example 7 Determination of E. coli BL21/pET-28a(+)-fdh formate dehydrogenase activity
  • the recombinant E.coli BL21/pET-28a(+)-fdh constructed in our laboratory was inoculated into 10 ml of LB medium containing kanamycin, and cultured overnight at 37 ° C with shaking, and the next day was adjusted to 1%.
  • the cells were cultured in 50 ml of LB medium and cultured at 37 ° C for 2 h until the OD 600 was 0.8. The final concentration was 0.8 mM IPTG.
  • the bacterial solution was centrifuged at 4 ° C, 10000 r / min for 10 min, (Na 2 HP0 4 -NaH 2 P0 4 ) was washed twice and finally suspended in 5 ml of pH 7.5 phosphate buffer.
  • the crude enzyme solution was prepared by ultrasonication treatment.
  • the 3-fluorenone 9 ⁇ -hydroxylase and formate dehydrogenase (FDH) crude enzyme solution obtained by ultrasonication were added to a 25 mL conversion system at a ratio of 1:0.5 enzyme activity: 0.1 M Tris-Hcl buffer Liquid, pH 7.0, containing 10 g ⁇ L -1 ammonium formate, 0.25 mmol NAD + or NADH, 10 g ⁇ L -1 substrate AD (first dissolved in ethyl acetate) and crude enzyme mixture (KSH and FDH ), the pH was adjusted with 20% formic acid and 50% ammonia water, the rotation speed was 160 r ⁇ min-1, and the transformation was carried out at 30 ° C for 20 h, and the substrate was added at the same time, and NAD+, NADH or FDH was not added as a control experiment, and the results are shown in Table 1.

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Abstract

提供了一种酶法转化生产9α-羟基雄甾-4-烯-3,17-二酮的方法，包括将来源于Mycobacterium sp.Strain VKMAc-1817D的3-甾酮9α-羟基化酶的氧化亚基KshA，还原亚基KshB，及未知活性亚基KshC在E.coli BL21中表达，并鉴定出KshC为氧化亚基，且酶活远高于KshA；利用本实验室已构建的BL21/pET-28a(+)-fdh表达甲酸脱氢酶FDH，以KSH(KshB+KshC)和FDH工程菌的粗酶液作为生物催化剂，以甾体化合物AD为底物，反应温度30℃，pH7.0。在最优条件下转化AD生成产物9-OH-AD，在20小时内，9-OH-AD的产量为4.7g/L，摩尔转化率达到96.7％。

