Abstract
A mutant L206F/P207F/L259F of Talaromyces thermophilus lipase (TTL) exhibited high hydrolytic activity towards 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) for synthesis of (S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid (S-CCMA), a key chiral intermediate of pregabalin. However, low conversion at high CNDE concentration and unreusability of the free TTL mutant restricted its industrial applications. In this study, the TTL mutant was immobilized onto epoxy resin and its catalytic properties for kinetic resolution of CNDE were investigated. Under the optimized conditions, the immobilized lipase exhibited an increased catalytic efficiency even at a CNDE concentration of 3 M with 49.7% conversion and 95% eep. The conversion retained higher than 46.3% even after 10 times repeated use of the immobilized lipase in n-heptane-water biphasic system. These results demonstrated great potential of the immobilized TTL mutant for industrial production of the chiral intermediate of pregabalin.
Highlights
Lipases (E.C. 3.1.1.3) have been widely employed in organic synthesis for hydrolysis, alcoholysis, acidolysis, esterification, and transesterification of carboxylic acid esters in aqueous or nonaqueous media [1,2,3,4]
Rhizopus arrhizus lipase covalently attached to epoxy resin SEPABEADS5 EC-EP exhibited much higher operational stability for synthesis of (S)-2-(1-hydroxy3-butenyl)-5-methylfuran compared to that of free enzyme [26]
The results indicated that the immobilized Talaromyces thermophilus lipase (TTL) mutant exhibited a specific activity of 413.2 U/g and the conversion of cyano-5-methylhexanoic acid ethyl ester (CNDE) reached 46.3% at a substrate loading of 1 M after 10 reaction cycles, demonstrating its excellent catalytic performance
Summary
Lipases (E.C. 3.1.1.3) have been widely employed in organic synthesis for hydrolysis, alcoholysis, acidolysis, esterification, and transesterification of carboxylic acid esters in aqueous or nonaqueous media [1,2,3,4]. Due to the high chemical stability and excellent mechanical strength, covalent binding interactions between enzyme and epoxy carrier can remarkably enhance the stability of free enzyme compared with physical or ionic binding and are employed as one of the most popular immobilization methods to improve catalytic performance [24]. Kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) using commercial lipase Lipolase5and Lipozyme TLIM5 as biocatalyst seems to be promising approaches to prepare S-CCMA [28]. Both of them exhibited inferior catalytic efficiency towards CNDE and could not be reused, leading to high cost of the bioprocess. The results indicated that the immobilized TTL mutant exhibited a specific activity of 413.2 U/g and the conversion of CNDE reached 46.3% at a substrate loading of 1 M after 10 reaction cycles, demonstrating its excellent catalytic performance
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