Department of Environmental Science, University of Kashmir, Srinagar, 190006, Jammu and Kashmir, India.
Microbiology Research Laboratory, Centre of Research for Development (CORD), University of Kashmir, Srinagar, 190006, Jammu and Kashmir, India.
Curr Genet. 2022 Apr;68(2):243-251. doi: 10.1007/s00294-021-01224-w. Epub 2021 Nov 27.
Lipases from Pseudomonas species are particularly useful due to their broader biocatalytic applications and temperature activity. In this study, we amplified the gene encoding wild-type cold-active lipase from the genome of psychrotrophic bacterium isolated from the Himalayan glacier. The isolated CRBC14 strain was identified as Pseudomonas sp. based on the 16S rRNA gene sequence. Lipase activity was determined by observing the hydrolysis zone on nutrient agar containing tributyrin (1%, v/v). The sequence analysis of cold-active lipase revealed a protein of 611 amino acids with a calculated molecular mass of 63.71 kDa. The three-dimensional structure of this lipase was generated through template-supported modeling. Distinct techniques stamped the model quality, following which the binding free energies of tributyrin and oleic acid in the complex state with this enzymatic protein were predicted through molecular mechanics generalized born surface area (MMGBSA). A relative comparison of binding free energy values of these substrates indicated tributyrin's comparatively higher binding propensity towards the lipase. Using molecular docking, we evaluated the binding activity of cold-active lipase against tributyrin and oleic acid. Our docking analysis revealed that the lipase had a higher affinity for tributyrin than oleic acid, as evidenced by our measurement of the hydrolysis zone on two media plates. This study will help to understand the bacterial diversity of unexplored Himalayan glaciers and the possible application of their cold-adapted enzymes.
由于其更广泛的生物催化应用和温度活性，假单胞菌属的脂肪酶特别有用。在这项研究中，我们扩增了从喜马拉雅冰川分离的嗜冷菌基因组中编码野生型冷活性脂肪酶的基因。根据 16S rRNA 基因序列，分离出的 CRBC14 菌株被鉴定为假单胞菌属。通过观察含有三丁酸甘油酯（1%，v/v）的营养琼脂上的水解区来确定脂肪酶活性。冷活性脂肪酶的序列分析揭示了一种 611 个氨基酸的蛋白质，计算分子量为 63.71 kDa。通过模板支持建模生成了该脂肪酶的三维结构。通过多种技术对模型质量进行了标记，随后通过分子力学广义 Born 表面积（MMGBSA）预测了该酶蛋白与三丁酸甘油酯和油酸复合物状态下的结合自由能。对这些底物的结合自由能值进行相对比较表明，三丁酸甘油酯对脂肪酶具有较高的结合倾向。通过分子对接，我们评估了冷活性脂肪酶对三丁酸甘油酯和油酸的结合活性。我们的对接分析表明，脂肪酶对三丁酸甘油酯的亲和力高于油酸，这可以从我们在两种培养基平板上测量的水解区得到证明。这项研究将有助于了解喜马拉雅冰川中未被探索的细菌多样性及其冷适应酶的可能应用。
