Nazir Arshia, Ijaz Maham, Rehman Hafiz Muzzammel, Sajjad Muhammad
School of Biological Sciences, University of the Punjab, Lahore, Pakistan.
School of Biochemistry and Biotechnology, University of the Punjab, Lahore, Pakistan.
Proteins. 2025 Feb;93(2):527-542. doi: 10.1002/prot.26748. Epub 2024 Sep 18.
High thermostability of the enzymes is one of the distinguishing characteristics that increase their industrial utility. In the current research work, rigidifying the flexible amino acid residues of a lysophospholipase (Pa-LPL) from Pyrococcus abyssi was used as a protein engineering approach to improve its thermostability. A truncated variant of Pa-LPL (t-LPL∆12) was constructed by trimming its 12 amino acid residues (50-61) through overlap extension PCR. The truncated enzyme worked optimally at 65°C and pH 6.5 with remarkable thermostability at 65°C-85°C. In comparison to wild-type Pa-LPL, 5.8 and 1.2-fold increase in half-life (t) of t-LPL∆12 was observed at 65 (optimum temperature) and 95°C, respectively. The activity of t-LPL∆12 was stimulated by 1 mM Cu followed by Ca, Ni, Co, and Mg. Both substrate docking and experimental results indicated that the truncated enzyme could hydrolyze a variety of p-nitrophenyl esters. K , V , and K for enzymatic hydrolysis of p-nitrophenyl butyrate were calculated to be 1 ± 0.087 mM, 1456 ± 36.474 U/mg, and 1.397 × 10 min, respectively. In short, broad substrate specificity and thermostability of t-LPL∆12 are some of the distinctive features that make it an ideal candidate for degumming of vegetable oils.
酶的高热稳定性是提高其工业实用性的显著特征之一。在当前的研究工作中，通过蛋白质工程方法，使来自深渊嗜热栖热菌的溶血磷脂酶（Pa-LPL）的柔性氨基酸残基刚性化，以提高其热稳定性。通过重叠延伸PCR去除其12个氨基酸残基（50-61）构建了Pa-LPL的截短变体（t-LPL∆12）。截短后的酶在65°C和pH 6.5时表现出最佳活性，在65°C至85°C时具有显著的热稳定性。与野生型Pa-LPL相比，t-LPL∆12在65°C（最适温度）和95°C下的半衰期（t）分别增加了5.8倍和1.2倍。1 mM Cu对t-LPL∆12的活性有促进作用，其次是Ca、Ni、Co和Mg。底物对接和实验结果均表明，截短后的酶能够水解多种对硝基苯酯。计算得出t-LPL∆12对丁酸对硝基苯酯的酶促水解的Km、Vmax和kcat分别为1±0.087 mM、1456±36.474 U/mg和1.397×10 min。简而言之，t-LPL∆12广泛的底物特异性和热稳定性是使其成为植物油脱胶理想候选物的一些显著特征。
