The chb1 gene, which encodes the unique lectin-like alpha-chitin-binding protein CHB1 of Streptomyces olivaceoviridis, was cloned. Transformants of Streptomyces lividans harbouring the plasmid pCHB10 overproduced the extracellular CHB1 protein; the protein showed neither enzymatic nor antifungal activity. Biochemical analyses and immunofluorescence microscopy indicated that CHB1 binds strongly to alpha-chitin, but neither to chitosan and beta-chitin, nor to various types of cellulose. Within hyphae of fungi, the relative location of crystalline chitin was visualized with fluorescein-labelled CHB1. These studies suggest that the new protein could serve as a tool to identify alpha-chitin within different organisms. The chb1 gene consists of a reading frame of 603 bp and its transcription occurred only if the Streptomyces strain was cultivated with chitin as the sole carbon source. The deduced mature CHB1 protein (18.7 kDa) shows no apparent similarity to any known protein. Within a region containing 100 residues of the deduced CHB1 protein, four tryptophan and two asparagine residues as well as one glycine and one cysteine residue were identified, the relative positions of which are analogous to those of several cellulose-binding domains of bacterial glycohydrolases. The results of spectroscopical studies suggest a possible involvement of tryptophan residues in the interaction of CHB1 with alpha-chitin.

Streptomyces olivaceoviridis efficiently degrades chitin. Shotgun cloning of partially Sau3A-cleaved DNA using the multicopy vector pIJ702 and Streptomyces lividans 66 as host resulted in the identification of the plasmid pCHI 01 which harbours an insert of 4.6 kb. In the presence of chitin as sole carbon source, transformants of S. Zividans 66 carrying pCHI 01 or its derivatives with smaller inserts overproduced an exochitinase which was purified to homogeneity. The chitininducible enzyme with an isoelectric point of 4.0 shows optimal activity at pH 7.3 and S°C , has an apparent molecular mass of 47 kDa and is competitively inhibited by the pseudosugar allosamidin. The enzyme was identified as an exochitinase since it generates exclusively chitobiose from chitotetraose, chitohexaose, and colloidal high-molecular mass chitin. Sequence analysis of a reading frame of 1794 base pairs and comparison of the deduced amino-acid sequence allowed the identification of the putative catalytic domain, one region with significant similarity to the type-I11 module of fibronectin and one domain of unknown function. Multiple sequence alignment and hydrophobiccluster analysis of 25 chitinolytic enzymes from bacteria, fungi and plants allowed the identification of their characteristic domains. The exochitinase from S. olivaceoviridis shares highest similarity with the chitinase D from Bacillus circulans.

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 crystalline substrate via its binding domain is a prerequisite for efficient hydrolysis.

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‐chi O1 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 immuno‐affinity chromatographies. Immunofluorescence microscopy revealed that the enzyme specifically binds to crystalline α‐chitin within fungi and other organisms as well as to β‐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°C) values. Thus a strong adhesion of the enzyme to its crystalline substrate via its binding domain is a prerequisite for efficient hydrolysis.