Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Thailand.
Appl Biochem Biotechnol. 2012 Apr;166(8):1969-82. doi: 10.1007/s12010-012-9624-9. Epub 2012 Mar 21.
The extracellular lipase from Streptomyces thermocarboxydus ME168 was purified to 9.5-fold with 20% yield, following concentration by acetone precipitation, ion exchange chromatography (Resource Q) and gel filtration chromatography (Superdex 200), respectively. The purified enzyme had an apparent molecular mass of 21 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The N-terminal sequence of the lipase was ASDFDDQILG and was different from most other reported lipase. The enzyme showed maximum activity at 50 °C with the half-life of 180 min at 65 °C. It showed high stability at a broad pH range of 5.5-9.5 and was thermostable at the temperature range of 25-60 °C. The K(m) and V(max) were 0.28 mM and 1,428 U/mg, respectively, using p-nitrophenyl palmitate as substrate. It was active toward p-nitrophenyl ester with medium to long acyl chain (C(8)-C(16)). Lipase activity was inhibited by Zn(2+), dithiothreitol (DTT), EDTA and some organic solvents, e.g., ethanol, acetone, dioxane, acetronitrile, tert-butanol and pyridine. Immobilized crude lipase of S. thermocarboxydus ME168 on celite could be used to synthesize sugar esters from glucose and vinyl acetate, vinyl butyrate or vinyl caproate in tert-butanol:pyridine (55:45 v/v) at 45 °C with conversion yields of 93, 67 and 55%, respectively.
从嗜热链霉菌 ME168 中提取的细胞外脂肪酶经丙酮沉淀、离子交换色谱（Resource Q）和凝胶过滤色谱（Superdex 200）分别浓缩 20%后，纯度提高到 9.5 倍。经十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）测定，纯化后的酶表观分子量为 21 kDa。脂肪酶的 N 端序列为 ASDFDDQILG，与大多数报道的脂肪酶不同。该酶在 50°C 时活性最高，半衰期为 180min，在 65°C 时半衰期为 65°C。该酶在 pH 值 5.5-9.5 的较宽范围内具有较高的稳定性，在 25-60°C 的温度范围内具有热稳定性。以对硝基苯棕榈酸酯为底物时，K(m)和 V(max)分别为 0.28 mM 和 1428 U/mg。它对具有中长链（C(8)-C(16)）的 p-硝基苯酯具有活性。脂肪酶活性被 Zn(2+)、二硫苏糖醇（DTT）、EDTA 和一些有机溶剂（如乙醇、丙酮、二恶烷、乙腈、叔丁醇和吡啶）抑制。固定在 celite 上的嗜热链霉菌 ME168 粗脂肪酶可用于在叔丁醇：吡啶（55:45 v/v）中，在 45°C 下，从葡萄糖和醋酸乙烯酯、醋酸丁酸酯或醋酸己酯合成糖酯，转化率分别为 93%、67%和 55%。
