Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan.
Appl Biochem Biotechnol. 2012 Sep;168(2):327-38. doi: 10.1007/s12010-012-9776-7. Epub 2012 Jun 30.
A psychrotrophic Pseudomonas sp. TK-3 was isolated from dirty and cool stream water in Toyama, Japan from which we cloned and characterized the bacterial lipase LipTK-3. The sequenced DNA fragment contains an open reading frame of 1,428 bp that encoded a protein of 476 amino acids with an estimated molecular mass of 50,132 Da. The lipase showed high sequence similarity to those of subfamily Ι.3 lipase and had a conserved GXSXG motif around the catalytic Ser residue. Its optimal temperature was 20-25 °C, lower than in most other subfamily Ι.3 lipases. The lipase exhibited about 30 % of maximal activity at 5 °C. The optimal pH value was 8.0. The activity was strongly inhibited by EDTA and was highly dependent on Ca(2+). Tricaprylin and p-nitrophenyl caprylate were the most favorable substrates among the triglycerides and p-nitrophenyl esters, respectively. LipTK-3 also showed high activity towards natural substrates including edible vegetable oils and animal fats. Furthermore, LipTK-3 was very active and stable in the presence of several detergents, metal ions, and organic solvents. This cold-adapted lipase may prove useful for future applications.
从日本富山的脏冷溪流水中分离到一株嗜冷假单胞菌 TK-3，我们对其进行了克隆和鉴定，得到了细菌脂肪酶 LipTK-3。测序的 DNA 片段包含一个 1428bp 的开放阅读框，编码一个由 476 个氨基酸组成的蛋白质，估计分子量为 50132Da。该脂肪酶与亚家族 Ι.3 脂肪酶具有很高的序列相似性，并且在催化丝氨酸残基周围具有保守的 GXSXG 模体。其最适温度为 20-25°C，低于大多数其他亚家族 Ι.3 脂肪酶。该脂肪酶在 5°C 时表现出约 30%的最大活性。最适 pH 值为 8.0。该活性被 EDTA 强烈抑制，并且高度依赖于 Ca(2+)。三辛酸甘油酯和对硝基苯辛酸酯分别是甘油三酯和对硝基苯酯中最有利于脂肪酶活性的底物。LipTK-3 对包括食用植物油和动物脂肪在内的天然底物也表现出很高的活性。此外，LipTK-3 在存在几种洗涤剂、金属离子和有机溶剂的情况下具有非常高的活性和稳定性。这种耐冷脂肪酶在未来的应用中可能具有很高的应用价值。
