CN101962661B - 一种羰酰还原酶在生产(s)-4-氯-3羟基丁酸乙酯中的应用 - Google Patents
一种羰酰还原酶在生产(s)-4-氯-3羟基丁酸乙酯中的应用 Download PDFInfo
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- CN101962661B CN101962661B CN2010102137246A CN201010213724A CN101962661B CN 101962661 B CN101962661 B CN 101962661B CN 2010102137246 A CN2010102137246 A CN 2010102137246A CN 201010213724 A CN201010213724 A CN 201010213724A CN 101962661 B CN101962661 B CN 101962661B
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Abstract
本发明公开了一种氨基酸序列如SEQ ID NO：2所示的羰酰还原酶在4-氯乙酰乙酸乙酯不对称还原制备(S)-4-氯-3羟基丁酸乙酯中的应用。即以氨基酸序列如SEQ ID NO：2所示的羰酰还原酶为催化剂，以4-氯乙酰乙酸乙酯为底物，以NADH为辅因子，不对称还原制备(S)-4-氯-3羟基丁酸乙酯。本发明首次将氨基酸序列如SEQ ID NO：2所示的羰酰还原酶应用于4-氯乙酰乙酸乙酯不对称还原制备(S)-4-氯-3羟基丁酸乙酯中，取得很好的效果，其酶活高达15U/mg，其对底物的得率高达91％，产物的对映体过量值为100％，且产量高，大大降低了生产成本。
Description
技术领域
本发明属于生物技术领域，涉及一种羰酰还原酶的应用，尤其涉及一种羰酰还原酶(Carbonyl Reductase CR)在以4-氯乙酰乙酸乙酯为底物不对称还原反应生产(S)-4-氯-3羟基丁酸乙酯中的应用。
背景技术
(S)-4-氯-3羟基丁酸乙酯(Ethyl 4-chloro-3-hydroxybutanoate，(S)-CHBE)是一种重要的手性中间体，可用于很多活性药物的合成，如他汀类药物——羟甲基戊二酰CoA(HMG-CoA)还原酶抑制剂和4-羟基吡啶烷酮(4-hydroxypyrrolidone)等[1]。
以4-氯乙酰乙酸乙酯(Ethyl 4-chloroacetoacetate，COBE)作为还原反应的潜手性底物，易于合成且价格低廉，以其为底物进行不对称还原反应获取(S)-CHBE是非常经济有效的制备途径。
迄今为止关于COBE不对称还原制备手性CHBE已进行了很多的研究报道，概括起来主要有化学法和生物法。
化学催化不对称还原法，所用催化剂包括价格昂贵的铑、釕等金属，采用化学法合成手性CHBE的缺点是产物的光学纯度不够高，且催化还原反应需要很高的氢气压，耗能高，污染大。
微生物法分为酶催化和全细胞催化法，Shimizu等用来自Sporobolomycessalmonicolor AKU 4429的NADPH依赖的醛基还原酶分别在单一水相体系[2]和水/有机溶剂两相体系[3]催化还原COBE制备手性CHBE，由于应用酶催化还原反应所用的酶要从微生物细胞中分离纯化得到，过程操作繁琐且酶容易失活，与全细胞催化相比，应用较少。全细胞法则分为采用野生酵母和基因工程菌催化COBE为(S)-CHBE两种，Yasohara等[4]从400株酵母菌中筛选得到了一株Candida magnoliae，在水/乙酸正丁酯体系中，在添加葡萄糖、NADP和葡萄糖脱氢酶以及反应过程中需要控制pH值的条件下产物(S)-CHBE在有机相的积累浓度可达90g/L，产物的光学纯度对映体过量值(enantiomeric excess e.e.)达到96％。由于采用野生酵母中往往含有多种能够催化COBE为不同构型CHBE的还原酶，因此采用野生酵母进行催化所获得的产物的光学活性往往很低，筛选到高立体选择性的优良微生物菌株非常困难，所以近来的研究着重集中于运用重组大肠杆菌不对称合成具有高立体选择性的(S)-CHBE。
Yasohara等[5]从木兰假丝酵母菌Candida magnoliae中分离得到了一个辅酶NADPH依赖型的羰基还原酶，将该酶与葡萄糖脱氢酶基因克隆到大肠杆菌中共表达，在定时添加适量的辅酶NADP和葡萄糖以及分批添加底物的条件下，催化COBE的不对称还原(S)-CHBE，其得率和光学纯度分别为85％e.e.和100％e.e.[6]。
