【作者】 黎小军；

【导师】 郑裕国；

【作者基本信息】 浙江工业大学 ， 生物化工， 2014， 博士

【摘要】 普瑞巴林(S-3-氨甲基-5-甲基己酸)是抑制性神经递质γ-氨基丁酸(GABA)的结构类似物。由于良好的神经病理性疼痛和癫痫治疗效果,普瑞巴林已成为“重磅炸弹药物”。由于S-型普瑞巴林具有更强的药理活性,不对称合成普瑞巴林成为近年来的研究热点。Pfizer公司开发的第二代普瑞巴林合成工艺引入商品化脂肪酶Lipolase(?),选择性拆分外消旋2-羧乙基-3-氰基-5-甲基己酸乙酯(CNDE),生成(3S)-2-羧乙基-3-氰基-5-甲基己酸。该手性中间体经脱羧、碱性水解、氢化制得普瑞巴林。但该工艺中最关键的生物催化剂,目前只有Novozymes 公司的 Lipolase(?)(来源于Thermomyces lanugnosus的商品化脂肪酶,TLL)能满足工业化生产的需要。因此,获得能够高效拆分CNDE的新催化剂,成为普瑞巴林化学酶法合成工艺的关键。本论文针对高效拆分CNDE制备普瑞巴林手性中间体新生物催化剂开发,从基因挖掘与克隆、异源表达、分子改造、催化工艺及应用等方面开展研究。以立体选择性拆分CNDE为目标反应,以高效催化CNDE水解的TLL的氨基酸序列为查询序列,在GenBank数据库进行同源序列比对,筛选出与TLL同源性为78%的脂肪酶LN,然后根据ln基因序列设计引物,从T.lanuginosus DSM 10635中克隆获得与ln高度同源的脂肪酶基因lip(GenBank no.KC479729),并在Escherichia coli中活性表达。重组大肠杆菌全细胞催化CNDE水解,产物ee值达98%,E>200,但活力偏低。此外,以真菌酯酶的保守序列设计简并引物进行 PCR 扩增,然后利用 5’-RACE PCR 获得了T.lanugino us DSM 10635 酯酶 TLE 的全长序列(945bp,GenBankno.KF305767)。分别在大肠杆菌和毕赤酵母系统实现该酯酶的活性表达,并利用Ni-NTA柱纯化TLE,研究TLE的基本酶学性质。TLE对CNDE表现出良好的选择性(E=95),与目前报道的用于CNDE拆分的其它酯酶相比,其选择性最高,说明该酶有一定的应用潜力。为了提高Lip对CNDE的水解活力,开展了基于定点饱和突变策略的分子改造研究。利用同源建模和分子对接等技术,构建了脂肪酶Lip的3D结构,分析了可能影响其活力的氨基酸残基,选择脂肪酶盖子结构附近及氧负离子洞组成氨基酸为突变位点。通过多轮点饱和突变,从突变库中筛选到活力明显提高的突变体S88T/A99N/V116D,其活力(2.35U/mg)是野生型Lip的60.3倍,与TLL(2.40U/mg,从Lipolase(?)中纯化)相当。通过同源建模、分子对接研究,揭示了酶的结构-功能的关系,分析了突变体活力提高的机理:S88T突变能更有效的稳定CNDE水解反应过程中的酶-底物过渡态;A99N和V116D突变新形成三对氢键,有利于稳定酶蛋白处于盖子打开状态,从而使酶活力提高。为了进一步提高Lip对CNDE的水解活力,以定点饱和突变获得的突变体Lip-T(S88T/A99N/V116D)为亲本,利用易错PCR构建随机突变库,筛选到突变体S63L/D232A,其活力是亲本Lip-T的2.0倍。为了验证这两个点突变在活力提高中的作用,利用定点突变技术分别构建单点突变体S63L和D232A,发现S63L和D232A突变对酶活的影响截然相反:S63L能有效提高酶活,单突变体S63L的活力是双突变体S63L/D232A的1.5倍;而D232A突变引起活力的降低,其活力仅为亲本的68%。为了考察其它18种氨基酸替换对活力的影响,分别构建了位点63和232的饱和突变文库,并筛选到活力更高的突变体 S63M/S88T/A99N/V116D,其活力达到 8.68 U/mg,为S88T/A99N/V116D的3.7倍,TLL的3.6倍,野生型Lip的222.6倍。通过同源建模发现,S63M突变引入疏水性残基,导致盖子打开程度更大,有利于底物、产物的出入,从而提高酶活。表达突变体S63M/S88T/A99N/V116D的大肠杆菌细胞(5%,w/v)能高效催化3 M(765 g/L)CNDE选择性水解,反应24 h,转化率45.2%,ee值98%,在催化剂使用量更少的情况下,已达到Lipolase(?)(8%,w/v)拆分CNDE的水平。脂肪酶催化CNDE水解过程中,产物抑制作用是影响脂肪酶催化效率的关键因素。论文进行了基于离子交换的原位吸附消除产物抑制、提高催化效率的研究,从7种代表性阴离子交换树脂中筛选出201×7,并考察了其用量对转化过程的影响。研究了加入阴离子交换树脂201×7进行原位吸附的分批转化过程,向转化体系中加入20%的树脂后,转化率达到45%所需的时间减半,催化效率加倍(3 M CNDE,反应12h,转化率>45%),时空产率从56.25mmol/L/h提高到 112.50 mmol/L/h,催化剂产率从 1.125 mmol/h/g 提高到2.250 mmol/h/g,分别是Pfizer工艺的2.0倍和3.2倍。分离转化液中的产物(3S)-2-竣乙基-3-氰基-5-甲基己酸钠,在105℃加热脱竣40 min,得到纯度>99%,ee值>99%的(S)-3-氰基-5-甲基-己酸乙酯。更多还原

【Abstract】 Pregabalin[(S)-3-(aminomethyl)-5-methylhexanoic acid]is a lipophilic derivative of the inhibitory neurotransmitter γ-aminobutyric acid(GABA).Due to its good curative effect on neuropathic pain and epilepsy,Pregabalin has been one of the fastest growing and block-buster drugs.Since(S)-Pregabalin is significantly more potent than the(R)-enantiomer and racemic-Pregabalin,more interest have been focused on the asymmetric synthesis of optical pure(S)-Pregabalin domestically as well as globally.The most successful scalable chemoenzymatic strategy for(S)-Pregabalin involved lipase-catalyzed resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester(CNDE)to(3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid,which was converted to Pregabalin after decarboxylation,hydrolysis and hydrogenation.The biocatalyst involved in Pfizer’s chemoenzymatic manufacturing process is Lipolase(?),a commercial available Thermomyces lanuginosus lipase(TLL)supplied by Novozymes,there’s no other robust biocatalyst used in industrial production of Pregabalin.Accordingly,development