Lipases from Bacillus thermocatenulatus are a member of superfamily of alpha/beta hydrolase, but there are structural differences between them. In this work, we focused on the alpha 5 helix of B. thermocatenulatus lipase (BTL2) which is absent in canonical alpha/beta hydrolase fold. In silico study showed that the alpha 5 helix is a region that causes disorder in BTL2 protein. So, the alpha 5 helix (residues 131 to 150) has been deleted from the B. thermocatenulatus lipase gene (BTL2) and the remain (Delta alpha 5-BTL2) has been expressed in Pichia pastoris yeast. The alpha 5 deletion results in increase of enzyme-specific activity in the presence of tributyrin, tricaproin, tricaprylin, tricaprin, trilaurin, and olive oil (C18) substrates by 1.4-, 1.7-, 2.0-, 1.2-, 1.75-, and 1.95-fold, respectively. Also, deletion leads to increase in enzyme activity in different temperatures and pHs, whereas it did not significantly affect on enzyme activity in the presence of organic solvents, metal ions, and detergents.