现有的羰酰还原酶大多为辅酶NADPH依赖型，包括来源于P.stipitis的PsCRI[7]，来源于Candida magnoliae的S 1[6]等。到目前，仅有来源于Kluyveromyces aestuarii的KaCR为辅酶NADH依赖型，其活力很低，仅3.06U/mg[8]。
综上所述，现有催化COBE为(S)-CHBE的技术存在底物得率低、产物光学活性低、成本高等问题。
本专利中涉及到的还原酶是羰酰还原酶的一种，其包含285个氨基酸，其在Genbank中的收录号为XP_001387285.1，ACCESSION XP_001387285(http://www.ncbi.nlm.nih.gov/protein/XP_001387285.1)，其氨基酸序列如SEQ ID NO：2所示。编码该蛋白的基因含有858bp，其在Genbank中的收录号为XM_001387248.1(http://www.ncbi.nlm.nih.gov/nuccore/126256851)，其基因序列如SEQ IDNO：1所示。至今未发现该羰酰还原酶用于COBE不对称还原制备(S)-CHBE的报道。
【参考文献】：
[1]Karanewsky DS，Badia MC，Ciosek CP Jr，Robl JF，Sofia MJ，Simpkins LM，DeLange B，Harrity TW，Biller SA，Gorden EM(1990)Phosphorus-containing inhibitors of HMG-CoAreductase.1.4-[2-arylethyl]-hydroxyphosphinyl]-3-hydroxybutanoic acids：a new class ofcell-selective inhibitors of cholesterol biosynthesis.J Med Chem 33：2925-2956。
[2]Shimizu S，Kataoka M，Morishita A，Katoh M，Morikawa T，Miyoshi T，Yamada H(1990a)Microbial asymmetric reduction of ethyl 4-chloro-3-oxobutaoate to optically active ethyl4-4-chloro-3-hydroxybutanoate.Biotechnol lett 12：593-596。
[3]Shimizu S，Kataoka M，Karoh M，Morikawa T，Miyoshi T，Yamada H(1990b)Stereoselective reduction of ethyl 4-chloro-3-oxobutaoate by a microbial aldehyde reductasein an organic solvent-water diphasic system.Appl Environ Microbiol 56：2374-2377。
[4]Yasohara Y，Kizaki N，Hasegawa J，Takahashi S，Wada M，Kataoka M，Shimizu S(1999)Synthesis of optically activeethyl 4-chloro-3-hydroxybutanoate by microbial reduction.ApplMicrobiol Biotechnol 51：847-851。
[5]Wada M，Kataoka M，Kawabata H，Yasohara Y，Kizaki N，Hasegawa J，Shimizu S(1998)Purification and characterization of NADPH-dependent carbonyl reductase involved instereoselective reduction of ethyl 4-chloro-3-oxobutanoate，from Candida magnoliae.BiosciBiotechnol Biochem 62：280-285。
[6]Yasohara Y，Kizaki N，Hasegawa J，Wada M，Kataoka M，Shimizu S(2000)Molecularcloning and overexpression of the gene encoding an NADPH-dependent carbonyl reductase，involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate，from Candidamagnoliae.Biosci Biotechnol Biochem 64：1430-1436。
