CLONING, EXPRESSION AND CHARACTERIZATION OF CUTINASE, A FUNGAL LIPOLYTIC ENZYME

Abstract

A cutinase from the fungus Fusarium solani pisi has been overproduced in E. coli by placing a phoA-signal/cutinase hybrid gene under the control of the tac promoter. Due to its periplasmic location the recombinant enzyme can be easily purified in large quantities. Assays using p-nitrophenylbutyrate suggest that the overproduced and authentic enzyme are catalytically equivalent. The specific activities on tributyrin (4000u/mg) and triolein (800u/mg) demonstrate the lipolytic nature of the enzyme. The cutinase, however, differs from classical lipases in that no measurable activation around the CMC of the tributyrin substrate is observed. We also provide evidence that the recombinant enzyme is quite thermostable.