CN103981160B - 疏绵状嗜热丝孢菌脂肪酶突变体、编码基因及其应用 - Google Patents
疏绵状嗜热丝孢菌脂肪酶突变体、编码基因及其应用 Download PDFInfo
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Abstract
本发明提供了一种疏绵状嗜热丝孢菌脂肪酶突变体、编码序列及其应用。所述一种疏绵状嗜热丝孢菌脂肪酶突变体,由序列如SEQ ID No.1的肽段经点突变获得,所述的点突变为下列之一或两者结合:(1)第63位的S突变为L或M;(2)第232位的D突变为A。本发明提供的疏绵状嗜热丝孢菌脂肪酶突变体较亲本活力大幅提高,可用于工业化生产(3S)‑2‑羧乙基‑3‑氰基‑5‑甲基己酸,从而进一步合成普瑞巴林。
Description
(一)技术领域
本发明涉及一组疏绵状嗜热丝孢菌脂肪酶突变体、编码基因,及其在水解外消旋2-羧乙基-3-氰基-5-甲基己酸乙酯制备普瑞巴林关键手性中间体(3S)-2-羧乙基-3-氰基-5-甲基己酸中的应用。
(二)背景技术
普瑞巴林(Pregabalin,简称PGB),化学名为(S)-(+)-3-氨甲基-5-甲基己酸(Ⅰ),属γ-氨基丁酸的3位异丁基取代物(Angew.Chem.Int.Ed.2008,47:3500-3504)。普瑞巴林是一种钙离子通道调节剂,能够有效抑制与神经病理性疼痛、癫痫相关的神经元过度兴奋及神经递质释放增加(Curr.Opin.Pharmacol.2006,6:108-113),已被FDA批准用于带状疱疹后神经痛、糖尿病外周神经痛、癫痫、纤维肌痛等。与传统药物相比,普瑞巴林的剂量更低、服用次数少、副作用小、持续时间长、耐受性强(Curr.Med.Res.Opin.2006,22:375-384),因此被国际疼痛学会(IASP)、欧洲神经病学学会联盟(EFNS)和英国NICE等多个国际指南推荐为一线治疗药物。自上市以来,普瑞巴林全球销售额快速增长,成为全球畅销药市场的“重磅炸弹”,2012年全球销售额已突破40亿美元,市场前景十分广阔。
国内外报道了大量有关普瑞巴林及其中间体化学合成工艺,可归为、不对称催化法、手性拆分法手性配体法和手性化合物直接合成法等四大类。化学法由于工艺步骤冗长、手性拆分试剂昂贵、反应条件苛刻、有毒有害溶剂用量大,而无法实现规模化生产。生物催化法由于反应条件温和、立体选择性高、环境友好等优点成为最受瞩目的医药及精细化学品合成技术之一。辉瑞公司开发的普瑞巴林生产工艺引入脂肪酶Lipolase催化拆分外消旋2-羧乙基-3-氰基-5-甲基己酸乙酯生成(S)-2-羧乙基-3-氰基-5-甲基己酸(Ⅱ)。该手性中间体经脱羧、碱性水解、氢化等步骤制得普瑞巴林(Ⅰ)(Org.Process Res.Dev.2008,12:392-398;US20050283023;US8134023B2)。
随着蛋白质工程技术和分子生物学的发展,运用定向进化和理性设计的手段对酶分子进行人工进化和改造已成为当前酶工程领域研究的热点。到目前为止,已有许多学者运用这项技术成功的改造了各种各样的酶,取得了令人瞩目的进展(Curr.Opin.Struc.Biol.2011,21:473-480)。获得能高效制备手性中间体Ⅱ的生物催化剂对制备普瑞巴林具有重要的意义。
(三)发明内容
本发明的目的在于通过易错PCR和定点突变技术对疏绵状嗜热丝孢菌脂肪酶进行改造,提供一种突变体蛋白,提高其对2-羧乙基-3-氰基-5-甲基己酸乙酯的活力,从而有利于该脂肪酶在普瑞巴林中间体制备中的应用。
本发明采用的技术方案是:
一种疏绵状嗜热丝孢菌脂肪酶突变体,由序列如下的肽段经点突变获得:RPVRRAVPQD LLDQFELFSQ YSAAAYCAAN NHAPVGSDVT CSENVCPEVD AADATFLYSF EDSGLGDVTGLLALDNTNKL IVLSFRGTRS VENWIANLNA DLTEISDICS GCEGHDGFVT SWRSVADTIR EQVQNAVNEHPDYRVVFTGH SLGGALATIA AAALRGNGYN IDVFSYGAPR VGNRAFAEFL TAQTGGTLYR ITHTNDIVPRLPPRDWGYSH SSPEYWVTSG NDVPVTANDI TVVEGIDSTD GNNQGNIPDI PSHLWYFGPI SECD(SEQ ID No.1);所述的点突变为下列之一或两者结合:(1)第63位的S突变为L或M;(2)第232位的D突变为A。