[7]Ye Q，Yan M，Xu L，Cao H，Li ZJ，Chen Y，Li SY，Ying HJ(2009)A novel carbonylreductase from Pichia stipitis for the production of(S)-4-chloro-3-hydroxybutanoate ethyl.Biotechnol Lett 31：537-542。
[8]Yamamoto H，Mitsuhashi K，Kimoto N，Matsuyama A，Esaki N，Kobayashi Y(2004)Anovel NADH-dependent carbonyl reductase from Kluyveromyces aestuarii and comparison ofNADH-regeneration system for the synthesis of ethyl(S)-4-chloro-3-hydroxybutanoate.Biosci Biotechnol Biochem 68：638-649。
发明内容
本发明所要解决的技术问题是提供一种羰酰还原酶在COBE不对称还原制备(S)-CHBE中的应用。
为解决上述技术问题，本发明所采用的技术方案如下：
一种氨基酸序列如SEQ ID NO：2所示的羰酰还原酶在4-氯乙酰乙酸乙酯(COBE)不对称还原制备(S)-4-氯-3羟基丁酸乙酯[(S)-CHBE]中的应用。
即以氨基酸序列如SEQ ID NO：2所示的羰酰还原酶为催化剂，以4-氯乙酰乙酸乙酯为底物，以还原型烟酰胺腺嘌呤二核苷酸(还原型辅酶Ⅰ，NADH)为辅因子，不对称还原制备(S)-4-氯-3羟基丁酸乙酯。
具体反应是将表达基因序列如SEQ ID NO：1所示的重组菌，经过破碎后与200mmol/L～2mol/L葡萄糖、1.6～320g/L的4-氯乙酰乙酸乙酯、50U～5KU的葡萄糖脱氢酶(glucose dehydrogenase，GDH)和0.05～0.5mmol/L的氧化型烟酰胺腺嘌呤二核苷酸(氧化型辅酶Ⅰ，NAD)，在pH6.0～7.5、20～30℃、150～300rpm条件下反应15～30h，得到(S)-4-氯-3羟基丁酸乙酯。其中，加入NAD+与GDH即能再生成NADH，且使反应循环进行，降低了加入量以减少了生产成本。
发明人基于现代生物信息学思想，结合分子生物学技术，采用基因工程的手段从毕赤酵母Pichia Stipitis CBS 6054克隆羰酰还原酶的基因，在大肠杆菌中表达后发现其在水相中能够高效催化COBE为(S)-CHBE，e.e.值为100％。同时，通过在水/有机相中反应、分批添加底物COBE等方式，解除了底物和产物对细胞和酶的抑制作用，显著的提高了转化效果。通过对羰酰还原酶的基因进行重组表达，获得了具有新型催化功能的酶蛋白，开发了该条基因的新功能——催化非天然底物COBE为高立体选择性的(S)-CHBE。
有益效果：本发明首次将氨基酸序列如SEQ ID NO：2所示的羰酰还原酶应用于COBE不对称还原制备(S)-CHBE中，取得很好的效果，其酶活高达15U/mg，而Yamamoto从海洋酵母菌Kluyveromyces aestuarii中分离得到的羰基还原酶，其酶活只有3.06U/mg[8]。氨基酸序列如SEQ ID NO：2所示的羰酰还原酶对底物COBE的得率高(大于91％)，产物CHBE的光学活性高(e.e.为100％)，且产量高，大大降低了生产成本。
本专利中涉及的辅酶NADH-依赖羰基还原酶是除了NADPH依赖羰基还原酶之外，在已报道还原酶中活力最高。而NADH比NADPH更为廉价，NADH价格约为300元/克，而NADPH约为8000元/克。氧化型辅酶NAD+价格约100元/克，NADP+价格约1500元/克。因此，使用本发明的羰酰还原酶应用于COBE不对称还原制备(S)-CHBE中，无疑可以极大的降低生产成本。
附图说明
图1为羰酰还原酶基因的构建图。
具体实施方式
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1：羰酰还原酶基因的获取
毕赤酵母Pichia Stipitis CBS 6054(购于Centraalbureau voor Schimmelcultures(CBS)Fungal Biodiversiry Centre)，培养基YPD(g·L-1)：酵母提取物10g，蛋白胨20g，葡萄糖20g，补蒸馏水至1L。
