Department of Biotechnology, School of Life Sciences, Pondicherry University, 605014, Puducherry, India.
Centre for Bioinformatics, School of Life Sciences, Pondicherry University, Pondicherry 605014, India.
Int J Biol Macromol. 2017 Oct;103:47-56. doi: 10.1016/j.ijbiomac.2017.04.111. Epub 2017 May 4.
A gene coding lipase from Bacillus sp. PU1 was cloned and expressed in E. coli BL21(DE3) pLysS. The purified lipase has a molecular weight of 23kDa, is highly alkaline (pH range 8-10) and mesophilic (20-50°C). Three dimensional structure of the lipase was modeled by comparative homology and identified as a typical serine lipase by the presence of conserved Ser77, Asp133, His156. The molecular stability and behavior of the lipase was carried out using MD simulation studies at different pH and temperature was performed in comparison with biochemical analysis. Structural modifications of the lipase under these conditions were trapped by dihedral based FEL analysis and the functional loops (loop-H5/B4 and loop-H6/B5 of lipase) are identified which would cause the catalytic behavior of the lipase by high flexibility. Further characteristic feature of lipase are observed as follows; SDS completely inhibits the lipase activity and enzyme activity is enhanced with non-ionic surfactants. The lipase was highly stable in different organic solvents and also it could tolerate NaCl (0.4-0.8M). This enzyme was found to disrupt the biofilm of tested pathogenic bacterial strains.
从芽孢杆菌 PU1 中克隆并在大肠杆菌 BL21(DE3)pLysS 中表达了编码脂肪酶的基因。纯化的脂肪酶分子量为 23kDa，呈高度碱性（pH 范围 8-10）和嗜温性（20-50°C）。通过比较同源性对脂肪酶的三维结构进行建模，并通过存在保守的 Ser77、Asp133 和 His156 将其鉴定为典型的丝氨酸脂肪酶。通过 MD 模拟研究在不同 pH 和温度下对脂肪酶的分子稳定性和行为进行了研究，并与生化分析进行了比较。通过基于二面角的 FEL 分析捕获了这些条件下脂肪酶的结构修饰，并确定了功能环（脂肪酶的 loop-H5/B4 和 loop-H6/B5），它们通过高灵活性导致脂肪酶的催化行为。还观察到脂肪酶的其他特征如下：SDS 完全抑制脂肪酶活性，非离子表面活性剂增强酶活性。脂肪酶在不同有机溶剂中高度稳定，也能耐受 0.4-0.8M 的 NaCl。该酶被发现可破坏测试的致病性细菌菌株的生物膜。
