APPLIED AND ENVIRONMENTAL MICROBlOLOGY, Sept. 1998, p. 3397-3402 Copyright @ 1998, American Society for Microbiology. All Rights Reserved. Purification and Characterization of Three Thermostable Endochitinases of a Noble Bacillus Strain, MH-1, Isolated from Chitin-Containing Compost KENJI SAKAI, MASAHARU NARIHARA, YOSHIKI KASAMA, MAMORU WAKAYAMA, AND MITSUAKI MORIGUCHI Department ofApplied Chemistry, Faculty of Engineering, Oita University, Oita 870-1192, and Center for Cellular and Molecular Research, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan Received 22 April 1998/Accepted 1 July 1998 A thermophilic and actinic bacterium strain, MH-1, which produced three different endochitinases in its culture fluid was isolated from chitin-containing compost. The microorganism did not grow in any of the usual media for actinomyces but only in colloidal chitin supplemented with yeast extract and (2,6-0-dimethyl)-P- cyclodextrin. Compost extract enhanced its growth. In spite of the formation of branched mycelia, other properties of the strain, such as the formation of endospores, the presence of meso-diaminopimelic acid in the cell wall, the percent G+C of DNA (55%), and the partial 16S ribosomal DNA sequence, indicated that strain MH-1 should belong to the genus Bacillus. Three isoforms of endochitinase (L, M, and S) were purified to homogeneity and characterized from Bacillus sp. strain MH-1. They had different molecular masses (71, 62, and 53 kDa), pIs (5.3, 4.8, and 4.7), and N-terminal amino acid sequences. Chitinases L, M, and S showed relatively high temperature optima (75, 65, and 75��) and stabilities and showed pH optima in an acidic range (pH 6.5, 5.5, and 5.5, respectively). When reacted with acetylchitohexaose [(GlcNAc)6], Chitinases L and S produced (GlcNAc)2 at the highest rate while chitinase M produced (GlcNAc)3 at the highest rate. None of the three chitinases hydrolyzed (GlcNAc)2. Chitinase L produced (GleNAc)2 and (GlcNAc)3 in most abundance from 66 and 11% partially acetylated chitosan. The p-nitrophenol (pNP)-releasing activity of chitinase L was highest toward pNP-(GlcNAc)2, and those of chitinases M and S were highest toward pNP-(GlcNAc),. All three enzymes were inert to pNP-GlcNAe. AgCl, HgCl2, and (GlcNAc)2 inhibited the activities of all three enzymes, while MnCl2 and CaCl2 slightly activated all of the enzymes. Back