将毕赤酵母Pichia Stipitis CBS 6054接种于5mLYPD液体培养基中30℃培养至对数生长期，使用基因组DNA提取试剂盒(北京天为生物工程有限公司酵母基因组提取试剂盒)提取基因组。
构建表达载体所用的引物加设酶切位点，引物序列如下：
上游引物(CRII-sense含Nde I)为：
5’-GGAATTCCATATGACTGTCGAAACCGCCACC-3’
下游引物(CRII-anti含BamH I)为：
5’-CGCGGATCCCTAGACAGAACAGTAACCACCT-3’
所有引物均由上海申能博彩公司合成。
基因的PCR条件：
94℃变性7min，按如下参数循环30次：94℃变性1min，60℃退火50s，72℃延伸1.5min。最后72℃延伸10min。
实施例2：基因的表达
用Nde I及BamH I分别酶切pET-22b(pET-22b购于Novagen(默克中国))及所扩增含有两个酶切位点的目的基因，分别胶回收已双酶切的目的片段和表达载体，将已双酶切的表达载体pET-22b与目的基因用T4连接酶进行连接过夜，将10uL的连接产物pET-22b-PsCRII加入100uL的Rosetta(DE3)感受态细胞中，冰上放置30min，42℃热激90sec。冰上放置2min。加入预热的0.45mL培养基。220rpm 37℃C 1h。将200uL菌液加入分别含有100μg/mL的氨苄青霉素和氯霉素的LB平板上，37℃过夜培养12-16h。构建图谱见图1。
实施例3：酶活的测定
挑取重组菌E.coli Rosseta(pET-22b-PsCR)及出发大肠杆菌Rosseta(DE3)至含抗生素的LB液体培养基中，37℃振荡培养过夜。然后按2％接种量分别接种到新鲜培养液中，37℃培养至OD600约为0.6时，加入IPTG至终浓度0.8mmol·L-1，25℃，220rpm，诱导表达10h后，离心(4℃，5000rpm，15min)，菌泥用100mM磷酸钾缓冲(pH7.0)重悬，超声破碎细胞(功率300W，超声5s，间歇5s，共5min)，离心(4℃，12000rpm，15min)，测定上清中的酶活。
酶反应体系包括100mM磷酸钾缓冲液(pH6.0)，5mM NADH，20mM COBE，30℃，340nm处测定吸光值的下降。酶活定义为每分钟内氧化1μmol NADH所需要的酶量为一个酶活单位U。蛋白采用Brandford法进行测定。
结果显示，出发大肠杆菌Rosseta(DE3)的比酶活为0.13U/mg，而重组菌E.coliRosseta(pET22b-PsCRII)的比酶活为15U/mg，高于能够催化该底物COBE为(S)-CHBE的羰基还原酶的最高报道(S1的比酶活为13.7U/mg[6])。
实施例4：重组大肠杆菌E.coli Rosseta(pET-22b-PsCRII)的发酵
挑取重组菌E.coli Rosseta(pET-22b-PsCRII)至含抗生素的LB培养液，37℃振荡培养过夜。然后按2％接种量分别接种到新鲜培养液中，37℃培养至OD600约为0.6时，加入IPTG至终浓度0.8mmol/L，25℃，220rpm，诱导表达10h后，8000rpm，4℃离心10min，弃上清，沉淀备用。
实施例5：
取实施例4的沉淀用磷酸钾缓冲(100mmol/L，pH 6.5)洗涤两次，称取0.5g(湿重)的大肠杆菌菌泥，悬浮于15mL的pH6.5磷酸钾缓冲中。超声处理细胞(功率300W，超声5s，间歇5s，共5min)，加入葡萄糖200mmol/L，COBE 1.5g/L，GDH 50U，NAD 0.05mmol/L，20℃，180rpm，16h。产物(S)-CHBE的产量为1.38g/L，产物的得率为：92.0％，光学纯度e.e.为100％。
实施例6：
取实施例4的沉淀用磷酸钾缓冲(100mmol/L，pH 7.5)洗涤两次，称取1g(湿重)的大肠杆菌菌泥，悬浮于15mL的pH 7.5磷酸钾缓冲中。超声处理细胞(功率300W，超声5s，间歇5s，共5min)，加入葡萄糖500mmol/L，COBE 25g/L(0、1、2、8、14h各5g/L)，GDH 200U，NAD 0.1mmol/L，25℃，220rpm，24h。产物(S)-CHBE的产量为23.1g/L，产物的得率为：92.4％，光学纯度e.e.为100％。
实施例7：
