Modification And Its Application Of Lipase From Talaromyces Thermophilus For Biosynthesis Of Chiral Intermediate Of Pregabalin

Pregabalin(Lyrica^TM),?S?-3-?Aminomethyl?-5-methylhexanoic acid,is a lipophilic analogue of 4-aminobutyric acid?GABA?.It is used as adjunctive therapy for partial seizures.The Pregabalin manufacturing process developed by Pfizer applied Lipolase^?from Thermomyces lanuginosus as biocatalyst,to enantioselectively hydrolyze 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester?CNDE?to?3S?-2-carboxyethyl-3-cyano-5-methylhexanoic acid.In this study,a novel lipase?TTL?was exploited from genome of Talaromyces thermophiles by gene mining for efficient synthesis of?3S?-2-carboxyethyl-3-cyano-5-methylhexanoic acid.TTL was overexpressed in Escherichia coli BL21and its specific activity at low temperature was more powerful to hydrolysis CNDE.TTL was further heterologous expressed Pichia pastoris with biological activity.1DTE was selected as the model temple to bulid 3D structure of TTL by Modeller,and docking technique was adopted to analyze amino acid residues which may affect its activity.The site-saturation mutagenesis libraries were created anda pH-indicator based high throughput screening method together with GC analysis were applied to screen TTL mutant with significant improved activity.The single mutants L147R and G91V demonstrated a 1.51-fold and 2.15-fold improvement in specific activity over the wild type and double mutants G91V/S58M and G91V/S83T demonstrated a 2.29-fold and 3.62-fold improvement,respectively.Enzymatic properties of the mutant G91V/S83T with highest activity were investigated.The optimum temperature of the wild type TTL and mutant G91V/S83T were determined as 50? and the half-life of G91V/S83T tat40?,50?,60? were 21.2 d,14.5 d,8.0 d,respectively.The optimum pH of G91V/S83T was determined as pH 8.0Molecular docking was used to analyze the varied of wild and mutants.When Leu147 was substituted by Phe,which developed the hydrogen bond with Leu147 and Ser116,and stable oxygen hole structure.The Val91 was located on the“lid”of TTL and its replacement by Gly91,was more conducive to open the“lid”.The distance of hydrogen bond formed between Thr83 and S-CNDE shorten from 3.2?to 2.6?,which was consistent with the result that the catalytic activity of TTL towards CNDE was improved when amino acid Ser83 was replaced by Thr83.When the Ser58 was replaced by Met58,the hydrogen bonding force and the protein polarity decreased and thus the activity of enzyme was increased.The kenetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester by G91V/S83T was carried out at pH 8.0,50?.A conversion of 42% with ee>94% was obtained after 8 h at a substrate loading of 100 mM.The obtained results demonstrated G91V/S83T as a promising biocatalyst in the pratical synthesis of pregabalin.