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Applied and Environmental Microbiology, November 2009, p. 6729-6735, Vol. 75, No. 21
0099-2240/09/$08.00+0     doi:10.1128/AEM.01563-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Cloning and Characterization of an Intracellular Esterase from the Wine-Associated Lactic Acid Bacterium Oenococcus oeni{triangledown} ,{dagger}

Krista M. Sumby, Angela H. Matthews,{ddagger} Paul R. Grbin, and Vladimir Jiranek*

School of Agriculture, Food and Wine, The University of Adelaide, PMB 1, Glen Osmond, South Australia 5064, Australia

Received 3 July 2009/ Accepted 26 August 2009

We report the cloning and characterization of EstB28, the first esterase to be so characterized from the wine-associated lactic acid bacterium, Oenococcus oeni. The published sequence for O. oeni strain PSU-1 was used to identify putative esterase genes and design PCR primers in order to amplify the corresponding region from strain Ooeni28, an isolate intended for inoculation of wines. In this way a 912-bp open reading frame (ORF) encoding a putative esterase of 34.5 kDa was obtained. The amino acid sequence indicated that EstB28 is a member of family IV of lipolytic enzymes and contains the GDSAG motif common to other lactic acid bacteria. This ORF was cloned into Escherichia coli using an appropriate expression system, and the recombinant esterase was purified. Characterization of EstB28 revealed that the optimum temperature, pH, and ethanol concentration were 40°C, pH 5.0, and 28% (vol/vol), respectively. EstB28 also retained marked activity under conditions relevant to winemaking (10 to 20°C, pH 3.5, 14% [vol/vol] ethanol). Kinetic constants were determined for EstB28 with p-nitrophenyl (pNP)-linked substrates ranging in chain length from C2 to C18. EstB28 exhibited greatest specificity for C2 to C4 pNP-linked substrates.

* Corresponding author. Mailing address: School of Agriculture, Food and Wine, The University of Adelaide, PMB 1, Glen Osmond, South Australia 5064, Australia. Phone: 61-8-8303-6651. Fax: 61-8-8303-7415. E-mail: vladimir.jiranek{at}adelaide.edu.au

{triangledown} Published ahead of print on 4 September 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Present address: Invitrogen Australia Pty. Limited, Level 3, 501 Blackburn Road, Mount Waverley, Victoria 3149, Australia.

Applied and Environmental Microbiology, November 2009, p. 6729-6735, Vol. 75, No. 21
0099-2240/09/$08.00+0     doi:10.1128/AEM.01563-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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