WO2017202193A1 - 重组酮还原酶在制备(r)-3-奎宁醇中的应用 - Google Patents

重组酮还原酶在制备(r)-3-奎宁醇中的应用 Download PDF

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WO2017202193A1
WO2017202193A1 PCT/CN2017/083434 CN2017083434W WO2017202193A1 WO 2017202193 A1 WO2017202193 A1 WO 2017202193A1 CN 2017083434 W CN2017083434 W CN 2017083434W WO 2017202193 A1 WO2017202193 A1 WO 2017202193A1
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recombinant
ketoreductase
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竺伟
高新星
吴会
王波
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尚科生物医药（上海）有限公司
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  • the invention belongs to the field of biopharmaceutics, and particularly relates to the application of a recombinant ketoreductase in the preparation of (R)-3-quinuclol.
  • (R)-3-quinuclol is an important intermediate for the treatment of urinary urinary incontinence drug solinasin.
  • the listed form of Solinas is an oral tablet, the original research company Anstelai, approved by the European Medicines Agency (EMA) in June 2004, and approved by the US Food and Drug Administration (FDA) in November 2004, 2006 In April, it was approved by the Japan Pharmaceutical and Medical Device Integration Agency (PMDA), and in September 2009, it was approved by the State Food and Drug Administration (CFDA). Since its launch, the product has become a heavyweight product in the pharmaceutical industry, with annual sales of 1.33 billion, 1.22 billion, and 1.13 billion in 2013-2015.
  • EMA European Medicines Agency
  • FDA US Food and Drug Administration
  • (R)-3-quinuclol is also a key intermediate in the treatment of chronic obstructive pulmonary disease.
  • the ampicillin-listed dosage form is an oral inhalation powder.
  • the original research company is Almirall and Forest Laboratories. It was approved by the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) in July 2012, and was awarded Japan in March 2015.
  • the Pharmaceutical Medical Device Integration Agency (PMDA) approved the listing.
  • the annual sales of the product in 2013-2015 are 170 million, 200 million, and 190 million, respectively. The future market prospects should not be underestimated.
  • the preparation method of (R)-3-quinuclol has attracted extensive attention of researchers.
  • the preparation methods of (R)-3-quinuclol mainly include chemical synthesis and biosynthesis.
  • the chemical synthesis method generally uses chiral resolution or the use of an expensive metal catalyst to complete the preparation of (R)-3-quinuclol, both of which have low reaction yield, low optical purity of the product, and high production cost. Disadvantages, difficult to industrialize production.
  • biosynthesis has the advantages of mild reaction conditions, high conversion rate and strong stereoselectivity.
  • Zhu Wenming et al. (Journal of Molecular Catalysis B: Enzymatic, 2013, 88, 14-19) used traditional soil screening methods to isolate two strains of microorganisms: Nocardia sp. WY1202 and Rhodococcus erythropolis. WY1406, wherein Nocardia WY1202 can catalyze the asymmetric reduction of 3-quinuctone to form (R)-3-quinuclol.
  • the yield of the product is 93%, the product ee is >99%, but the substrate is the highest.
  • the concentration is only 99 mM, which seriously affects its industrial application.
  • Nobuya Itoh et al. (Appl. Environ. Microbiol. 2013, 79, 1378-84) screened a strain of Microbacterium luteolum JCM9174, which is capable of reducing 3-quinucdinone production (R)-3-quine Ning alcohol, and digging into two NADH-dependent reductases QNR and BacC, the conversion rate of the method is 100% and 94%, respectively, the product ee>99.9%, but the substrate concentration is low, the highest is 313mM, which is not suitable for industrialization. produce.