取实施例4的沉淀用磷酸钾缓冲(100mmol/L，pH 7.0)洗涤两次，称取2g(湿重)的大肠杆菌菌泥，悬浮于50mL的pH 7.0磷酸钾缓冲中。超声处理细胞(功率300W，超声5s，间歇5s，共5min)，加入葡萄糖600mmol/L，COBE 35g/L(0、1、2、8、14h各7g/L)，GDH500U，NAD 0.15mmol/L，30℃，240rpm，28h。产物(S)-CHBE的产量为32.3g/L，产物的得率为：92.3％，光学纯度e.e.％为100％。
实施例8：
取实施例4的沉淀用磷酸钾缓冲(100mmol/L，pH 6.0)洗涤两次，称取4g(湿重)的大肠杆菌菌泥，悬浮于50mL的pH 6.0磷酸钾缓冲中。超声处理细胞(功率300W，超声5s，间歇5s，共5min)，加入葡萄糖1.5mol/L，50mL乙酸正丁酯(可促进COBE的溶解并解除底物和产物对酶和细胞的抑制作用)，加入COBE 100g/L(0、2、4、6、10各20g/L)，GDH 3KU，NAD 0.3mmol/L，20℃，240rpm，28h。产物(S)-CHBE的产量为95.2g/L，产物的得率为：95.2％，光学纯度e.e为100％。
实施例9：
取实施例4的沉淀用磷酸钾缓冲(100mmol/L，pH 6.5)洗涤两次，称取2g(湿重)的大肠杆菌菌泥，悬浮于15mL的pH 6.5磷酸钾缓冲中。超声处理细胞(功率300W，超声5s，间歇5s，共5min)，加入15mL乙酸正丁酯(可促进COBE的溶解并解除底物和产物对酶和细胞的抑制作用)，加入葡萄糖1mol/L，COBE 50g/L(0、2、4、6、10各10g/L)，GDH 2KU，NAD 0.2mmol/L，20℃，240rpm，28h。产物(S)-CHBE的产量为46.9g/L，产物的得率为：93.8％，光学纯度e.e为100％。
实施例10：
取实施例4的沉淀用磷酸钾缓冲(100mmol/L，pH 6.5)洗涤两次，称取10g(湿重)的大肠杆菌菌泥，悬浮于200mL的pH 6.5磷酸钾缓冲中。超声处理细胞(功率300W，超声5s，间歇5s，共5min)，加入200mL乙酸正丁酯(可促进COBE的溶解并解除底物和产物对酶和细胞的抑制作用)，加入葡萄糖2mol/L，COBE 300g/L(0、2、4、6、10各60g/L)，GDH 5KU，NAD 0.5mmol/L，25℃，280rpm，32h。产物(S)-CHBE的产量为276.6g/L，产物的得率为：92.2％，光学纯度e.e.为100％。
实施例11：产物的检测方法
对于水相反应：反应结束后，加入等体积乙酸乙酯，剧烈振荡10min然后放置两小时，8000rpm离心10min分离有机层和水层。小心吸取上层乙酸乙酯过有机膜，加入内标，保存测样。
对于水/有机两相反应：反应结束后8000rpm离心10min分离有机层和水层。小心吸取上层乙酸乙酯过有机膜，加入内标，保存测样。
PEG-20M毛细管柱，内标物为萘。程序为：检测器FID，温度210℃，汽化室温度210℃，柱温150℃，柱头压0.03MPa，氢气0.05MPa，空气0.1MPa，尾吹0.08MPa。用HPLC对(S)-4-氯-3-羟基丁酸乙酯的旋光性进行分析(手性柱Chiralcel OB，4.6×250mm；Daicel Chemical Industries，日本)，检测条件：流动相为正己烷∶正己烷(9∶1)，波长214nm，流量为0.8mL/min，R型和S型CHBE的出峰时间分别为：10.5min和11.6min。
核苷酸或氨基酸序列表
SEQUENCE LISTING
<110>南京工业大学
<120>一种羰酰还原酶在生产(S)-4-氯-3羟基丁酸乙酯中的应用
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<170>PatentIn version 3.3
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Asp Val Arg Lys Ala Asp Gln Val Lys Ser Val Ile Asp Lys Ile Glu
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Trp Thr Ser Gly Pro Met Ile Asp Gln Glu Thr Asp Asp Asp Trp His
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Cys Gly Ala Tyr Leu Tyr Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr
