Cloning and characterization of an intracellular esterase from the wine-associated lactic acid bacterium Oenococcus oeni.
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 degrees C, pH 5.0, and 28% (vol/vol), respectively. EstB28 also retained marked activity under conditions relevant to winemaking (10 to 20 degrees 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 C(2) to C(18). EstB28 exhibited greatest specificity for C(2) to C(4) pNP-linked substrates.
Sumby
Krista M
KM
School of Agriculture, Food and Wine, The University of Adelaide, PMB 1, Glen Osmond, South Australia 5064, Australia.
Matthews
Angela H
AH
Grbin
Paul R
PR
Jiranek
Vladimir
V
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D056585:Oenococcus / Q000201:enzymology* / Q000235:genetics; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D004950:Esterases