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Applied and Environmental Microbiology, February 1999, p. 477-482, Vol. 65, No. 2
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

V. Khalameyzer,1 I. Fischer,2 U. T. Bornscheuer,1,* and J. Altenbuchner2

Institute for Technical Biochemistry,1 and Institute for Industrial Genetics,2 University of Stuttgart, 70569 Stuttgart, Germany

Received 11 September 1998/Accepted 2 November 1998

A genomic library of Pseudomonas fluorescens DSM 50106 in a lambda RESIII phage vector was screened in Escherichia coli K-12 for esterase activity by using alpha -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 gamma -butyrolactone (18.1 U/mg) to varepsilon -caprolactone (21.8 U/mg) to delta -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.

* Corresponding author. Mailing address: Institute for Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. Phone: 49 711 685 4523. Fax: 49 711 685 3196. E-mail: bornscheuer{at}po.uni-stuttgart.de.

Applied and Environmental Microbiology, February 1999, p. 477-482, Vol. 65, No. 2
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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