A new esterase showing similarity to putative dienelactone hydrolase from a strict marine bacterium, Vibrio sp. GMD509.
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.
Park
Sang-Yi
SY
Korea Ocean Research and Development Institute, Ansan P.O. Box 29, Seoul, South Korea.
Kim
Jun-Tae
JT
Kang
Sung Gyun
SG
Woo
Jung-Hee
JH
Lee
Jung-Hyun
JH
Choi
Hyoung-Tae
HT
Kim
Sang-Jin
SJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature; D014733:Vibrio / Q000145:classification / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D004950:Esterases; D002265:Carboxylic Ester Hydrolases; C046130:carboxymethylenebutenolidase