Screening, Nucleotide Sequence, and Biochemical Characterization of an Esterase from Pseudomonas fluorescens with High Activity towards Lactones

Author:

Khalameyzer V.1,Fischer I.2,Bornscheuer U. T.1,Altenbuchner J.2

Affiliation:

1. Institute for Technical Biochemistry,1 and

2. Institute for Industrial Genetics,2 University of Stuttgart, 70569 Stuttgart, Germany

Abstract

ABSTRACT A genomic library of Pseudomonas fluorescens DSM 50106 in a λRESIII phage vector was screened in Escherichia coli K-12 for esterase activity by using α-naphthyl acetate and Fast Blue RR. A 3.2-kb DNA fragment was subcloned from an esterase-positive clone and completely sequenced. Esterase EstF1 was encoded by a 999-bp open reading frame (ORF) and exhibited significant amino acid sequence identity with members of the serine hydrolase family. The deduced amino acid sequences of two other C-terminal truncated ORFs exhibited homology to a cyclohexanone monooxygenase and an alkane hydroxylase. However, esterase activity was not induced by growing of P. fluorescens DSM 50106 in the presence of several cyclic ketones. The esterase gene was fused to a His tag and expressed in E. coli . The gene product was purified by zinc ion affinity chromatography and characterized. Detergents had to be added for purification, indicating that the enzyme was membrane bound or membrane associated. The optimum pH of the purified enzyme was 7.5, and the optimum temperature was 43°C. The showed highest purified enzyme activities towards lactones. The activity increased from γ-butyrolactone (18.1 U/mg) to ɛ-caprolactone (21.8 U/mg) to δ-valerolactone (36.5 U/mg). The activities towards the aliphatic esters were significantly lower; the only exception was the activity toward ethyl caprylate, which was the preferred substrate.

Publisher

American Society for Microbiology

Subject

Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology

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