  • the present invention discloses the use of a recombinant ketoreductase for the preparation of (R)-3-quinuclol, thereby improving the production of (R)-3-quinolol. effectiveness.
  • the present invention discloses a use of a recombinant ketoreductase for preparing (R)-3-quinolol, wherein the amino acid sequence of the recombinant ketoreductase is SEQ ID No. 2, 4, 6, As shown in any of 8, 10 .
  • nucleotide sequence encoding the amino acid sequence of the recombinant ketoreductase is as shown in any one of SEQ ID No. 1, 3, 5, 7, and 9.
  • the concentration of the recombinant ketoreductase in the reaction mixture is 5 to 10 g/L
  • the concentration of the recombinant glucose dehydrogenase is 1-1.5 g/L
  • the concentration of 3-quinuclidinone or its hydrochloride is 100 to 500 g/
  • the concentration of L and the cofactor is 0.1 to 0.15 g/L
  • the glucose concentration is 120 to 600 g/L
  • the concentration of the buffer is 50 to 100 mM.
  • diatomaceous earth is added to the reaction liquid, stirred, filtered, and the filtrate is adjusted to pH about 13 with NaOH, extracted with n-butanol, concentrated and crystallized, and the molar yield of the target product (R)-3-quinuclidin is more than 90. %, ee value is greater than 99%.
  • the recombinant ketoreductase is a mutant of a ketoreductase derived from Lactobacillus americana, which is obtained by screening a mutant library of Lactobacillus ketol reductase in the company.
  • the wild type ketoreductase derived from Lactobacillus of Caucasian is available under the accession number AY267012.1 from NCBI. All of the above gene sequences can be made by commercialized whole gene synthesis services.
  • the recombinant glucose dehydrogenase is supplied by Sangke Biomedical (Shanghai) Co., Ltd. under the number GDH105.
  • the cofactor is any one selected from the group consisting of NAD, NADH, NADP, and NADPH, or a combination thereof, and preferably NADP.
  • the buffer is preferably a phosphate buffer.
  • the recombinant ketoreductase is obtained by fermentation of a genetically engineered bacterium selected from the group consisting of recombinant Escherichia coli or recombinant Pichia pastoris, of which recombinant Escherichia coli BL21 (DE3) is preferred.
  • the recombinant ketoreductase is added in the form of an enzyme powder, a cell disruption solution containing ketoreductase or whole cells, preferably in the form of an enzyme powder.
  • the recombinant ketoreductase enzyme powder can be obtained by a molecular biology, ultrasonication, and vacuum freeze-drying method conventional in the art.
  • the invention Compared with the prior art, the invention has the advantages that the reaction conditions are mild, the product yield and the ee value are high, and the substrate concentration is up to 500 g/L, which greatly improves the (R)-3-quinolol. The preparation efficiency and the production cost are reduced, and have important industrial application value.
  • Example 1 is a GC chart of the reaction conversion ratio and the product ee value detected in Example 2 of the present invention.
  • the cells were collected by centrifugation, and the cells were resuspended in 40 mL of phosphate buffer (10 mM, pH 7.5), sonicated in an ice water bath for 15 min, and the supernatant was collected by centrifugation, pre-frozen at -20 ° C, vacuum freeze-dried for 48 h, and then crushed. That is, a recombinant ketoreductase enzyme powder is obtained.