本发明使用多轮易错PCR方法对脂肪酶Lip-T编码基因进行随机突变,连接表达载体后转化宿主大肠杆菌,诱导表达后再通过高通量筛选方法将正突变检出,然后利用定点突变方法,得到活力进一步提高的突变体。获得的突变体能催化高浓度(2.0~3.0mol/L)的2-羧乙基-3-氰基-5-甲基己酸乙酯制备普瑞巴林关键手性中间体(3S)-2-羧乙基-3-氰基-5-甲基己酸。
具体方法如下:来源于Thermomyces lanuginosus DSM 10635的脂肪酶Lip(GenBank no.KC479729)的三突变体Lip-T(S88T/A99N/V116D,见SEQ ID NO.1)由274氨基酸残基组成,已成功构建表达载体pET28-Lip-T,功能正常的酶蛋白在大肠杆菌BL21(DE3)中实现过量表达(Appl.Microbiol.Biotechnol.DOI 10.1007/s00253-013-5136-y)。采用多轮易错PCR方法对其进行随机突变,重组到表达载体pET-28b(+),再将重组质粒转入表达宿主E.coli BL21(DE3)中,构建突变库。挑选突变库中单克隆于96孔培养板中诱导表达酶蛋白,离心重悬后,取菌液加入底物(2-羧乙基-3-氰基-5-甲基己酸乙酯)和溴百里香酚蓝指示剂,反应适当时间后,初筛出活力明显高于对照的菌株,然后对这些阳性突变菌株进行活力测定,将筛选得到的高活力突变菌株测序,获得突变体DNA序列信息。经对上述构建的突变文库筛选以及理性设计方法,获得3个活力提高的突变体,即S63L/D232A,S63L和S63M。
所述嗜热丝孢菌脂肪酶突变体S63L/D232A(该酶的第63位的丝氨酸突变为亮氨酸,第232位的天冬氨酸突变为丙氨酸)序列如下(SEQ ID No.3):RPVRRAVPQD LLDQFELFSQYSAAAYCAAN NHAPVGSDVT CSENVCPEVD AADATFLYSF EDLGLGDVTG LLALDNTNKL IVLSFRGTRSVENWIANLNA DLTEISDICS GCEGHDGFVT SWRSVADTIR EQVQNAVNEH PDYRVVFTGH SLGGALATIAAAALRGNGYN IDVFSYGAPR VGNRAFAEFL TAQTGGTLYR ITHTNDIVPR LPPRDWGYSH SSPEYWVTSGNAVPVTANDI TVVEGIDSTD GNNQGNIPDI PSHLWYFGPI SECD。
所述嗜热丝孢菌脂肪酶突变体S63L(该酶的第63位的丝氨酸突变为亮氨酸)序列如下(SEQ ID No.5):RPVRRAVPQD LLDQFELFSQ YSAAAYCAAN NHAPVGSDVT CSENVCPEVDAADATFLYSF EDLGLGDVTG LLALDNTNKL IVLSFRGTRS VENWIANLNA DLTEISDICS GCEGHDGFVTSWRSVADTIR EQVQNAVNEH PDYRVVFTGH SLGGALATIA AAALRGNGYN IDVFSYGAPR VGNRAFAEFLTAQTGGTLYR ITHTNDIVPR LPPRDWGYSH SSPEYWVTSG NDVPVTANDI TVVEGIDSTD GNNQGNIPDIPSHLWYFGPI SECD。
所述嗜热丝孢菌脂肪酶突变体S63M(该酶的第63位的丝氨酸突变为甲硫氨酸)序列如下(SEQ ID No.7):RPVRRAVPQD LLDQFELFSQ YSAAAYCAAN NHAPVGSDVT CSENVCPEVDAADATFLYSF EDMGLGDVTG LLALDNTNKL IVLSFRGTRS VENWIANLNA DLTEISDICS GCEGHDGFVTSWRSVADTIR EQVQNAVNEH PDYRVVFTGH SLGGALATIA AAALRGNGYN IDVFSYGAPR VGNRAFAEFLTAQTGGTLYR ITHTNDIVPR LPPRDWGYSH SSPEYWVTSG NDVPVTANDI TVVEGIDSTD GNNQGNIPDIPSHLWYFGPI SECD。