Description

一种酶法转化生产9α-羟基雄甾-4-烯-3,17-二酮的方法 技术领域
本发明涉及一种酶法转化生产9α-羟基雄甾-4-烯-3,17-二酮的方法，属于基因工程和酶工程领域。
背景技术
甾体化合物是广泛存在于生物体组织内的一类重要的天然有机化合物，由于甾体类药物具有多种生理功能并发挥独特疗效，故在临床上有广泛的应用，是仅次于抗生素的第二大类药物，年增长率在15％以上。甾类化合物通常具有一系列独特的生理功能，这主要是由于甾体化合物母核上取代基、双键位置或立体构型的不同。甾类化合物拥有类似的结构且结构复杂数目繁多，普遍存在于动、植物组织和某些微生物中，比较常见的有动物组织中的胆固醇、胆酸、性激素、肾上腺皮质激素、孕酮和雄酮等植物中的薯预皂素和谷甾醇等以及酵母细胞中的麦角固醇等。
9α-羟基雄甾-4-烯-3,17-二酮(9α-OH-AD)化学结构上9-羟基的存在，可借助常规甾体化学合成手段形成C9，11-双键体系，从而很方便地在C9位引入一个卤素原子形成糖皮质激素必不可缺少的功能羟基。9α-OH-AD是一种重要的甾体药物中间体，其9α位作为被羟基化的位点，在进行简单的卤化反应后便可引入F或Cl等卤素取代基，从而有效提升某些皮质类激素(如地塞米松、倍他米松、糠酸莫米松及氯地米松等药物)的药效，工艺流程的实现可根本解决目前工业生产中存在的C11α-羟基化转化率低、副产物多的问题，具有极高的商业价值。
3-甾酮 9α-羟基化酶(3-ketosteroid-9-alpha-hydroxylase(KSH))是甾体微生物代谢的一个关键酶，该酶在微生物中广泛存在，例如红球菌属Rhodococcus，诺卡氏菌属Nocardia，节杆菌属Arthrobacter，分枝杆菌属Mycobacteriu。3-甾酮 9α-羟基化酶系统是一个双组分酶，由KshA(3-甾酮 9α-羟基化酶氧化酶)和KshB(3-甾酮 9α-羟基化酶还原酶)组成，由基因kshA和kshB分别编码KshA和KshB，这一点最早在Rhodococcus erythropolis SQ1中获得证实，且对Rhodococcus erythropolis SQ1所进行的基因敲除研究表明，kshA和kshB都是KSH表达所必需的组分。KshB是3-甾酮 9α-羟基化酶系统的还原组件，它负责将来自NADH的还原力传递给KshA，使其从氧化态重新回到还原态并不断地在甾体的相应位置产生羟基化反应，另外，KshB是一个多功能酶，它不仅仅在3-甾酮 9α-羟基化酶系统中发挥作用，在生物体内其他需要还原力的地方也发挥着重要的作用。
甲酸脱氢酶(FDH)能催化甲酸分解生成CO 2，同时将NAD +还原生成NADH，可与3-甾酮9α-羟基化酶系统结合。大量研究表明，FDH是最好的NADH再生酶，在手性化合物的合成中具有独特的优势：(1)FDH催化的反应不可逆，主反应产物回收率可达99％以上；(2)底物甲酸根离子较廉价，产物CO 2易从体系中逃逸，简化分离纯化步骤；(3)FDH在较宽的耐受性，此反应只产生一种副产物-CO 2，而CO 2对任何酶的活性均没有任何影响，且反应完全，同时CO 2作为一种气体，很容易从反应体系中逃逸出来。
发明内容
为解决上述问题，本发明基于KSH和FDH的不同催化功能，成功建立了用粗酶液将雄甾-4-烯-3,17-二酮(AD)转化为9-OH-AD的方法，实现了羟化体系和辅酶再生体系的偶联。
本发明的第一个目的是提供一种转化雄甾-4-烯-3,17-二酮生成9α-羟基雄甾-4-烯-3,17-二酮的组合物，所述组合物包括由3-甾酮 9α-羟基化酶还原亚基KshB和3-甾酮 9α-羟基化酶氧化亚基KshC组成的3-甾酮 9α-羟基化酶混合酶液。
在本发明的一种实施方式中，所述组合物还包括辅酶NAD +、NADH、甲酸脱氢酶或三者的任意组合。
在本发明的一种实施方式中，所述3-甾酮 9α-羟基化酶还原亚基KshB的氨基酸序列为SEQ ID NO.4。
在本发明的一种实施方式中，所述3-甾酮 9α-羟基化酶氧化亚基KshC的氨基酸序列为SEQ ID NO.5。
在本发明的一种实施方式中，所述组合物为由3-甾酮 9α-羟基化酶还原亚基KshB和3-甾酮 9α-羟基化酶氧化亚基KshC组成的3-甾酮 9α-羟基化酶混合酶液、NADH和甲酸脱氢酶。