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ggt tct gac atc aga gtc gac ggt ggt tac tgt tct gtc taa 858
Gly Ser Asp Ile Arg Val Asp Gly Gly Tyr Cys Ser Val
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<210>2
<211>285
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Met Thr Val Glu Thr Ala Thr Ala Pro Gln Ser Met Cys Asn Thr Asp
1 5 10 15
Ile Gly Ser Leu Pro Ala Ala Asp Pro Val Leu Pro Thr Asn Val Leu
20 25 30
Asp Phe Phe Lys Leu Asp Gly Lys Thr Ala Ala Ile Thr Gly Gly Ala
35 40 45
Arg Gly Ile Gly Tyr Ala Ile Ser Glu Ala Tyr Leu Gln Ala Gly Ile
50 55 60
Ser Lys Leu Ala Ile Ile Asp Tyr Ala Pro Asn Glu Ala Ala Leu Asp
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Glu Leu Arg Ser Arg Phe Leu Lys Ser Thr Ile Val Tyr His Asn Cys
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Ala Ser Met Ser Ala His Ile Val Asn Val Pro Gln Leu Gln Ala Ala
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Tyr Asn Ala Ala Lys Ala Gly Val Leu His Leu Gly Lys Ser Leu Ala
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Val Glu Trp Ala Pro Phe Ala Arg Val Asn Thr Val Ser Pro Gly Tyr
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Ile Ser Thr Glu Leu Ser Asp Phe Val Pro Thr Glu Met Lys Asn Lys
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Cys Gly Ala Tyr Leu Tyr Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr
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Gly Ser Asp Ile Arg Val Asp Gly Gly Tyr Cys Ser Val
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Claims (3)
1.一种氨基酸序列如SEQ ID NO：2所示的羰酰还原酶在4-氯乙酰乙酸乙酯不对称还原制备(S)-4-氯-3羟基丁酸乙酯中的应用。
2.根据权利要求1所述的应用，其特征在于以氨基酸序列如SEQ ID NO：2所示的羰酰还原酶为催化剂，以4-氯乙酰乙酸乙酯为底物，以NADH为辅因子，不对称还原制备(S)-4-氯-3羟基丁酸乙酯。
3.根据权利要求2所述的应用，其特征在于表达基因序列如SEQ ID NO：1所示的重组菌，经过破碎后与200mmol/L～2mol/L葡萄糖、1.6～320g/L的4-氯乙酰乙酸乙酯、50U～5KU的葡萄糖脱氢酶和0.05～0.5mmol/L的NAD，在pH6.0～7.5、20～30℃、150～300rpm条件下反应15～30h，得到(S)-4-氯-3羟基丁酸乙酯。
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