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Abstract

提供一种重组酮还原酶在制备(R)-3-奎宁醇中的应用，其中重组酮还原酶的氨基酸序列如SEQ ID No.2、4、6、8、10中任一项所示，将重组酮还原酶、3-奎宁环酮或3-奎宁环酮盐酸盐、辅因子、缓冲液、重组葡萄糖脱氢酶、葡萄糖按一定比例混合反应得到产物。

Description

重组酮还原酶在制备(R)-3-奎宁醇中的应用 技术领域
本发明属于生物制药领域，具体涉及一种重组酮还原酶在制备(R)-3-奎宁醇中的应用。
背景技术
(R)-3-奎宁醇为白色晶体，化学名：(R)-(-)-1-氮杂双环[2.2.2]辛-3-醇，分子式为C7H13NO，分子量为127.18，CAS号为25333-42-0，熔点为217-224℃，沸点为120℃，比旋光度为-44.5°(c＝3g/100ml，1N HCl)，在水中的溶解度为1g/mL。
(R)-3-奎宁醇是治疗尿频尿失禁药物索利那辛的重要中间体。索利那辛上市剂型为口服片剂，原研公司安斯泰来，2004年6月获欧洲药物管理局(EMA)批准上市，2004年11月获美国食品药品管理局(FDA)批准上市，2006年4月获日本医药品医疗器械综合机构(PMDA)批准上市，2009年9月获国家食品药品监督管理总局(CFDA)批准上市。该产品自上市以来成为医药界的一个重磅产品，2013-2015年的年销售额分别为13.3亿、12.2亿、11.3亿。
同时(R)-3-奎宁醇还是治疗慢性阻塞性肺病阿地溴铵的关键中间体。阿地溴铵上市剂型为口服吸入性粉剂，原研公司为Almirall和Forest Laboratories，2012年7月获欧洲药物管理局(EMA)和美国食品药品管理局(FDA)批准上市，2015年3月获日本医药品医疗器械综合机构(PMDA)批准上市。该产品2013-2015年的年销售额分别为1.7亿、2.0亿、1.9亿，未来市场前景不容小觑。
因此(R)-3-奎宁醇的制备方法受到科研工作者的广泛关注，目前(R)-3-奎宁醇的制备方法主要有化学合成法和生物合成法。其中化学合成法一般采用手性拆分或借助昂贵的金属催化剂来完成(R)-3-奎宁醇的制备，此两种方法都存在反应收率低、产物光学纯度低、生产成本高的缺点，难以工业化生产。与化学合成法相比，生物合成法具有反应条件温和、转化率高和立体选择性强等优点。
Sakayu Shimizu(Appl.Microbiol.Biotechnol.2009,83,617-626)用从深红酵母(Rhodotorula rubra)中克隆得到的酮还原酶催化3-奎宁酮不对称还原得到(R)-3-奎宁醇， 底物浓度最高达到618mM，且对映体过量值(ee)>99.9％，但此酶的Km值高达145mM，这一高Km值表明该酶对底物的亲和力较弱，底物浓度较低时反应速率较慢，导致反应时间大大延长，生产成本提高。
朱敦铭等人(Journal of Molecular Catalysis B:Enzymatic,2013,88,14-19)利用传统的土壤筛选法分离到两株微生物：诺卡氏菌(Nocardia sp.)WY1202和红串红球菌(Rhodococcuserythropolis)WY1406，其中诺卡氏菌WY1202可催化3-奎宁酮的不对称还原生成(R)-3-奎宁醇，该方法产物收率为93％，产物ee为>99％，但是底物最高浓度仅有99mM，严重影响其工业化应用。
Nobuya Itoh等人(Appl.Environ.Microbiol.2013,79,1378-84)筛选到一株淡黄微杆菌(Microbacterium luteolum)JCM9174，该菌能够还原3-奎宁酮生成(R)-3-奎宁醇，并从中挖掘到两个NADH依赖性还原酶QNR和BacC，该方法转化率分别为100％和94％，产物ee>99.9％，但是底物浓度较低，最高为313mM，不适合工业化生产。
发明内容