在本发明的一种实施方式中，所述3-甾酮 9α-羟基化酶氧化亚基KshC、3-甾酮 9α-羟基化酶还原亚基KshB与甲酸脱氢酶按酶活添加比例为1～10:1～10:1～10。
在本发明的一种实施方式中，所述NADH的添加量为0.01～1mol/L。
本发明的第二个目的是所述组合物的制备方法，所述方法具体是：
(1)以SEQ ID NO.2所示核酸序列为模板，扩增得到编码3-甾酮 9α-羟基化酶还原亚基KshB的基因，将其克隆到大肠杆菌表达载体pET-28a上，获得重组质粒pET-28a-kshB；
(2)以SEQ ID NO.3所示核酸序列为模板，扩增得到编码3-甾酮 9α-羟基化酶氧化亚基KshC的基因，将其克隆到大肠杆菌表达载体pET-Duet1(+)上，获得重组质粒pET-Duet1(+)-kshC；
(3)将步骤(1)和步骤(2)的重组质粒分别转化至大肠杆菌中获得重组大肠杆菌 BL21/pET-28a(+)-kshB和BL21/pET-Duet1(+)-kshC；
(4)利用步骤(3)的重组大肠杆菌发酵获得3-甾酮 9α-羟基化酶还原亚基KshB和3-甾酮 9α-羟基化酶氧化亚基KshC组成的3-甾酮 9α-羟基化酶混合酶液。
本发明的第三个目的是提供一种提高雄甾-4-烯-3,17-二酮转化生成9α-羟基雄甾-4-烯-3,17-二酮效率的方法，所述方法是利用上述组合物作为催化剂，进行生物转化。
本发明的第四个目的是提供所述组合物在医药领域的应用。
本发明的有益效果：
本发明利用分子技术克隆了来自Mycobacterium sp.Strain VKM Ac-1817D的3-甾酮 9α-羟基化酶的氧化亚基基因kshA，还原亚基基因kshB，及未知活性亚基基因kshC，构建重组表达载体pET-28a(+)-kshA，pET-28a(+)-kshB，pET-Duet1(+)-kshC并通过化学方法将其转化至E.coli BL21中，成功构建了基因工程菌BL21/pET-28a(+)-kshA，BL21/pET-28a(+)-kshB，BL21/pET-Duet1(+)-kshC，鉴定出KshC为氧化亚基。并利用本实验室已构建的BL21/pET-28a(+)-fdh，酶活测定结果发现KSH(KshB+KshC)和FDH的酶活分别为5.66U/mL和0.25U/mL。基于获得的基因工程菌，通过细胞破碎获得每株工程菌的粗酶液，用于转化AD产生9-OH-AD进行了初步研究，在20小时内，9-OH-AD的产量为4.7g/L，摩尔转化率达到96.7％。
具体实施方式
KSH测定体系包括：105μM NADH，200μM底物AD(溶于100％异丙醇)，50m Tris-HCL(200μL，pH7.0)，以及KSH酶。因此其活性定义为，1min内氧化甾体底物AD所需要的酶量，表示为1min内氧化1nmol NADH所需要的酶量，比活力单位为nmol min -1mg -1(U/mg)。
FDH酶活测定方法：配制17mg·mL -1的甲酸钠溶液(pH7.5)，3mg·mL -1的NAD +溶液,取1.48mL的甲酸钠溶液置于石英比色皿中，依次加入80μL NAD +溶液和10μL粗酶液，在340nm下检测每分钟的吸光值变化，酶活定义单位为1min催化1μmol NAD +转化成NADH所需的酶量。
HPLC分析：AD与9-OH-AD在254nm紫外波长下均有特征吸收峰，所以采用HPLC法制定10μL，流速：1.0ml/min。
LB培养基：蛋白胨10g/L，酵母膏5g/L，NaCl 10g/L(固体培养基加入2％琼脂粉)
实施例1：3-甾酮 9α-羟基化酶引物设计
根据NCBI中Mycobacterium sp.Strain VKM Ac-1817D的全基因组核酸序列中kshA基因序列(SEQ ID NO.1)，设计3-甾酮 9α-羟基化酶氧化亚基KshA的PCR引物P1和P2(SEQ ID NO.6和SEQ ID NO.7)。
P1：5’-CG GGATCCATGACGACTGAGCACGCCGG-3’(BamH I)
P2：5’-CCC AAGCTTTCAGCTTGATTGAGCGGTTTC-3’(Hind III)
