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Study of the effect of F 17 A mutation on characteristics of B acillus thermocatenulatus lipase expressed in P ichia pastoris using in silico and experimental methods
Author(s) -
Karimi Esmat,
Karkhane Ali Asghar,
Yakhchali Bagher,
Shamsara Mehdi,
Aminzadeh Saeed,
Torktaz Ibrahim,
Hosseini Mostafa,
Safari Zahra
Publication year - 2014
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1164
Subject(s) - lipase , oxyanion hole , pichia pastoris , active site , tributyrin , chemistry , enzyme , biochemistry , mutant , stereochemistry , gene , recombinant dna
B acillus thermocatenulatus lipase 2 ( BTL 2), a thermoalkalophilic lipase, is the best studied enzyme for its particular properties, which make it useful in different industries. Displacement of conserved phenylalanine 17 ( P he‐17) residue in the active site of BTL 2 has a critical role in oxyanion hole formation, which is important for enzyme activity. In this study, to facilitate oxyanion hole formation, P he‐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 P ichia 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.