本发明还涉及编码所述疏绵状嗜热丝孢菌脂肪酶突变体的基因。
具体的,所述基因为下列之一:SEQ ID NO.4(编码SEQ ID No.3所示突变体),SEQID NO.6(编码SEQ ID No.5所示突变体),SEQ ID NO.8(编码SEQ ID No.7所示突变体)。
本发明还涉及所述的疏绵状嗜热丝孢菌脂肪酶突变体在水解外消旋2-羧乙基-3-氰基-5-甲基己酸乙酯制备普瑞巴林关键手性中间体(3S)-2-羧乙基-3-氰基-5-甲基己酸中的应用。
所述的脂肪酶突变体可以以工程菌全细胞形式使用,也可以是未经纯化的粗酶形式使用,也可以是经部分纯化的或完全纯化的酶的形式使用。如果需要,还可以利用本领域已知的固定化技术将本发明的脂肪酶突变体制成固定化酶或者固定化细胞形式的固化酶。
本发明的有益效果主要体现在:本发明提供的疏绵状嗜热丝孢菌脂肪酶突变体较亲本活力大幅提高,可用于工业化生产(3S)-2-羧乙基-3-氰基-5-甲基己酸,从而进一步合成普瑞巴林。
(四)附图说明
图1为脂肪酶突变体全细胞催化2-羧乙基-3-氰基-5-甲基己酸乙酯(2.0M)制备(3S)-2-羧乙基-3-氰基-5-甲基己酸反应进程;
图2为脂肪酶突变体全细胞催化2-羧乙基-3-氰基-5-甲基己酸乙酯(3.0M)制备(3S)-2-羧乙基-3-氰基-5-甲基己酸反应进程。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
实施例1:易错PCR(error-prone PCR)构建脂肪酶突变文库
以克隆有Thermomyces lanuginosus DSM 10635脂肪酶Lip三突变体Lip-T(S88T/A99N/V116D,氨基酸序列SEQ ID NO.1,核酸序列SEQ ID NO.2)编码基因的质粒pET28-Lip-T为模板,利用T7promoter引物和T7terminator为引物(表1)进行易错PCR扩增,随机引入突变。PCR体系包括:不含Mg2+的1×Taq Buffer,0.2mM dGTP,0.2mM dATP,1.0mM dCTP,1.0mMdTTP,7mM MgCl2,0.2mM MnCl2,T7promoter引物和T7terminator引物各0.2μM,质粒pET28-Lip-T 50ng,Taq DNA聚合酶5U,补水至100μl。PCR条件为94℃预变性5min,30个循环:94℃30s,55℃30s,72℃1min,循环结束后延伸72℃10min。经0.9%琼脂糖凝胶电泳分析PCR为阳性后,取PCR溶液1μl为模板,进行下一轮PCR扩增,PCR体系和条件同第一轮,连续进行三轮易错PCR。第三轮得到的PCR反应液进行琼脂糖凝胶电泳和切胶回收,然后用NcoI和HindⅢ双酶切,与同样酶切处理的表达载体pET28b(+)连接,连接液电击法转化E.coli BL21(DE3)感受态细胞,涂布含卡那霉素(50μg/ml)的LB平板,37℃培养过夜,得到超过2000个克隆的突变体库。
实施例2:脂肪酶突变文库的筛选
突变文库的筛选的高通量筛选技术参考(Bioorgan.Med.Chem.,1999,7:2183-2188)的描述。具体过程如下:
挑取实施例1中突变体库中的单克隆于96孔板,每孔加有200μl的LB培养基(含有50μg/ml卡纳霉素),37℃培养3h左右,至OD600约为0.6,加入0.1mM IPTG(异丙基-β-D-硫代吡喃半乳糖苷),于28℃诱导10h。4000g,4℃,离心10min,弃去上清,用PBS缓冲液(pH7.2,10mM)重悬细胞,加入含有底物2-羧乙基-3-氰基-5-甲基己酸乙酯和指示剂溴百里香酚蓝的反应液,以突变前的工程菌细胞为参照,在96孔板上初筛活力提高的阳性克隆。初筛出的阳性克隆经重复筛选证实,筛选到活力最高的突变体1,其活性是对照出发菌株的2倍,并挑取该单克隆送上海桑尼生物技术公司测序,该突变体为S63L/D232A,其氨基酸序列见序列表中的SEQ ID No.3,核酸序列见序列表中的SEQ ID No.4。
实施例3:S63L和D232A的定点突变
定点突变技术参考(Current Protocols in Protein Science 2011,26.6.1-26.6.10;Anal.Biochem.2008,375:376-378)的描述,具体过程如下:
为了将Lip-T分别定点突变为单点突变体S63L和D232A,分别设计突变引物S63L-F和S63L-R,D232A-F和D232A-R(表1),以质粒pET28-Lip-T为模板,进行全质粒扩增进行定点突变。PCR体系为:5×PS Buffer 10μl,dNTP(2.5mM each)4μl,突变引物S63L-F和S63L-R(或D232A-F和D232A-R)各0.5μl,质粒pET28-Lip-T 0.5μl,PrimeSTAR DNA聚合酶0.5μl,补水至50μl。PCR条件为98℃预变性2min,25个循环:98℃10s,65℃10s,72℃6min,最后72℃10min。经0.9%琼脂糖凝胶电泳分析PCR为阳性后,取各PCR溶液20μl,加入1μl Dpn Ⅰ,37℃酶切2h去除模板质粒,65℃灭活10min,转化感受态细胞E.coli BL21(DE3),涂布含卡那霉素(50μg/ml)的LB平板。挑取单克隆经菌落PCR验证,阳性克隆保存并送上海桑尼生物技术公司测序,证实获得单点突变体为S63L和D232A。将单点定突变体S63L和D232A活力与突变体为S63L/D232A比较,获得活力较S63L/D232A提高的S63L,其氨基酸序列见序列表中的SEQID No.5,核酸序列见序列表中的SEQ ID No.6。
实施例4:S63的定点饱和突变与筛选
定点饱和突变技术参考(Current Protocols in Protein Science 2011,26.6.1-26.6.10;Anal.Biochem.2008,375:376-378)的描述,具体方法和过程如下:
为了将脂肪酶Lip-T氨基酸序列中的第63位点的Ser(S)进行饱和突变,设计引物S63-F和S63-R(见表1),以质粒pET28-S63L为模板(见实施例3),进行全质粒扩增。PCR体系为:5×PS Buffer 10μl,dNTP(2.5mM each)4μl,突变引物S63-F和S63-R各0.5μl,质粒pET28-S63L 0.5μl,PrimeSTAR DNA聚合酶0.5μl,补水至50μl。PCR条件为98℃预变性2min,25个循环:98℃10s,65℃10s,72℃6min,最后72℃10min。经0.9%琼脂糖凝胶电泳分析PCR为阳性后,取PCR溶液20μl,加入1μl Dpn Ⅰ,37℃酶切2h去除模板质粒DNA,65℃灭活10min,转化感受态细胞E.coli BL21(DE3),涂布含卡那霉素(50μg/ml)的LB平板,37℃培养过夜,获得约300个克隆的突变体库。定点饱和突变库的筛选同实施例2,不同是对照为实施例3获得的pET28-S63L突变体细胞。筛选出的阳性克隆再经活力测定证实后从阳性克隆中抽提质粒,经DNA测序确定引入的点突变,活力最高的阳性克隆DNA测序显示63位的Ser突变成了Met(S63M)。突变体S63M的氨基酸序列见序列表中的SEQ ID No.7,其中其中一种编码基因序列见序列表中的SEQ ID No.8。
表1:引物
引物名称 | 引物序列 |
T7promoter | 5′-TAATACGACTCACTATAGGG-3′ |
T7terminator | 5′-TGCTAGTTATTGCTCAGCGG-3′ |
S63L-F | 5′-CTATTCTTTTGAAGATTTGGGATTAGGCGATGTTAC-3′ |
S63L-R | 5′-GTAACATCGCCTAATCCCAAATCTTCAAAAGAATAG-3′ |
D232A-F | 5′-CACGTCTGGTAACGCCGTCCCAGTGACCGC-3′ |
D232A-R | 5′-GCGGTCACTGGGACGGCGTTACCAGACGTG-3′ |
S63-F | 5′-CTATTCTTTTGAAGATNNKGGATTAGGCGATGTTAC-3′ |
S63-R | 5′-GTAACATCGCCTAATCCMNNATCTTCAAAAGAATAG-3′ |
实施例5:亲本脂肪酶和脂肪酶突变体工程菌的诱导表达