根据NCBI中Mycobacterium sp.Strain VKM Ac-1817D的全基因组核酸序列中kshB基因序列(SEQ ID NO.2)，设计3-甾酮 9α-羟基化酶还原亚基KshB的PCR引物P3和P4(SEQ ID NO.8和SEQ ID NO.9)。
P3：5’-CG GGATCCATGACTGATGAACCGTTAGGTAG-3’(BamH I)
P4：5’-CCC AAGCTTTCACTCGTCGTAGGTCACCTC-3’(Hind III)
根据NCBI中Mycobacterium sp.Strain VKM Ac-1817D的全基因组核酸序列中kshC基因序列(SEQ ID NO.3)，设计3-甾酮 9α-羟基化酶KshC的PCR引物P5和P6(SEQ ID NO.10和SEQ ID NO.11)。
P5：5’-CG GGATCCGATGGCCGGTCTGAACAACGATAG-3’(BamH I)
P6：5’-CCC AAGCTTTCAGCCGCTGGCCGGGGCGGCC-3’(Hind III)
实施例2：3-甾酮 9α-羟基化酶基因kshA，kshB，kshC的克隆
以合成的DNA为模板，利用上面提供的引物做PCR扩增，扩增条件为：94℃预变性，5min，一个循环；94℃变性，lmin，58℃返火，lmin，72℃延伸，90s，34个循环；72℃终延伸10min。PCR扩增体系：模板1μL，上下游引物各0.4μL，dNTP Mix 4μL，10 x Ex Taq Buffer 5μL，灭菌的双蒸水37μL，Ex Taq DNA聚合酶1μL。采用凝胶回收试剂盒对PCR产物进行纯化和回收，电泳检验回收产物的浓度。回收产物存放在1.5ml的离心管中，20℃冰箱保存备用。回收产物与pMD18-T Vector连接，连接产物转化E.coil JM109，转化产物涂布含氨苄抗性的LB平板，经37℃培养过夜，挑取菌落到10ml液体LB培养基中，37℃摇床过夜培养后提取质粒，命名为pMD18 T-kshA，pMD18 T-kshB，pMD18 T-kshC，经酶切验证连接成功后，加入甘油至终浓度15％～20％(w/v)，-70℃冰箱保藏。
实施例3：重组表达载体pET-28a(+)-kshA，pET-28a(+)-kshB，pET-Duet1(+)-kshC的构建
提取保存于E.coli jM109中的质粒pMD18 T-kshA，pMD18 T-kshB，pMD18 T-kshC和pET-28a(+)，pET-Duet1(+)并分别用BamH I/Hind III进行双酶切，利用凝胶回收试剂盒回收后进行连接，连接体系:目的基因酶切产物7μL，pET-28a(+)或pET-Duet1(+)酶切产物1μL，T4DNA连接酶buffer 111L，T4DNA连接酶1μL，16℃过夜连接。将连接好的重组质粒pET-28a(+)-kshA，pET-28a(+)-kshB，pET-Duet1(+)-kshC转化到感受态E.coil jM109，用LB卡那霉素抗性和氨苄抗性培养基，挑取阳性菌落。37℃摇床过夜培养后提取质粒，命名为pET-28a(+)-kshA，pET-28a(+)-kshB，pET-Duet1(+)-kshC，酶切验证正确后，加入甘油至终浓度15％-20％(w/v)，-70℃冰箱保藏备用。
实施例4：重组质粒pET-28a(+)-kshA，pET-28a(+)-kshB，pET-Duet1(+)-kshC转化E.coli BL21
E.coli BL21感受态制备：(1)从LB平板上挑取新活化的单菌落，接种于10mL的LB液体培养基中培养12h左右，再将该菌悬液以1％的接种量接种于50mL的LB液体培养基培养2-3h，直至OD 600达到0.5左右。(2)将菌液置于冰水浴中15min，使菌液迅速冷却。(3)分装菌液于若干个1.5mL离心管中，8000rpm离心1min后弃去上洁，用。.1M的CaCl 2溶液洗细胞2-3次。(4)经过清洗的菌液用80μL Ca Cl 2和40μL50％的甘油悬浮后，于-40℃长期保存。
