Purification and Characteristics of an Autolytic Chitinase of Piromyces communis OTS1 from Culture Medium Masaru Sakurada, 1, * Diego P. Morgavi, 2 Kenji Komatani, 1 Yoshifumi Tomita, 1 Ryoji Onodera 1 1 Laboratory of Animal Nutrition and Biochemistry, Animal Science Division, Faculty of Agriculture, Miyazaki University, Nishi 1-1, Gakuen Kibana-dai, Miyazaki 889-21, Japan 2 Rowett Research Institute, Bucksburn, Aberdeen, AB2 9SB, Scotland, UK Received: 3 December 1996 / Accepted: 28 January 1997 Abstract. An autolysis chitinase was purified from the cultural medium of the anaerobic fungus Piromyces communis OTS1 by ammonium sulfate precipitation, affinity chromatography with regenerated chitin, chromato-focusing, gel filtration, and chromato-focusing again. The optimal pH and temperature were 6.0 and 50°C, respectively, for a 20-min assay. The chitinase was stable from pH 6.0 to 8.0, but was unstable at 70°C for 20 min. The molecular mass of chitinase was estimated by SDS-PAGE to be 44.9 kDa, and its pI was 4.4. The enzyme activity, which was of the ‘endo’ type, was inhibited by Hg 21 and allosamidin. The chitinase hydrolyzes chitin powder and fungal cell walls at a higher rate than an artificial chitin substrate. It can be concluded that extracellular chitinase is similar to cytosolic chitinase, but they are not the same protein. Chitin is one of the most abundant polysaccharides in the world, second only to cellulose. Chemically, it is a (1-4)-b-linked homopolymer of N-acetyl-D-glucosamine [3, 4]. The complete hydrolysis of chitin to free N-acetyl- D-glucosamine is performed by a chitinolytic system consisting of two hydrolases, chitinase (EC 3.2.1.14) and N-acetyl-b-glucosaminidase (EC 3.2.1.52), that act con- secutively. The former hydrolyzes the polymers of N- acetyl glucosamine, and the latter hydrolyzes diacetylchi- tobiose [7]. Microorganisms with an ability to degrade chitin are found in many different ecosystems. The role of the chitinolytic system can be pathogenic, nutritive, or auto- lytic and morphogenetic in chitin-containing organisms [5]. The anaerobic rumen fungi that inhabit the rumen of ruminant animals have chitin-containing cell walls [12] and chitinase as an autolytic enzyme for their morphologi- cal maintenance [20]. The chitinolytic activities (crude enzymes) of Piromyces communis OTS1 were studied [18], the cytosolic chitinase of the fungus was purified, and its characteristics were investigated [19]. The charac- teristics of the fungal cytosolic chitinase were: molecular mass 42 kDa, isoelectric point 4.9, optimal pH 6.2, and optimal temperature 60°C. In this study, to investigate derivation of autolysis enzyme, extracellular chitinase as an autolysis enzyme was purified and its characteristics were compared with those of chitinase with cytosolic chitinase. Materials and Methods Chemicals. Regenerated chitin was produced by the method of Molano [9]. Glycol chitin was obtained by acetylation of glycol chitosan [10]. Colloidal chitin was prepared from flaked chitin by the method of Shimahara and Takiguchi [21]. Allosamidin was provided by A. Suzuki (The University of Tokyo) and A. Isogai (Nara Institute of Science and Technology). Fungal cell walls for the substrate specificity test were prepared from the mycelium of a 4-day-old culture of P. communis OTS1 (autoclaved, dried at 60°C, and ground by a mortar). The other chemical reagents used were commercial products of maximum grade. Organism. The rumen fungus OTS1 was used for this study. The methods of isolation and cultivation [17], identification and storage [18] were described in a previous study. Enzyme assay and protein measurement. The methods of enzyme assay were described in detail in a previous study [18]. The enzyme solution was mixed with 33 mM sodium citrate-phosphate buffer (pH 5.5) containing 60 mM 4-methylumbelliferyl N,N8,N9-triacetylchitotri- ose as the substrate. The mixture was incubated at 39°C for 20 min, and * Present address: Laboratory of Bioorganic Chemistry, Department of Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. Correspondence to: M. Sakurada CURRENT MICROBIOLOGY Vol. 35 (1997), pp. 48–51 An International Journal R Springer-Verlag New York Inc. 1997