Purification and characterization of a novel chitinase from the entomopathogenic fungus, Metarhizium anisopliae

A novel chitinase was detected in extracellular culture fluids of the entom opathogenic fungus Metarhizium anisopliae (ATCC 20500) grown in liquid medi um containing chitin as a sole carbon source. A chitinase was purified to n ear homogeneity from culture broth of M. anisopliae by DEAE-Sephacel, CM-Se pharose CL-GB ion-exchange chromatography, and gel filtration with Superose 12HR. The molecular mass of the enzyme determined by SDS-polyacrylamide ge l electrophoresis was approximately 60 kDa and the optimum pH of the enzyme was 5.0. This molecular mass is different from values of 33, 43.5, and 45 kDa for endochitinases and 110 kDa for an exochitinase (N-acetylglucosamini dase) from M. anisopliae ME-1 published previously. In addition, N-terminal sequences of 60-kDa chitinase are different from those of 43.4- and 45-kDa endochitinases. The purified enzyme showed high chitinolytic activity agai nst colloidal, crystalline chitin of crab shells as well as against p-nitro phenyl-beta-D-N-acetylglucosamide, p-nitrophenyl-beta-D-N,N'-diacetylchitob iose, and p-nitrophenyl-N, N'-N "-triacetylchitotriose, indicating that thi s enzyme has both endo- and exochitinase activity. (C) 1999 Academic Press.