The chitinolytic system of Streptomyces olivaceoviridis

Streptomyces olivaceoviridis produces several chitinases and degrades chitin efficiently. 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 pCHI01. In the presence of chitin as sole carbon source, transformants of Streptomyces lividans 66 carrying pCHI01 secreted large quantities of a chitin-inducible exochitinase of 59 kDa which was found to bind strongly to the crystalline medium. In the course of cultivation, the 59 kDa enzyme was processed proteolytically to a truncated 47 kDa, still active enzyme, which was then released to the culture filtrate. The purified 47 kDa enzyme has an isoelectric point of 4.0, shows optimal activity at pH 7.3 and 55 degrees Celsius 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, comparison of the deduced aminoacid sequence, and biochemical studies of the mature protein (59 kDa), and the proteolytically processed form (47 kDa) allowed the identification of the C-terminal catalytic domain, one central region with significant similarity to the type III module of fibronectin, and one N-terminal chitin-binding domain (12 kDa). During cultivation in the presence of chitin as sole carbon source, Streptomyces olivaceoviridis secretes several different chitinases and a 18.7 kDa chitin-binding protein (CHB1). If grown in the presence of crab chitin, transformants of Streptomyces lividans 66 harbouring the cloned chb1 gene on a multicopy vector overproduced large quantities of the 18.7 kDa protein which was purified to homogeneity. Biochemical studies and immunofluorescence microscopy revealed that the CHB1 protein binds strongly to alpha-chitin from crab shell or fungi, but neither to beta-chitin nor to various cellulose types. A reading frame of 606 bp was shown to encode the CHB1 protein. Aminoacid sequence comparisons and biochemical studies allowed the identification of aminoacids which appear to be involved in the interaction with chitin. The role of this novel lectin-like protein is at present being investigated.