转化：将10μL重组质粒pET-28a(+)-kshA，pET-28a(+)-kshB，pET-Duet1(+)-kshC各加入到两管E.coli BL21感受态细胞中，轻轻混匀后置于冰上45min，然后42℃水浴热激90s后再置于冰上5min，然后加入800μL LB液体培养基后置于37℃培养1h后，8000rpm离心1min弃去上洁，将剩余菌液涂布于含卡那霉素抗性和氨苄抗性的LB平板上，370℃培养后挑取阳性菌落，提取质粒酶切验证，得到重组菌BL21/pET-28a(+)-kshA，BL21/pET-28a(+)-kshB和BL21/pET-Duet1(+)-kshC。
实施例5：KshC的功能鉴定及3-甾酮 9α-羟基化酶活力测定
将实施例4构建的重组菌BL21/pET-28a(+)-kshA，BL21/pET-28a(+)-kshB与出发菌株BL21/pET-28a(+)分别接种于10ml含卡那霉素的LB培养基中，BL21/pET-Duet1(+)-kshC接种于10ml含氨苄的LB培养基中37℃振荡培养过夜，次日按1％的接种量转接于50ml LB培养基中，37℃培养2h后至OD 600为0.8时加入终浓度为0.8mM的IPTG，28℃诱导9h后将菌液于4℃，10000r/min离心10min，pH7.5的磷酸盐缓冲液(Na 2HP0 4-NaH 2P0 4)清洗2次，最后用5ml pH7.5磷酸盐缓冲液悬浮。超声波破碎处理制备粗酶液。
KSH测定体系为105μM NADH，200μM底物AD(溶于100％异丙醇)，50m Tris-HCL(200μL，pH7.0)，以及KSH酶(包括氧化亚基与还原亚基)。通过验证，KshC为氧化亚基，且在保证诱导培养细胞浓度相同的情况下，相同添加量的KshB粗酶液+KshC粗酶液一起作用的酶活为30.7U/mL，远高于KshA与KshB粗酶液共同作用的KSH酶活6.1U/mL，前者是后者的5.4倍，而原始菌E.coli BL21/pET-28a(+)并没有检测到KSH的酶活，从而实现了在E.coli BL21中3-甾酮 9α-羟基化酶酶活力的从无到有。
实施例6：利用3-甾酮 9α-羟基化酶转化AD生成9-OH-AD
将实施例5获得的KshA或KshC粗酶液和KshB粗酶液以酶量添加量为1:1.5的比例加入到10mL转化体系：0.1M Tris-Hcl缓冲液，pH7.0，0.32mMol NADH，1g·L-1底物AD(先溶于乙酸乙酯)和粗酶混合液(KshA+KshB或KshC+KshB)，用20％甲酸和50％氨水调节pH，转速160r·min-1，30℃转化20h，且需要适时补加底物AD及辅酶NADH，结果表 明，KshA+KshB粗酶液转化体系中9-OH-AD的产量为0.98g/L,而KshC+KshB粗酶液转化体系的9-OH-AD产量为7.8g/L。由此看出3-甾酮 9α-羟基化酶的氧化亚基KshC比KshA有更好的效果。
实施例7：E.coli BL21/pET-28a(+)-fdh甲酸脱氢酶活力测定
将本实验室构建的重组菌E.coli BL21/pET-28a(+)-fdh接种于10ml含卡那霉素的LB培养基中，37℃振荡培养过夜，次日按1％的接种量转接于50ml LB培养基中，37℃培养2h后至OD 600为0.8时加入终浓度为0.8mM的IPTG，28℃诱导9h后将菌液于4℃，10000r/min离心10min，(Na 2HP0 4-NaH 2P0 4)清洗2次，最后用5ml pH7.5磷酸盐缓冲液悬浮。超声波破碎处理制备粗酶液。
配制17mg/mL的甲酸纳溶液(pH7.5)，3mg/mL的NAD +溶液，取1.48mL的甲酸纳溶液置于石英比色皿中，依次加入80μL NAD +溶液和10μL粗酶液，在340nm下检测每分钟的吸光值变化，酶活定义单位为1min催化1umol NAD+转化成NADH所需的酶量。结果表明重组菌E.coli BL21/pET-28a(+)-fdh的酶活为0.25U/mL。
实施例8：利用3-甾酮 9α-羟基化酶和甲酸脱氢酶转化AD生成9-OH-AD