为了克服现有技术所存在的上述缺陷，本发明公开了一种重组酮还原酶在制备(R)-3-奎宁醇中的应用，以此提高(R)-3-奎宁醇的生产效率。
具体生物酶催化制备(R)-3-奎宁醇工艺路线如下所示：
Figure PCTCN2017083434-appb-000001
为实现上述发明目的，本发明公开了一种重组酮还原酶在制备(R)-3-奎宁醇中的应用，其中重组酮还原酶的氨基酸序列如SEQ ID No.2、4、6、8、10中任一项所示。
进一步说，编码重组酮还原酶的氨基酸序列的核苷酸序列如SEQ ID No.1、3、5、7、9中任一项所示。
进一步说，(R)-3-奎宁醇由以下步骤制备得到：
将氨基酸序列如SEQ ID No.2、4、6、8、10中任一项所示的重组酮还原酶、3-奎宁环酮或其盐酸盐、辅因子、缓冲液、重组葡萄糖脱氢酶、葡萄糖按一定比例混合，在pH＝6～7、温度为25～35℃下反应20～48h。反应混合液中重组酮还原酶的浓度为5～10g/L、重组葡萄糖脱氢酶的浓度为1-1.5g/L、3-奎宁环酮或其盐酸盐的浓度为100～500g/L、辅因子的浓度为0.1～0.15g/L、葡萄糖浓度为120-600g/L和缓冲液的浓度为50～100mM。反应结束后向反应液中加入硅藻土搅拌，过滤，滤液加NaOH调pH至13左右，用正丁醇萃取、浓缩和结晶，目标产物(R)-3-奎宁醇摩尔收率大于90％、ee值大于99％。
进一步说，重组酮还原酶为来源于高加索乳杆菌的酮还原酶的突变体，是通过对本公司内部的高加索乳杆菌酮还原酶的突变体文库进行筛选后得到的。来源于高加索乳杆菌的野生型酮还原酶在NCBI的登录号为AY267012.1。上述所有基因序列均可通过商业化的全基因合成服务制得。
进一步说，所述重组葡萄糖脱氢酶由尚科生物医药(上海)有限公司提供，编号为GDH105。
进一步说，所述辅因子为选自NAD、NADH、NADP和NADPH的任意一种或它们的组合，其中优选为NADP。
进一步说，缓冲液优选为磷酸盐缓冲液。
进一步说，重组酮还原酶由基因工程菌发酵得到，所述基因工程菌选自重组大肠杆菌或重组毕赤酵母菌，其中优选为重组大肠杆菌BL21(DE3)。
进一步说，重组酮还原酶以酶粉形式、含酮还原酶的细胞破碎液或全细胞的形式加入，优选以酶粉形式加入。
进一步说，所述重组酮还原酶酶粉可采用本领域常规的分子生物学、超声破碎及真空冷冻干燥方法制得。
与现有技术相比，本发明的优点在于反应条件温和，产物收率及ee值均较高，同时底物浓度最高可达500g/L，大大提高了(R)-3-奎宁醇的制备效率，降低其生产成本，具有重要的工业应用价值。
附图说明
图1为本发明实施例2中检测的反应转化率及产物ee值的GC图谱。
具体实施方式
下面结合具体实施例对本发明的技术内容作进一步的阐述，其目的是为了更好的理解本发明的内容，但本发明的保护范围不限于此。
实施例1重组酮还原酶的制备
将含有酮还原酶编码基因(SEQ ID No.1、3、5、7或9)的基因工程菌(载体pET24a，宿主细胞E.Coli BL21(DE3))接种至5mL含卡那霉素的LB试管培养基中活化培养(37℃培养12h)，按1％接种量转接活化培养物至400mL含卡那霉素的LB液体培养基中，37℃培养OD至0.6-0.8，加入IPTG(终浓度0.1mM)于25℃诱导培养16h。离心收集菌体，用40mL磷酸盐缓冲液(10mM，pH 7.5)重悬菌体后，于冰水浴中超声破碎15min，离心收集上清，-20℃预冻后真空冷冻干燥48h后碾碎，即得重组酮还原酶酶粉。
实施例2克级(R)-3-奎宁醇的制备
于25mL反应容器中加入100mM的磷酸盐缓冲液(10mL，pH＝6.5)、葡萄糖(3.6g)及底物3-奎宁环酮盐酸盐(3.0g)，搅拌均匀后加入重组酮还原酶酶粉(120mg)、重组葡萄糖脱氢酶酶粉(15mg)及辅酶NADP(1.5mg)，定容至15mL，30℃下磁力搅拌反应，NaOH(0.5M)控制反应pH在6.5左右，TLC检测反应进程。反应结束后取样离心，上清加NaOH调节pH至13左右，等体积正丁醇萃取三次，合并有机相，无水硫酸钠干燥，减压旋干，即得产品。GC检测转化率及产物ee值，图谱见图1(底物保留时间为13.1min，S型产物保留时间为18.8min，R型产物保留时间为19.5min)转化率>99％，R型产物ee值>99％。
