Lipases (triacylglycerol acyl hydrolase EC 3.1.1.3) are the most important industrial enzymes that are used in various industries including food, detergents, pharmaceutical products, leather, textile, cosmetic and paper. Bacterial lipases are member of a/b hydrolase family that hydrolyz triacylglycerols at the water-lipid interface. The comparision of Bacillus lipases with a/b hydrolase structure showed that a5 helix enter in Bacillus lipases during the evolution. Improvement of catalytic activity and substrate specificity by using protein engineering is significant in commercial applications. In this study, after bioinformatic researches, a5 helix from btl 2 gene of Bacillus thermocatenulatus was deleted by SOE-PCR method and mutant gene was cloned into TA vector, Then was transformed into E.coli bacteria. After the confirmation of cloning, mutant gene was cloned into pPICZaB expression vector; afterwards, it was transformed to Pichia pastoris yeast. Moreover, after the confirmation of expression, enzyme's activity of lipase purified with DE52 resin was measured with pH-stat device. The effect of various substrate, temperature, thermostability, pH, metal ions, detergents and organic solvents on normal and mutant enzymes was examined. The results indicated that the mutant lipase’s activity is higher than the native lipase in a wider range of pH. The mutation has dramatically increased enzyme’s activity in the presence of substrate and mainly C8 substrate. Mutant lipase tolerates 45-60° C temperatures and this feature leads to various applications in detergents, food and oleochemical industries. The results demonstrated that the mutant enzymes’ activity decreased in presence of different organic solvents, detergents and some ions in comparison with native enzyme. In terms of thermostability, mutant enzyme did not change perceptibly comparing to the native one. Probably, the deletion of a5 helix causes less exposure of hydrophobic molecules to the surface and in some cases this turn may result the increasing of enzyme activity. Furthermore, it is supposed that the mutation changed the active site of mutant enzyme and provided the ability of hydrolyzing substrate with longer hydrocarbon chain. Generally, the result proves that the structure of a5 helix which belongs to Bacillus thermocatenulatus lipase is not necessary and the deletion of it did not change the folding of enzyme. In addition, this deletion, in some cases, increased the activity of enzyme which may lead to more application of this enzyme in different industries. Keywords : Bacillus themocatenulatus, lipase, Pichia pastoris, protein engineering,a/bhydrolase,a5 helix.