将出发菌株E.coli BL21(DE3)/pET28-Lip-T、突变菌株E.coli BL21(DE3)/pET28-S63L/D232A(见实施例1)、E.coli BL21(DE3)/pET28-S63L(实施例3)和E.coli BL21(DE3)/pET28-S63M(实施例4)分别接种到含有50mg/L卡那霉素的LB液体培养基中37℃培养12h,再以1%接种量(v/v)接种到新鲜的含有50mg/L卡那霉素的LB液体培养基中,培养至菌体浓度OD600约0.6左右,再向LB液体培养基加入终浓度为0.1mM的IPTG,28℃诱导培养10h后,4℃、8000g离心10min,收集含有重组脂肪酶的菌体细胞,可用于酶活测定和生物催化制备(3S)-2-羧乙基-3-氰基-5-甲基己酸。
实施例6:亲本脂肪酶和脂肪酶突变体的活性测定
实施例5中获得的突变体菌株和出发菌株细胞分别测定活力。反应体系组成:100mM 2-羧乙基-3-氰基-5-甲基己酸乙酯,50mM Ca(OAc)2,Tris-HCl(pH7.5,100mM)和一定量的细胞,40℃、150r/min反应1h,取样200μl,并加50μl 1M的HCl终止反应,乙酸乙酯萃取、无水硫酸钠处理后气相测定转化率和产物的对映体过量值(e.e.)。采用外标定量法来测定转化液中2-羧乙基-3-氰基-5-甲基己酸乙酯、(3S)-2-羧乙基-3-氰基-5-甲基己酸的含量。各成分的量用气相色谱岛津GC-14C测定,色谱柱类型:G-TA毛细管柱;色谱条件:柱温135℃,进样室温度220℃,FID检测器220℃,氦流量为1ml/min,分流比为30:1。酶活单位(U)定义为:在40℃、pH 7.5条件下,在1min内催化2-羧乙基-3-氰基-5-甲基己酸乙酯生成1μmol(3S)-2-羧乙基-3-氰基-5-甲基己酸所需要的酶量定义为1U。
表2:脂肪酶突变体与亲本脂肪酶比活性的比较
实施例7:脂肪酶突变体全细胞在(3S)-2-羧乙基-3-氰基-5-甲基己酸制备中的应用
转化体系组成及转化操作如下:200ml反应体系中,加入98ml水,脂肪酶突变体S63M湿细胞(见实施例4和实施例5)10g,Ca(OAc)25.2g,底物2-羧乙基-3-氰基-5-甲基己酸乙酯102g(底物浓度2.0M,即510g/l);反应温度40℃,反应过程中通过滴加NaOH的方式控制反应液的pH值7.0,期间取样用气相色谱监测反应进程(见图1),气相检测条件同实施例5。由图1可知,5%(W/v)的脂肪酶突变体S63M湿细胞能催化2M的CNDE选择性水解,反应16h,转化率达到46.3%,并获得ee值为99%以上的产物(3S)-2-羧乙基-3-氰基-5-甲基己酸。离心或者过滤除去大肠杆菌细胞,减压蒸馏至1/3体积,通过两次甲苯萃取除去剩余底物,再减压蒸馏去除残留甲苯,得到目标产物(3S)-2-羧乙基-3-氰基-5-甲基己酸(e.e.>99%)。
实施例8:脂肪酶突变体全细胞在(3S)-2-羧乙基-3-氰基-5-甲基己酸制备中的应用
转化体系:200ml反应体系中,加入47ml水,脂肪酶突变体S63M湿细胞(见实施例4和实施例5)10g,Ca(OAc)25.2g,底物2-羧乙基-3-氰基-5-甲基己酸乙酯153g(底物浓度3.0M,即765g/l);转化操作、转化后的处理方式同实施例7,反应24h(进程见图2)。从图2可知,脂肪酶突变体S63M的底物耐受性非常好,即使在非常高的底物浓度(3M),5%(W/v)的脂肪酶突变体S63M湿细胞做催化剂,转化24h,转化率可达41.1%,产物e.e.>99%。
本发明不受上述具体文字描述的限制,本发明可在权利要求书所概括的范围内做各种改变,这些改变菌在本发明的范围之内。
Claims (4)
1.一种疏绵状嗜热丝孢菌脂肪酶突变体,由序列如下的肽段经点突变获得: 所述的点突变为第63位的S突变为L或M;所述嗜热丝孢菌脂肪酶突变体序列如下列中的一种:
①
②
2.编码权利要求1所述疏绵状嗜热丝孢菌脂肪酶突变体的基因。
3.如权利要求2所述的基因,其特征在于所述基因为SEQ ID NO.6或SEQ ID NO.8。
4.如权利要求1所述的疏绵状嗜热丝孢菌脂肪酶突变体在水解外消旋2-羧乙基-3-氰基-5-甲基己酸乙酯制备普瑞巴林关键手性中间体(3S)-2-羧乙基-3-氰基-5-甲基己酸中的应用。
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