实施例3公斤级(R)-3-奎宁醇的制备
向带夹套的玻璃反应釜(25L)中加入100mM的磷酸盐缓冲液(10L，pH＝6.0)、葡萄糖(3.6kg)及底物3-奎宁环酮盐酸盐(3.0kg)，搅拌均匀后加入重组酮还原酶酶粉(120g)、重组葡萄糖脱氢酶酶粉(15g)及辅酶NADP(1.5g)，定容至15L，30℃搅拌反应，NaOH(0.5M)控制反应pH在6.5左右，TLC检测转化率达到99％后结束反应，调节pH至2.0左右，60℃保温1h使蛋白变性，加入硅藻土搅拌15min后过滤，滤液加 NaOH调节pH至13左右，等体积正丁醇萃取三次，合并有机相，无水硫酸钠干燥，减压旋干，即得产品2.26kg，摩尔收率为91％，产品纯度>95％，产物ee值>99％。
实施例4公斤级(R)-3-奎宁醇的制备
向带夹套的玻璃反应釜(25L)中加入50mM的磷酸盐缓冲液(10L，pH＝7.0)、葡萄糖(1.8kg)及底物3-奎宁环酮盐酸盐(1.5kg)，搅拌均匀后加入重组酮还原酶酶粉(75g)、重组葡萄糖脱氢酶酶粉(15g)及辅酶NADP(1.5g)，定容至15L，25℃搅拌反应，NaOH(0.5M)控制反应pH在6.0左右，TLC检测转化率达到99％后结束反应，调节pH至2.0左右，60℃保温1h使蛋白变性，加入硅藻土搅拌15min后过滤，滤液加NaOH调节pH至13左右，等体积正丁醇萃取三次，合并有机相，无水硫酸钠干燥，减压旋干，即得产品1.13kg，摩尔收率为92％，产品纯度>95％，产物ee值>99％。
实施例5公斤级(R)-3-奎宁醇的制备
向带夹套的玻璃反应釜(25L)中加入100mM的磷酸盐缓冲液(10L，pH＝6.5)、葡萄糖(9.0kg)及底物3-奎宁环酮盐酸盐(7.5kg)，搅拌均匀后加入重组酮还原酶酶粉(150g)、重组葡萄糖脱氢酶酶粉(22g)及辅酶NADP(2.0g)，定容至15L，35℃搅拌反应，NaOH(0.5M)控制反应pH在6.5左右，TLC检测转化率达到99％后结束反应，调节pH至2.0左右，60℃保温1h使蛋白变性，加入硅藻土搅拌15min后过滤，滤液加NaOH调节pH至13左右，等体积正丁醇萃取三次，合并有机相，无水硫酸钠干燥，减压旋干，即得产品5.84kg，摩尔收率为94％，产品纯度>95％，产物ee值>99％。
实施例6公斤级(R)-3-奎宁醇的制备
向带夹套的玻璃反应釜(25L)中加入100mM的磷酸盐缓冲液(10L，pH＝6.5)、葡萄糖(7.2kg)及底物3-奎宁环酮(6kg)，搅拌均匀后加入重组酮还原酶酶粉(150g)、重组葡萄糖脱氢酶酶粉(22g)及辅酶NADP(2.0g)，定容至15L，35℃搅拌反应，NaOH(0.5M)控制反应pH在6.5左右，TLC检测转化率达到99％后结束反应，调节pH至2.0左右，60℃保温1h使蛋白变性，加入硅藻土搅拌15min后过滤，滤液加NaOH调节pH至13左右，等体积正丁醇萃取三次，合并有机相，无水硫酸钠干燥，减压旋干，即得产品5.84kg，摩尔收率为92％，产品纯度>95％，产物ee值>99％。
Figure PCTCN2017083434-appb-000002
Figure PCTCN2017083434-appb-000003
Figure PCTCN2017083434-appb-000004
Figure PCTCN2017083434-appb-000005
Figure PCTCN2017083434-appb-000006
Figure PCTCN2017083434-appb-000007
Figure PCTCN2017083434-appb-000008
Figure PCTCN2017083434-appb-000009