of novel biocatalyst with high process efficiency will be of great significance and value for Pregabalin production.In this thesis,we focused on the development a novel robust biocatalyst for kinetic resolution of CNDE.The gene mining and cloning,heterologous expression,molecular engineering of the biocatalyst,and its application in efficient biosynthesis of chiral intermediate of Pregabalin were investigated in details.The amino-acid sequence of known TLL,the robust biocatalyst used in industrial production of Pregabalin,was used to search for homologues in GenBank.Lipase LN,the amino acid sequence with 78%identity to that of TLL,was discovered by gene mining for lipase.The lip gene(GenBank accession no.KC479729),with 99%identity to that of In,was cloned from T.lanuginosus DSM 1065 by RT-PCR using the primers that designed from the available In sequence.The lip gene was cloned into the expression vector pET-28b,and overexpressed in biologically active in Escherichia coli BL21(DE3).The kinetic resolution of CNDE by recombinant E.coli cell overexpressing Lip suggested that the enzyme showed excellent enantioselectivity(98%eep,E>200)for CNDE.However,the catalytic activity of the recombinant Lip was too low to fulfill the industrial application.Meanwhile,a novel esterase encoding gene,tle,was successfully cloned from T.lanuginosus DSM10635 with degenerate RT-PCR and RACE-PCR methods.The tle(GenBank accession no.KF305767)had an open reading frame of 945 bp encoding TLE of 314 amino acids.TLE was heterologous expressed in E.coli and Pichia pastoris in biologically active,respectively.Recombinant TLE was purified to homogeneity by single-step affinity chromatography on a Ni-NTA column.Several biochemical properties of TLE were studied.Among the reported esterases,TLE showed the highest enantioselectivity(E=95)in the kinetic resolution of CNDE.The unique properties of the esterase indicate that TLE is a potential candidate for industrial application.The site-saturation mutagenesis technique was used to improve the hydrolytic activity of Lip towards CNDE.The three-dimensional homology modelof Lip was generated by modeling,and series key residues were revealed by modeling and docking studies.The site-saturation mutagenesis libraries were created at oxyanion hole and the lid hinge region.The saturation mutagenesis libraries were screened using the high-throughput colorimetric activity assay on a 96-well plate format.The most active variant,S88T/A99N/V116D(2.35 U/mg),demonstrated a 60.3-fold improvement over the wild-type Lip(0.039 U/mg)in specific activity for CNDE,which almost matches TLL’s performance(2.40 U/mg).Modeling and docking studies demonstrated that the mutant could more effectively stabilize oxygen anions in transition states and the lid of Lip in the open conformation.The increased activity for S88T might be explained by the increase in steric bulk introduced in oxyanion hole,result in more effectively stabilized oxygen anions in transition states in the hydrolytic reaction.The substitution of A99N and V116D exhibited higher hydrolytic activity towards CNDE due to three new hydrogen bonds were formed to stabilize the lid in the maximally open position,one hydrogen bond was between Asp101 and Asp 116,and the other two was between Asn99 and Asp 116.In order to develop a novel biocatalyst