BINDING AND SUBSTRATE SPECIFICITIES OF A STREPTOMYCES-OLIVACEOVIRIDIS CHITINASE IN COMPARISON WITH ITS PROTEOLYTICALLY PROCESSED FORM

Abstract

Streptomyces olivaceoviridis is an efficient chitin degrader. One of its genes encoding an exochitinase (exo-ChiO1) was previously characterized. The transcription was found to be inducible by chitin, but not by glucose. The transcriptional start site is situated 38 bp upstream of the start codon. S. olivaceoviridis as well as transformants of S. vinaceus and S. lividans carrying the exo-chiO1 gene on a multicopy vector secrete a 59-kDa chitinase which adheres strongly and under most conditions irreversibly to the substrate chitin. After having released the enzyme from the crystalline substrate in the presence of high concentrations of guanidine hydrochloride, it was purified to homogeneity by consecutive chitin- and immunoaffinity chromatographies. Immunofluorescence microscopy revealed that the enzyme specifically binds to crystalline alpha-chitin within fungi and other organisms as well as to beta-chitin, but not to colloidal chitin, chitosan, various types of cellulose, or other polysaccharides. The amino acids deduced from the highly specific binding domain (12 kDa) of this enzyme do not share significant similarity with any known region interacting with chitin or another substrate. During cultivation with chitin, the 59-kDa enzyme is proteolytically processed to a 47-kDa truncated chitinase lacking the chitin-binding domain. The 47-kDa enzyme hydrolyses crystalline chitin considerably less efficiently than the 59-kDa enzyme, whereas colloidal chitin and low-molecular-mass substrates are quite equally degraded by both enzymes at identical optimal pH (7.3) and temperature (45-55 degrees C) values. Thus a strong adhesion of the enzyme to its crystal line substrate via its binding domain is a prerequisite for efficient hydrolysis.