Claims (11)

  1. 一种重组酮还原酶，其氨基酸序列如SEQ ID No.2、4、6、8、10中任一项所示。
  2. 编码如权利要求1所述重组酮还原酶的基因。
  3. 如权利要求2所述的基因，其特征在于：编码氨基酸序列为SEQ ID No.2、4、6、8和10的重组酮还原酶的基因的核苷酸序列分别如SEQ ID No.1、3、5、7和9所示。
  4. 表达如权利要求2所述基因的微生物。
  5. 如权利要求4所述的微生物，其特征在于：其为重组大肠杆菌或重组毕赤酵母菌，优选为重组大肠杆菌BL21(DE3)。
  6. 如权利要求1所述重组酮还原酶或者如权利要求4所述微生物在制备(R)-3-奎宁醇中的应用。
  7. 如权利要求6所述的应用，其特征在于，所述(R)-3-奎宁醇由以下步骤制备：将氨基酸序列如SEQ ID No.2、4、6、8、10中任一项所示的重组酮还原酶、3-奎宁环酮或3-奎宁环酮盐酸盐、辅因子、缓冲液、重组葡萄糖脱氢酶、葡萄糖按一定比例混合反应得到产物。
  8. 如权利要求7所述的应用，其特征在于：所述重组酮还原酶的浓度为5～10g/L、所述重组葡萄糖脱氢酶的浓度为1～1.5g/L、所述3-奎宁环酮或3-奎宁环酮盐酸盐的浓度为100～500g/L、所述辅因子的浓度为0.1～0.15g/L、所述葡萄糖的浓度为120～600g/L和所述缓冲液的浓度为50～100mM。
  9. 如权利要求7所述的应用，其特征在于：所述反应在pH＝6～7、温度为25～35℃下进行。
  10. 如权利要求7所述的应用，其特征在于：所述辅因子为选自NAD、NADH、NADP和NADPH的任意一种或它们的组合，其中优选为NADP。
  11. 如权利要求7所述的应用，其特征在于：所述缓冲液为磷酸盐缓冲液。
PCT/CN2017/083434 2016-05-26 2017-05-08 重组酮还原酶在制备(r)-3-奎宁醇中的应用 WO2017202193A1 (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112725389A (zh) * 2019-10-29 2021-04-30 广东东阳光药业有限公司 一种米拉贝隆中间体的制备方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109456949A (zh) * 2018-12-27 2019-03-12 尚科生物医药（上海）有限公司 一种用于制备r型去氧肾上腺素的酮还原酶突变体

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102186972A (zh) * 2008-08-29 2011-09-14 科德克希思公司 用于立体选择性生产(4s)-3-[(5s)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1，3-噁唑烷-2-酮的酮还原酶多肽
WO2012175119A1 (en) * 2011-06-22 2012-12-27 Isochem Process for the preparation of solifenacin and salts thereof
CN103555608A (zh) * 2013-09-16 2014-02-05 华东理工大学 一种奎宁酮还原酶及其在不对称合成(r)-3-奎宁醇中的应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102186972A (zh) * 2008-08-29 2011-09-14 科德克希思公司 用于立体选择性生产(4s)-3-[(5s)-5-(4-氟苯基)-5-羟基戊酰基]-4-苯基-1，3-噁唑烷-2-酮的酮还原酶多肽
WO2012175119A1 (en) * 2011-06-22 2012-12-27 Isochem Process for the preparation of solifenacin and salts thereof
CN103555608A (zh) * 2013-09-16 2014-02-05 华东理工大学 一种奎宁酮还原酶及其在不对称合成(r)-3-奎宁醇中的应用

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112725389A (zh) * 2019-10-29 2021-04-30 广东东阳光药业有限公司 一种米拉贝隆中间体的制备方法
CN112725389B (zh) * 2019-10-29 2023-07-18 广东东阳光药业有限公司 一种米拉贝隆中间体的制备方法

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