Study of the effect of F17A mutation on characteristics of Bacillus thermocatenulatus lipase expressed in Pichia pastoris using in silico and experimental methods

• Published in: Biotechnology and applied biochemistry Vol. 61; no. 3; pp. 264 - 273
• Main Authors: Karimi, Esmat, Karkhane, Ali Asghar, Yakhchali, Bagher, Shamsara, Mehdi, Aminzadeh, Saeed, Torktaz, Ibrahim, Hosseini, Mostafa, Safari, Zahra
• Format: Journal Article
• Language: English
• Published: United States Blackwell Publishing Ltd 01-05-2014; Wiley Subscription Services, Inc
• Subjects: Alanine; Bacillus; Bacillus - enzymology; Bacillus - genetics; Bacillus thermocatenulatus; Crystal structure; docking; Enzymes; Gene Expression - genetics; in silico; Lipase; Lipase - chemistry; Lipase - genetics; Lipase - metabolism; Methods; Molecular Docking Simulation; Mutation; Mutation - genetics; Mutations; oxyanion hole; Phenylalanine - genetics; Pichia - genetics; Pichia pastoris; Residues; docking; in silico; oxyanion hole; Bacillus thermocatenulatus
• ISSN: 0885-4513; 1470-8744
• DOI: 10.1002/bab.1164

Bacillus thermocatenulatus lipase 2 (BTL2), a thermoalkalophilic lipase, is the best studied enzyme for its particular properties, which make it useful in different industries. Displacement of conserved phenylalanine 17 (Phe‐17) residue in the active site of BTL2 has a critical role in oxyanion hole formation, which is important for enzyme activity. In this study, to facilitate oxyanion hole formation, Phe‐17 was substituted with Alanine residue (F17A). The best structures of the opened form of the native and mutated lipases were garnered based on the crystal structures of 2W22. To evaluate catalytic activity, both lipases were docked to a set of ligands using Hex 6.3 software. Following in silico study, both native and mutant btl2 genes were cloned and expressed in Pichia pastoris. Based on the results obtained, the mutation increased lipase lipolytic activity against most of the applied substrates, especially for tributyrin and tricaprylin, by 1.9 and 2.15 fold, respectively. However, optimum temperature and pH were the same for both lipases (60 °C and pH 8.0). As previously reported, it is believed that F17A mutation simplifies oxyanion hole formation and declines steric hindrance in the enzyme active site, which might ultimately lead to more efficient accessibility of substrates.