Abstract：
Appropriate amino acid substitutions are critical for protein engineering to redesign catalytic properties of industrially important enzymes like lipases. The present study aimed for improving the environmental stability of lipase from Pseudomonas plecoglossicida S7 through site-directed mutagenesis driven by computational studies. lipA gene was amplified and sequenced. Both wild type (WT) and mutant type (MT) lipase genes were expressed into the pET SUMO system. The expressed proteins were purified and characterized for pH and thermostability. The lipase gene belonged to subfamily I.1 lipase. Molecular dynamics revealed that Y12F-palmitic acid complex had a greater binding affinity (-6.3 Kcal/mol) than WT (-6.0 Kcal/mol) complex. Interestingly, MDS showed that the binding affinity of WT-complex (-130.314 ± 15.11 KJ/mol) was more than mutant complex (-108.405 ± 69.376 KJ/mol) with a marked increase in the electrostatic energy of mutant (-26.969 ± 12.646 KJ/mol) as compared to WT (-15.082 ± 13.802 KJ/mol). Y12F mutant yielded 1.27 folds increase in lipase activity at 55 °C as compared to the purified WT protein. Also, Y12F mutant showed increased activity (~ 1.2 folds each) at both pH 6 and 10. P. plecoglossicida S7. Y12F mutation altered the kinetic parameters of MT (Km- 1.38 mM, Vmax- 22.32 µM/min) as compared to WT (Km- 1.52 mM, Vmax- 29.76 µM/min) thus increasing the binding affinity of mutant lipase. Y12F mutant lipase with better pH and thermal stability can be used in biocatalysis.
摘要：
适当的氨基酸取代对于蛋白质工程重新设计工业上重要的酶如脂肪酶的催化性质是关键的。本研究旨在通过计算研究驱动的定点诱变来改善PseudomonasplecoglossicidaS7脂肪酶的环境稳定性。对lipA基因进行扩增和测序。野生型（WT）和突变型（MT）脂肪酶基因均表达到pETSUMO系统中。纯化表达的蛋白质并表征pH和热稳定性。脂肪酶基因属于I.1脂肪酶亚家族。分子动力学显示Y12F-棕榈酸复合物具有比WT(-6.0Kcal/mol)复合物更大的结合亲和力(-6.3Kcal/mol)。有趣的是,MDS显示，WT复合物的结合亲和力（-130.314±15.11KJ/mol）高于突变体复合物（-108.405±69.376KJ/mol），突变体的静电能显着增加（-26.969±12.646KJ/mol）与WT（-15.082±13.802KJ/mol）相比。与纯化的WT蛋白相比，Y12F突变体在55°C下的脂肪酶活性增加了1.27倍。此外，Y12F突变体在pH为6和10时显示出增加的活性（每个〜1.2倍）。P.plecoglossicidaS7。Y12F突变改变了MT的动力学参数(Km-1.38mM，Vmax-22.32µM/min）与WT（Km-1.52mM，Vmax-29.76µM/min），从而增加突变脂肪酶的结合亲和力。Y12F突变体脂肪酶具有较好的pH值和热稳定性，可用于生物催化。