将通过超声破碎所获得的3-甾酮 9α-羟基化酶和甲酸脱氢酶(FDH)粗酶液按照酶活添加量为1:0.5的比例加入到25mL转化体系：0.1M Tris-Hcl缓冲液，pH7.0，包含10g·L -1甲酸铵，0.25mmol NAD +或者NADH任一种，10g·L -1底物AD(先溶于乙酸乙酯)和粗酶混合液(KSH和FDH)，用20％甲酸和50％氨水调节pH，转速160r·min-1，30℃转化20h，且需要适时补加底物，同时以不添加NAD+，NADH或FDH作为对照实验，结果如表1所示，3-甾酮 9α-羟基化酶为KshC+KshB粗酶液时，且添加FDH+NADH参与辅酶再生时，9-OH-AD的产量为9.7g/L，摩尔转化率达到97％。
综上所述，可得产量结果如下表：
表1不同酶液和辅酶添加合成9α-羟基雄甾-4-烯-3,17-二酮
Figure PCTCN2018081871-appb-000001
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2018081871-appb-000002
Figure PCTCN2018081871-appb-000003
Figure PCTCN2018081871-appb-000004
Figure PCTCN2018081871-appb-000005
Figure PCTCN2018081871-appb-000006
Figure PCTCN2018081871-appb-000007
Figure PCTCN2018081871-appb-000008
Figure PCTCN2018081871-appb-000009

Claims (10)

  1. 一种转化雄甾-4-烯-3,17-二酮生成9α-羟基雄甾-4-烯-3,17-二酮的组合物，其特征在于，所述组合物包括由3-甾酮9α-羟基化酶还原亚基KshB和3-甾酮9α-羟基化酶氧化亚基KshC组成的3-甾酮9α-羟基化酶混合酶液。
  2. 根据权利要求1所述的组合物，其特征在于，所述组合物还包括辅酶NAD +、NADH、甲酸脱氢酶或三者的任意组合。
  3. 根据权利要求1所述的组合物，其特征在于，所述3-甾酮9α-羟基化酶还原亚基KshB的氨基酸序列为SEQ ID NO.4。
  4. 根据权利要求1所述的组合物，其特征在于，所述3-甾酮9α-羟基化酶氧化亚基KshC的氨基酸序列为SEQ ID NO.5。
  5. 根据权利要求1所述的组合物，其特征在于，所述组合物为由3-甾酮9α-羟基化酶还原亚基KshB和3-甾酮9α-羟基化酶氧化亚基KshC组成的3-甾酮9α-羟基化酶混合酶液、NADH和甲酸脱氢酶组成。
  6. 根据权利要求5所述的组合物，其特征在于，所述3-甾酮9α-羟基化酶氧化亚基KshC、3-甾酮9α-羟基化酶还原亚基KshB与甲酸脱氢酶按酶活添加比例为1～10:1～10:1～10。
  7. 根据权利要求5所述的组合物，其特征在于，所述NADH的添加量为0.01～1mol/L。
  8. 权利要求1所述的组合物的制备方法，其特征在于，所述方法具体是：
    (1)以SEQ ID NO.2所示核酸序列为模板，扩增得到编码3-甾酮9α-羟基化酶还原亚基KshB的基因，将其克隆到大肠杆菌表达载体pET-28a上，获得重组质粒pET-28a-kshB；
    (2)以SEQ ID NO.3所示核酸序列为模板，扩增得到编码3-甾酮9α-羟基化酶氧化亚基KshC的基因，将其克隆到大肠杆菌表达载体pET-Duet1(+)上，获得重组质粒pET-Duet1(+)-kshC；
    (3)将步骤(1)和步骤(2)的重组质粒分别转化至大肠杆菌中获得重组大肠杆菌BL21/pET-28a(+)-kshB和BL21/pET-Duet1(+)-kshC；
    (4)利用步骤(3)的重组大肠杆菌发酵获得3-甾酮9α-羟基化酶还原亚基KshB和3-甾酮9α-羟基化酶氧化亚基KshC组成的3-甾酮9α-羟基化酶混合酶液。
  9. 一种提高雄甾-4-烯-3,17-二酮转化生成9α-羟基雄甾-4-烯-3,17-二酮效率的方法，所述方法是利用权利要求1所述的组合物作为催化剂，进行生物转化。
  10. 权利要求1所述的组合物在医药领域的应用。
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