Park Sang-Yi, Kim Jun-Tae, Kang Sung Gyun, Woo Jung-Hee, Lee Jung-Hyun, Choi Hyoung-Tae, Kim Sang-Jin
Korea Ocean Research and Development Institute, Ansan P.O. Box 29, Seoul, South Korea.
Appl Microbiol Biotechnol. 2007 Nov;77(1):107-15. doi: 10.1007/s00253-007-1134-2. Epub 2007 Aug 22.
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.
从海兔卵中分离出的海洋细菌弧菌属GMD509在三丁酸甘油酯（TBN）平板上表现出脂解活性，并克隆了代表脂解活性的基因。结果，发现了一个由1017个碱基对（338个氨基酸）组成的开放阅读框（ORF），该ORF推导的氨基酸序列与具有保守G-X(1)-S-X(2)-G序列的α/β水解酶如二烯内酯水解酶和酯酶/脂肪酶的相似性较低（<20%）。系统发育分析表明，该蛋白与各种假定蛋白一起属于酯酶/脂肪酶的一个新家族。该酶在大肠杆菌中过量表达并纯化至同质。纯化后的酶（Vlip509）在各种对硝基苯酯（C(2)至C(18)）中对丁酸对硝基苯酯（C(4)）表现出最佳水解活性，Vlip509的最佳活性分别出现在30℃和pH 8.5时。测定了对丁酸对硝基苯酯的动力学参数，分别为K (m)（307 μM）、k (cat)（5.72 s(-1)）和k (cat)/K (m)（18.61 s(-1) mM(-1)）。此外，Vlip509优先水解外消旋氧氟沙星酯的S-对映体。尽管其与二烯内酯水解酶具有序列同源性，但Vlip509没有表现出二烯内酯水解酶活性。本研究鉴定了一种来自海洋环境的新型脂解酶。
