Two extracellular chitinases (FI and FII) were purified from the culture supernatant of Pseudomonas aeruginosa K-187. The molecular weights of FI and FII were 30,000 and 32,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 60,000 and 30,000, respectively, by gel filtration. The pIs for FI and FII were 5.2 and 4.8, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of FI were pH 8, 50؇C, pH 6 to 9, and 50؇C; those of FII were pH 7, 40؇C, pH 5 to 10, and 60؇C. The activities of both enzymes were activated by Cu 2؉ ; strongly inhibited by Mn 2؉ , Mg 2؉ , and Zn 2؉ ; and completely inhibited by glutathione, dithiothreitol, and 2-mercaptoethanol. Both chitinases showed lysozyme activity. The purified enzymes had antibacterial and cell lysis activities with many kinds of bacteria. This is the first report of a bifunctional chitinase/lysozyme from a prokaryote. Chitin, a homopolymer of N-acetyl-D-glucosamine (Glc-NAc) residues linked by 1-4 bonds, is the most abundant renewable natural resource after cellulose (4). It is widely distributed in nature as the integuments of insects and crustaceans and as a component of fungi and algae (23). It is estimated that the worldwide annual recovery of chitin from the processing of marine invertebrates, for example, is 37,300 metric tons (32). Chitin and its derivatives are of interest because they have various biological activities, such as those of an immunoadjuvant and a flocculant of wastewater sludge, and agrochemical uses (7). Chitooligosaccharides are prepared by partial hydrolysis of chitin with hydrochloric acid or enzymatically by degradation and transglycosylation. This pretreated chitin is then mixed with a chitinolytic enzyme to hydrolyze it to the monomer or oligomer of N-acetylglucosamine. Chitinases, a group of enzymes capable of degrading chitin directly to low-molecular-weight products, have been shown to be produced by a number of microorganisms. Almost all of the reported chitinase-producing strains will use chitin or colloidal chitin as a carbon source (42). Commercial interest in the utilization of chitin and its derivatives has led to the need for inexpensive, reliable sources of active and stable chitinase preparations. The production of inexpensive chitinolytic enzymes is an important element in the utilization of shellfish wastes that not only solves environmental problems but also promotes the economic value of the marine products (42). Some animal and higher plant chitinases have lysozyme activity (chitinase/lysozyme), while bifunctional chitinases have not been isolated from microorganisms. We reported the medium composition and the character of crude chitinase from Pseudomonas aeruginosa K-187 in a previous paper (42). In the present study, we isolated two kinds of bifunctional chitinases/ lysozymes from K-187 cell-free culture broth. This paper describes the purification and characterization of these enzymes. MATERIALS AND METHODS Materials. The shrimp and crab shell powder (SCSP) ...
