Rigidifying Flexible Sites: A Promising Strategy to Improve Thermostability of Lysophospholipase From Pyrococcus abyssi

• Published in: Proteins, structure, function, and bioinformatics structure, function, and bioinformatics, 2024-09
• Main Authors: Nazir, Arshia, Ijaz, Maham, Rehman, Hafiz Muzzammel, Sajjad, Muhammad
• Format: Article
• Language: English
• ISSN: 0887-3585; 1097-0134; 1097-0134
• DOI: 10.1002/prot.26748

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.