Department of Biotechnology, Panjab University, Sector 14, Chandigarh 160014, India.
Mol Biol Rep. 2012 Mar;39(3):2795-804. doi: 10.1007/s11033-011-1038-1. Epub 2011 Jun 16.
A gene encoding extracellular lipase was cloned and characterized from metagenomic DNA extracted from hot spring soil. The recombinant gene was expressed in E. coli and expressed protein was purified to homogeneity using hydrophobic interactions chromatography. The mature polypeptide consists of 388 amino acids with apparent molecular weight of 43 kDa. The enzyme displayed maximum activity at 50 °C and pH 9.0. It showed thermal stability up to 40 °C without any loss of enzyme activity. Nearly 80% enzyme activity was retained at 50 °C even after incubation for 75 min. However above 50 °C the enzyme displayed thermal instability. The half life of the enzyme was determined to be 5 min at 60 °C. Interestingly the CD spectroscopic study carried out in the temperature range of 25-95 °C revealed distortion in solution structure above 35 °C. However the intrinsic tryptophan fluorescence spectroscopic study revealed that even with the loss of secondary structure at 35 °C and above the tertiary structure was retained. With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K ( m ), V ( max ) and K ( cat ) of 0.73 ± 0.18 μM, 239 ± 16 μmol/ml/min and 569 s(-1) respectively. Enzyme activity was strongly inhibited by CuCl(2), HgCl(2) and DEPC but not by PMSF, eserine and SDS. The protein retained significant activity (~70%) with Triton X-100. The enzyme displayed 100% activity in presence of 30% n-Hexane and acetone.
从温泉土壤中提取的宏基因组 DNA 中克隆并表征了一种编码细胞外脂肪酶的基因。该重组基因在大肠杆菌中表达，并用疏水相互作用色谱法将表达的蛋白质纯化至均一性。成熟多肽由 388 个氨基酸组成，表观分子量为 43 kDa。该酶在 50°C 和 pH 9.0 时显示出最大活性。它在 40°C 下表现出热稳定性，没有任何酶活性损失。即使在孵育 75 分钟后，在 50°C 下仍保留近 80%的酶活性。然而，高于 50°C 时，该酶表现出热不稳定性。该酶的半衰期在 60°C 下测定为 5 分钟。有趣的是，在 25-95°C 的温度范围内进行的 CD 光谱研究表明，在 35°C 以上时溶液结构发生扭曲。然而，内源性色氨酸荧光光谱研究表明，即使在 35°C 及以上失去二级结构，三级结构仍得以保留。以对硝基苯棕榈酸酯为底物，该酶表现出 K ( m )、V ( max ) 和 K ( cat ) 分别为 0.73 ± 0.18 μM、239 ± 16 μmol/ml/min 和 569 s(-1)。酶活性强烈抑制 CuCl(2)、HgCl(2)和 DEPC，但不受 PMSF、依色林和 SDS 抑制。该蛋白质在 Triton X-100 存在下保留了显著的活性（~70%）。该酶在 30%正己烷和丙酮存在下显示出 100%的活性。
