Ryu H S, Kim H K, Choi W C, Kim M H, Park S Y, Han N S, Oh T K, Lee J K
Korea Research Institute of Bioscience & Biotechnology, P.O. Box 115, Taejon, 305-600, South Korea.
Appl Microbiol Biotechnol. 2006 Apr;70(3):321-6. doi: 10.1007/s00253-005-0058-y. Epub 2005 Aug 9.
A Photobacterium strain, M37, showing lipolytic activity, was previously isolated from an intertidal flat of the Yellow Sea in Korea and identified as Photobacterium lipolyticum sp. nov. In the present study, the corresponding gene was cloned using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (1,023 bp) corresponded to a protein of 340 amino acid residues with a molecular weight of 38,026. No sequence similarity was found with any known bacterial lipases/esterases; instead, the most similar enzymes were several filamentous fungal lipases. Although the similarity was very low (less than 16%), there were many conserved regions over the entire sequence and N-terminal oxyanion hole (RG) region, a signature sequence of filamentous fungal lipases. The novel protein M37 was produced in both a soluble and insoluble form when the Escherichia coli cells harboring the gene were cultured at 18 degrees C. The soluble protein exhibited lipase activity in a pH-stat assay using an olive oil emulsion. The M37 lipase also displayed a maximum activity at 25 degrees C and maintained its activity at a low temperature range (5-25 degrees C) with an activation energy (E(a)) of 2.07 kcal/mol. Accordingly, these results indicate that the M37 lipase from P. lipolyticum sp. nov. is a new cold-adapted enzyme.
一株具有脂肪分解活性的发光杆菌菌株M37，先前从韩国黄海的潮间带分离得到，并被鉴定为新种解脂发光杆菌。在本研究中，采用鸟枪法克隆了相应基因。从核苷酸序列（1023 bp）推导的氨基酸序列对应于一个由340个氨基酸残基组成、分子量为38026的蛋白质。未发现与任何已知细菌脂肪酶/酯酶有序列相似性；相反，最相似的酶是几种丝状真菌脂肪酶。尽管相似性很低（低于16%），但在整个序列和N端氧阴离子孔（RG）区域有许多保守区域，这是丝状真菌脂肪酶的一个特征序列。当携带该基因的大肠杆菌细胞在18℃培养时，新型蛋白质M37以可溶和不溶两种形式产生。在使用橄榄油乳液的pH计测定中，可溶性蛋白质表现出脂肪酶活性。M37脂肪酶在25℃时也表现出最大活性，并在低温范围（5-25℃）保持其活性，活化能（E(a)）为2.07千卡/摩尔。因此，这些结果表明，来自解脂发光杆菌新种的M37脂肪酶是一种新的冷适应酶。
