Purification and Immobilization of a Novel Enantioselective Lipase from Tsukamurella tyrosinosolvents for Efficient Resolution of Ethyl 2-(2-oxopyrrolidin-1-yl) Butyrate.

Abstract

A highly enantioselective lipase from Tsukamurella tyrosinosolvents E105 was purified via ultrasonic extraction, precipitation, and chromatographic steps. The enzyme was purified about 38-fold with the recovery yield of 9% and was confirmed as a dimer protein consisting of two identical subunits with a molecular mass of 24kDa. The purified lipase was used to catalyze resolution of racemic ethyl 2-(2-oxopyrrolidin-1-yl) butyrate to (S)-2-(2-oxopyrrolidin-1-yl) butyric acid. The maximum activity of such lipase was obtained at pH7.5, 35°C, and the highest relative activity (156.80%) was observed in the presence of 0.5mM Co2+. Subsequently, the lipase was encapsulated within a mixture of 3% sodium alginate and 0.8% carrageenan, and then cross-linked with 0.6% glutaraldehyde to enhance its biocatalytic capability and stability. Comparing with 36.9% product yield and 97.5% product ee of free lipase, the highest product yield of 46.3% and ee of 98.5% for immobilized lipase were achieved with the presence of 20mM substrate. In addition, the reusability of immobilized lipase was also investigated, which could maintain 63.7% of its initial conversion yield after seven repeated batch reactions. Thus, the evaluated enantioselective lipase in this work has a good potential for further industrial application.