Screening, nucleotide sequence, and biochemical characterization of an esterase from Pseudomonas fluorescens with high activity towards lactones

A genomic library of Pseudomonas fluorescens DSM 50106 in a lambda RESIII p hage vector was screened in Escherichia coil K-12 for esterase activity by using cy-naphthyl acetate and Fast Blue RR. A 3.2-kb DNA fragment was subcl oned from an estrase-positive clone and completely sequenced. Esterase EstF 1 was encoded by a 999-bp open reading frame (ORF) and exhibited significan t amino acid sequence identity with members of the serine hydrolase family. The deduced amino acid sequences of two other C-terminal truncated ORFs ex hibited homology to a cyclohexanone monooxygenase and an alkane hydroxylase . However, esterase activity was not induced by growing of P. fluorescens D SM 50106 in the presence of several cyclic ketones. The esterase gene was f used to a His tag and expressed in E. coli. The gene product was purified b y zinc ion affinity chromatography and characterized. Detergents had to be added for purification, indicating that the enzyme was membrane bound or me mbrane associated. The optimum pH of the purified enzyme was 7.5, and the o ptimum temperature was 43 degrees C. The showed highest purified enzyme act ivities towards lactones. The activity increased from gamma-butyrolactone ( 18.1 U/mg) to epsilon-caprolactone (21.8 U/mg) to delta-valerolactone (36.5 U/mg). The activities towards the aliphatic esters were significantly lowe r; the only exception was the activity toward ethyl caprylate, which was th e preferred substrate.