Abstract |
Vibrio sp. GMD509, a marine bacterium isolated from eggs of the sea hare, exhibited lipolytic activity on tributyrin (TBN) plate, and the gene representing lipolytic activity was cloned. As a result, an open reading frame (ORF) consisting of 1,017 bp (338 aa) was found, and the deduced amino acid sequence of the ORF showed low similarity (< 20%) to alpha/beta hydrolases such as dienelactone hydrolases and esterase/lipase with G-X(1)-S-X(2)-G sequence conserved. Phylogenetic analysis suggested that the protein belonged to a new family of esterase/lipase together with various hypothetical proteins. The enzyme was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme (Vlip509) showed the best hydrolyzing activity toward p-nitrophenyl butyrate (C(4)) among various p-nitrophenyl esters (C(2) to C(18)), and optimal activity of Vlip509 occurred at 30 degrees C and pH 8.5, respectively. Kinetic parameters toward p-nitrophenyl butyrate were determined as K (m) (307 muM), k (cat) (5.72 s(-1)), and k (cat)/K (m) (18.61 s(-1) mM(-1)). Furthermore, Vlip509 preferentially hydrolyzed the S-enantiomer of racemic ofloxacin ester. Despite its sequence homology to dienelactone hydrolase, Vlip509 showed no dienelactone hydrolase activity. This study represents the identification of a novel lipolytic enzyme from marine environment.
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Authors | Sang-Yi Park, Jun-Tae Kim, Sung Gyun Kang, Jung-Hee Woo, Jung-Hyun Lee, Hyoung-Tae Choi, Sang-Jin Kim |
Journal | Applied microbiology and biotechnology
(Appl Microbiol Biotechnol)
Vol. 77
Issue 1
Pg. 107-15
(Nov 2007)
ISSN: 0175-7598 [Print] Germany |
PMID | 17712554
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Chemical References |
- Bacterial Proteins
- DNA, Bacterial
- Recombinant Proteins
- Esterases
- Carboxylic Ester Hydrolases
- carboxymethylenebutenolidase
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Topics |
- Amino Acid Sequence
- Bacterial Proteins
(chemistry, genetics, metabolism)
- Carboxylic Ester Hydrolases
(chemistry, genetics, metabolism)
- Cloning, Molecular
- DNA, Bacterial
(chemistry, genetics)
- Electrophoresis, Polyacrylamide Gel
- Esterases
(chemistry, classification, metabolism)
- Hydrogen-Ion Concentration
- Molecular Sequence Data
- Phylogeny
- Recombinant Proteins
(chemistry, isolation & purification, metabolism)
- Sequence Analysis, DNA
- Sequence Homology, Amino Acid
- Stereoisomerism
- Substrate Specificity
- Temperature
- Vibrio
(classification, enzymology, genetics)
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