Department of Biotechnology, BMS Block 1, South Campus, Panjab University, Sector 25 Chandigarh 160014, India.
Int J Biol Macromol. 2018 Mar;108:845-852. doi: 10.1016/j.ijbiomac.2017.10.176. Epub 2017 Oct 31.
Thermostability of enzymes is an important issue in protein engineering and has been studied in detail. Still there is no hard and fast rule to define the conditions which will provide thermal stability. Understanding the various factors and mechanism responsible for thermal stability will add on new insights into our present knowledge in this area. Pro247-Ser variant was constructed based on homology modelling and rational design. It exhibited 60 fold increase in thermal stability at 60°C and+0.7M shift in C value for urea denaturation as compared to WT. Variant displayed noticeable tolerance to organic solvents. With decrease in K, catalytic efficiency of Pro247-Ser variant was increased by 12 fold. The activity and stability assay including circular dichroism and fluorescence spectroscopy favoured increased thermal performance of variant. Hydrolytic activity of variant was found to be high in comparison to control for all p-nitrophenol esters investigated. The immobilized variant enzyme demonstrated nearly two fold enhanced conversion of methyl oleate than WT enzyme. The additional molecular interactions of variant residue might contribute to increased thermostability of lipase. The homology modeling predicted formation of additional hydrogen bonds between Ser247/O-Thr251/OG1 as well as Ser247/O-Glu250/N.
酶的热稳定性是蛋白质工程中的一个重要问题，已经进行了详细的研究。尽管没有明确的规则来定义提供热稳定性的条件，但理解导致热稳定性的各种因素和机制将为我们目前在这一领域的知识增添新的见解。Pro247-Ser 变体是基于同源建模和合理设计构建的。与 WT 相比，它在 60°C 时的热稳定性提高了 60 倍，在脲变性的 C 值上提高了+0.7M。变体表现出对有机溶剂的明显耐受性。随着 K 的降低，Pro247-Ser 变体的催化效率提高了 12 倍。包括圆二色性和荧光光谱在内的活性和稳定性测定支持变体的热性能提高。与对照相比，变体的水解活性在所有研究的对硝基苯酚酯中都较高。固定化变体酶的转化率比 WT 酶高近两倍。变体残基的额外分子相互作用可能有助于脂肪酶的热稳定性提高。同源建模预测 Ser247/O-Thr251/OG1 以及 Ser247/O-Glu250/N 之间形成额外的氢键。
