Purification and properties of enantioselective ester hydrolase from a strain of Trichosporon species (DSMZ 11829)

Abstract An isolated strain of Trichosporon species (DSMZ 11829) was capable of producing a novel membrane-bound, enantioselective ester hydrolase. Its application in the kinetic resolution of racemic methyl ester of 6-methoxy-α-methyl-2-naphthaleneacetic acid (naproxen) into ( S )-(+)-naproxen (>99% ee, E  ∼ 500) and acyloxy derivatives of 1-chloro-3-(1-naphthyloxy)-2-propanol into ( R )-alcohol (>99% ee, E  ∼ 550) have already been reported by us. The enzyme designated as TSL was extracted from the cells by toluenization and purified to homogeneity from cell free extract with 8.5% overall yield. The purified enzyme with a specific activity of 831 U/mg protein was achieved by column chromatography using DEAE-Sepharose, phenyl-Sepharose and Sephadex G-100 resins. The purified enzyme exhibited hydrolytic activity without any noticeable decrease in the rate of hydrolysis or its enantioselectivty in the resolution of racemic naproxen into ( S )-(+)-naproxen or in the resolution of 1-chloro-3-(1-naphthyloxy)-2-propanol and 1-(6-methoxy-2-naphthyl)ethanol. Purified enzyme is a glycosylated monomer with a molecular weight of ∼40 kD and isoelectric point (p I ) ∼ 4.1. TSL was maximally active at 30 °C and pH 8.0. The enzyme was insensitive to serine reactive agent PMSF, metal chealator EDTA, non-ionic detergent Triton X-100 and reducing agent mercaptoethanol. The N -terminal amino acid sequence of TSL determined as Thr-Val-Thr-Thr-Pro-Thr-Leu-Ser-Ala-Asp-Ala-Lys-Lys-Gly- did not reveal any homology with the N -terminal amino acid sequence of any other known microbial ester hydrolases.