The effect of substitution of Phe181 and Phe182 with Ala on activity, substrate specificity and stabilization of substrate at the active site of Bacillus thermocatenulatus lipase

Abstract Steric hindrance leads to limitation in the access of substrate into the enzyme active site. In order to decrease steric hindrance, two conserved residues, Phe 181 and Phe 182 , in the lid domain of Bacillus thermocatenulatus lipase were substituted with alanine by using site-directed mutagenesis. As a result, three mutant lipases were produced. Circular dichroism (CD) spectroscopy showed that the secondary structure of all lipases is similar to one another. F181A mutation increased the distance between phe 181 and catalytic ser 114 , which is buried in the active site by 3.24 A. It can be suggested that such an increase in distance may lead to a decrease in steric hindrance. F181A mutation increased overall lipase activity by up to 2.6-fold (4670 U mg −1 ) toward C8 substrate. It also resulted in optimal lipase activity at 65 °C rather than 55 °C. F182A mutation increased the distance between phe 182 and catalytic ser 114 by 1.54 A but failed to induce any significant effect on lipase activity. However, F181A–F182A mutation significantly decreased the activity due to decreased van der Waals interactions between the phenyl group of phenylalanines and the acyl chain of triacylglycerol. These results indicate that presence of one of the two residues, Phe 181 or Phe 182 , is important for stabilizing triacylglycerols in active site.