Verma M L, Kanwar S S
Himachal Pradesh University, Department of Biotechnology, Shimla 171 005, India.
Acta Microbiol Immunol Hung. 2010 Sep;57(3):191-207. doi: 10.1556/AMicr.57.2010.3.4.
A low molecular mass alkaliphilic extra-cellular lipase of Bacillus cereus MTCC 8372 was purified 35-fold by hydrophobic interaction (Octyl-Sepharose) chromatography. The purified enzyme was found to be electrophoretically pure by denaturing gel electrophoresis and possessed a molecular mass of approximately 8 kDa. It is a homopentamer of 40 kDa as revealed by native-PAGE. The lipase was optimally active at 55 °C and retained approximately half of its original activity after 40 min incubation at 55 °C. The enzyme was maximally active at pH 8.5. Mg2+, Cu2+, Ca2+, Hg2+, Al3+ and Fe3+ at 1 mM enhanced hydrolytic activity of the lipase. Interestingly, Hg2+ ions synergized and Zn2+ and Co2+ ions antagonized the lipase activity. Among surfactants, Tween 80 promoted the lipase activity. Phenyl methyl sulfonyl fluoride (PMSF, 15 mM) decreased 98% of original activity of lipase. The lipase was highly specific towards p-nitrophenyl palmitate and showed a Vmax and Km of 0.70 mmol.mg⁻¹.min⁻¹ and 32 mM for hydrolysis of pNPP.
蜡样芽孢杆菌MTCC 8372的一种低分子量嗜碱胞外脂肪酶通过疏水相互作用（辛基琼脂糖）色谱法纯化了35倍。通过变性凝胶电泳发现纯化后的酶在电泳上是纯的，其分子量约为8 kDa。天然聚丙烯酰胺凝胶电泳显示它是一种40 kDa的同五聚体。该脂肪酶在55℃时活性最佳，在55℃孵育40分钟后保留了约一半的原始活性。该酶在pH 8.5时活性最高。1 mM的Mg2+、Cu2+、Ca2+、Hg2+、Al3+和Fe3+增强了脂肪酶的水解活性。有趣的是，Hg2+离子具有协同作用，而Zn2+和Co2+离子则拮抗脂肪酶的活性。在表面活性剂中，吐温80促进脂肪酶活性。苯甲基磺酰氟（PMSF，15 mM）使脂肪酶的原始活性降低了98%。该脂肪酶对棕榈酸对硝基苯酯具有高度特异性，水解对硝基苯磷酸酯的Vmax和Km分别为0.70 mmol·mg⁻¹·min⁻¹和32 mM。
