Biochemical and structural characterization of Amy1: an alpha-amylase from Cryptococcus flavus expressed in Saccharomyces cerevisiae

Abstract

An extracellular alpha-amylase (Amy1) whose gene from Cryptococcus flavus was previously expressed in Saccharomyces cerevisiae was purified to homogeneity (67 kDa) by ion-exchange and molecular exclusion chromatography. The enzyme was activated by NH 4 + and inhibited by Cu +2 and Hg +2 . Significant biochemical and structural discrepancies between wild-type and recombinant α- amylase with respect to K m values, enzyme specificity, and secondary structure content were found. Far-UV CD spectra analysis at pH 7.0 revealed the high thermal stability of both proteins and the difference in folding pattern of Amy1 compared with wild-type amylase from C. flavus, which reflected in decrease (10-fold) of enzymatic activity of recombinant protein. Despite the differences, the highest activity of Amy1 towards soluble starch, amylopectin, and amylase, in contrast with the lowest activity of Amy1 w , points to this protein as being of paramount biotechnological importance with many applications ranging from food industry to the production of biofuels.