Department of Microbiology, N.E.S. Science college , Nanded - 431 605 , India ; School of Life Sciences, Biotechnology Research Laboratory, Swami Ramanand Teerth Marathwada University , Nanded - 431 606 , India.
Braz J Microbiol. 2009 Apr;40(2):358-66. doi: 10.1590/S1517-838220090002000028. Epub 2009 Jun 1.
An extra cellular lipase was isolated and purified from the culture broth of Pseudomonas aeruginosa SRT 9 to apparent homogeneity using ammonium sulfate precipitation followed by chromatographic techniques on phenyl Sepharose CL- 4B and Mono Q HR 5/5 column, resulting in a purification factor of 98 fold with specific activity of 12307.8 U/mg. The molecular weight of the purified lipase was estimated by SDS-PAGE to be 29 kDa with isoelectric point of 4.5. Maximum lipase activity was observed in a wide range of temperature and pH values with optimum temperature of 55ºC and pH 6.9. The lipase preferably acted on triacylglycerols of long chain (C14-C16) fatty acids. The lipase was inhibited strongly by EDTA suggesting the enzyme might be metalloprotein. SDS and metal ions such as Hg(2+), Zn(2+), Cu(2+), Ag(2+) and Fe(2+) decreased the lipase activity remarkedly. Its marked stability and activity in organic solvents suggest that this lipase is highly suitable as a biotechnological tool with a variety of applications including organo synthetic reactions and preparation of enantiomerically pure pharmaceuticals. The Km and Vmax value of the purified enzyme for triolein hydrolysis were calculated to be 1.11 mmol/L and 0.05 mmol/L/min respectively.
从铜绿假单胞菌 SRT 9 的发酵液中分离和纯化了一种细胞外脂肪酶，采用硫酸铵沉淀和苯基 Sepharose CL-4B 和 Mono Q HR 5/5 柱层析技术，将其纯化至明显均一，得到 98 倍的纯化倍数和 12307.8 U/mg 的比活性。通过 SDS-PAGE 测定纯化脂肪酶的分子量为 29 kDa，等电点为 4.5。该脂肪酶在较宽的温度和 pH 值范围内表现出最大的酶活性，最适温度为 55°C，最适 pH 值为 6.9。该脂肪酶优先作用于长链（C14-C16）脂肪酸的三酰基甘油。该脂肪酶强烈被 EDTA 抑制，表明该酶可能是金属蛋白酶。SDS 和金属离子，如 Hg(2+)、Zn(2+)、Cu(2+)、Ag(2+)和 Fe(2+)，显著降低了脂肪酶的活性。其在有机溶剂中的显著稳定性和活性表明，该脂肪酶非常适合作为生物技术工具，具有多种应用，包括有机合成反应和制备对映体纯药物。纯化酶对三油酸甘油酯水解的 Km 和 Vmax 值分别计算为 1.11 mmol/L 和 0.05 mmol/L/min。