with high process efficiency compatible with the industrial prerequisites,we tried to further enhance its activity toward CNDE of Lip-T(S88T/A99N/V116D)by complementary protein engineering strategy,including error-prone PCR,site-directed mutagenesis and site-saturation mutagenesis.By screening approximately 2,500 clones from random-mutant libraries created by epPCR,the mutant S63L/D232A with 2.0-fold improved activity toward CNDE was obtained.To identify whether both substitutions in S63L/D232A are necessary for activity improvement,site-directed mutagenesis was performed to construct the single-point mutants S63L and D232A,respectively.Interestingly,the mutant S63L and D232A showed opposite effect on activity,S63L exhibited a significant improvement on activity,whereas D232A exerted a slight inhibitory effect.To enhance the effectiveness of directed evolution,the saturation mutagenesis libraries at site S63 and D232 were created and screened for mutants with improved activity,respectively.The mutant S63M/S88T/A99N/V116D with the highest activity(8.68 U/mg)was obtained from the site-saturation mutagenesis library at the site S63.It is worth noting that the specific activity of mutant S63M/S88T/A99N/V116D was about 3.7-fold,3.6-fold and 222.6-fold that of the S88T/A99N/V116D,TLL and wild-type Lip,respectively.Structural changes resulting from the mutations were analyzed and the mechanism responsible for the enhanced activity was discussed.Moreover,the engineered lipase was used as a robust biocatalyst for enantioselective hydrolysis of CNDE at a very high substrate loading(3 M,765 g/L).As only 5%(w/v)resting cells were used,the bioprocess is much more cost-effective than Pfizer’s process using 8%(w/v)commercially available lipase Lipolase(?).During the kinetic resolution of CNDE by whole cells,2-carboxyethyl-3-cyano-5-methylhexanoic acid was found to inhibit its own production.To enhance catalytic efficiency,a new biocatalytic process of in situ product adsorption(ISPA)has been developed utilizing anion-exchange resin.To optimize the bioconversion of 2-carboxyethyl-3-cyano-5-methylhexanoic acid from CNDE,seven anion-exchange resins were examined,and the resin 201×7 was selected.In fed-batch biotransformation with ISPA with 20%resin 201×7 and 3 M CNDE was performed,and>45%conversion was achieved after 12 h.As compared to the conventional fed-batch mode,this approach reduced the reaction times by about 50 percent(from 24 h to 12 h),and allowed(3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid space-time yield and biocatalyst productivity to be increased from 56.25 mmol/L/h and 1.125 mmol/h/g catalyst to 112.50 mmol/L/h and 2.250 mmol/h/g catalyst,respectively.The batch biotransformation with ISPA demonstrated 2.0-fold and 3.2-fold improvement over Pfizer’s process in space-time yield and biocatalyst productivity,respectively.The ISPA method by addition of resin 201×7 was proven to be an effective way to eliminate the product inhibition and enhance process productivity.After phase separation of the aqueous solution from the enzymatic reaction,a rapid chemical decarboxylation of(3S)-2-carboxyethyl-3-cyano-5-methyl-hexanoic acid into(S)-3-cyano-5-methylhexanoic acid ethyl ester took place in 105℃ reflux(40 min).(S)-3-cyano-5-methylhexanoic acid ethyl ester with 99%purity and 99%ee was obtained.更多还原