Engineering Of Thermomyces Lanuginosus Lipase And Its Application In Biosynthesis Of Chiral Intermediates Of Pregabalin

Thermomyces lanuginosus lipase(TLL)is a basophilic and noticeably thermostable enzyme,displaying highly catalytic activity compared with other thermophilic fungi lipase.Currently,TLL has been broadly applied in food,detergents,biodiesel and fine chemical industry with the form of soluble(Lipolase(?))and immobilized(Lipozyme TL IM(?))that supplied by Novozymes.Recently,with the fast growing green chemistry,TLL was more frequently used in chiral pharmaceutical intermediates production.Lipolase(?)was applied in the kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester(CNDE)to produce(3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid in industrial scale.Certainly,improving the catalytic activity towards CNDE of TLL will be of great significance for Pregabalin production.In this study,based on the crystal structure of TLL,E.coli BL21(DE3)/pET28b-TLL was selected as the parent strain and enzyme-substrate interaction was simulated by computer technology.The six(Glu56,Ser58,Trp89,Ile202,Val203,Leu259)residues nearby the active site,three residues(Ser83?Asn94,Leu96)in the hinge regions of lib and two residues(Ala20,Tyr21),which may influence the forming of disulfide bond,were selected as target mutation sites.The recombinant plasmid pET28b-TLL was used as the template for saturation mutagenesis using polymerase chain reaction.From the constructed mutaion clone libraries,two mutants S83T and S85L/S83T which possessed significantly higher activity towards CNDE were obtained using a pH-based high-throughput screening and further gas chromatography analysis.Enzymatic properties of the mutant S58L/S83T indicated that:the optimum temperature of mutant S58L/S83T increased slightly from 45?to 50? and exhibited noticeably thermal stability at 40? with a half-life of 22.5 days.The optimum pH(pH 9.5)of TLL and mutant S58L/S83T is almost the same.The mutant S58L/S83T showed excellent pH stability in high pH conditons and remained 60%of the initial activity after stored at pH 9.0 for 26 days.Homology modeling and molecular docking studies were conducted to analyze and discuss the mechanism for the enhanced activity.The catalytic activity of TLL towards CNDE was notably improved when amino acid Ser83 was replaced by Thr,the distance of hydrogen bond formed between Thr83 and CNDE shorten from 2.8 A to 1.9 A.The residue Leu5 8,substituted for the amino acid Thr,which made the hydrogen bond(Arg84 NH-OD Asp57)broken easier.The enzymatic kinetics was researched and the specific activity was detected.The specific activity of mutant S83T and S58L/S83T was 2.69-fold and 5.46-fold relative to the parental enzyme,and the apparent kcat/Km values were also notably increased(2.97-fold and 7.18-fold,respectively).The results indicated that the mutant S58L/S83T displayed better catalytic activity than the parental enzyme.The biotransformation of racemic 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl esters using whole cells of mutant S58L/S83T as biocatalysts was investigated.The optimal reaction conditions were as follows:150mM calcium acetate,pH 8.0 and 40?.Whole cells of mutant S58L/S83T(5%w/v)was used to catalyze 3 M CNDE under the optimal reaction condition,the product reached 1344 mM with e.e.p value over 96%after 24 h.The catalytic performance is equivalent to that of Lipolase(?)(8%w/v).