BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS A novel goose-type lysozyme gene with chitinolytic activity from the moderately thermophilic bacterium Ralstonia sp. A-471: cloning, sequencing, and expression Mitsuhiro Ueda & Konomi Ohata & Toshiaki Konishi & Aji Sutrisno & Hitomi Okada & Masami Nakazawa & Kazutaka Miyatake Received: 9 July 2008 / Revised: 19 August 2008 / Accepted: 21 August 2008 / Published online: 30 September 2008 # Springer-Verlag 2008 Abstract In this study, we cloned the gene encoding goose-type (G-type) lysozyme with chitinase (Ra-ChiC) activity from Ralstonia sp. A-471 genomic DNA library. This is the first report of another type of chitinase after the previously reported chitinases ChiA (Ra-ChiA) and ChiB (Ra-ChiB) in the chitinase system of the moderately thermophilic bacterium, Ralstonia sp. A-471 and also the first such data in Ralstonia sp. G-type lysozyme gene. It consisted of 753 bp nucleotides, which encodes 251 amino acids including a putative signal peptide. This ORF was modular enzyme composed of a signal sequence, chitin- binding domain, linker, and catalytic domain. The catalytic domain of Ra-ChiC showed homologies to those of G-type lysozyme (glycoside hydrolases (GH) family 23, 16.8%) and lysozyme-like enzyme from Clostridium beijerincki (76.1%). Ra-ChiC had activities against ethylene glycol chitin, carboxyl methyl chitin, and soluble chitin but not against the cell wall of Micrococcus lysodeikticus. The enzyme produced α-anomer by hydrolyzing β-1,4-glycosidic linkage of the substrate, indicating that the enzyme catalyzes the hydrolysis through an inverting mechanism. When N-acetylglucosamine hexasaccharide [(GlcNAc)6] was hydrolyzed by the enzyme, the second and third glycosidic linkage from the non-reducing end were split producing (GlcNAc)2+(GlcNAc)4 and (GlcNAc)3+ (GlcNAc)3 of almost the same concentration in the early stage of the reaction. The G-type lysozyme hydrolyzed (GlcNAc)6 in an endo-splitting manner, which produced (GlcNAc)3+(GlcNAc)3 predominating over that to (GlcNAc) 2+(GlcNAc)4. Thus, Ra-ChiC was found to be a novel enzyme in its structural and functional properties. Keywords Goose-type lysozyme . Ralstonia . Chitinase . Oligosaccharides . Molecular cloning Introduction Chitin, a β-1,4 polymer of N-acetyl-D-glucosamine (GlcNAc), is the second abundant biopolymer found in nature after cellulose. At least, 10 gigatons (1×10 13 kg) of chitin are synthesized and degraded each year in the biosphere (Muzzarelli 1999). This natural resource is relatively easily accessible, e.g., as crab and shrimp shell waste. N-acetyl-chitooligosaccharides and chitooligosacchar- ides have varied biological functions and many potential applications in a wide range of fields (Tokoro et al. 1988; Hirano and Nagano 1989). To obtain enzyme that can be applied to develop environmentally friendly techniques in chitin-oligosaccharide production, we have isolated a thermophilic strain belonging to the genus Ralstonia, which is capable of degrading chitin in a chitin-containing waste (Sutrisno et al. 2004). Chitinase A (Ra-ChiA) was excreted into culture medium and was constantly produced until the colloidal chitin was wholly degraded. The other chitinase, chitinase B (Ra-ChiB), showed a trace amount of protein in the culture medium, and had weaker activity than Ralstonia chitinase A. We have isolated and purified Ra-ChiA and Appl Microbiol Biotechnol (2009) 81:1077–1085 DOI 10.1007/s00253-008-1676-y The sequence data reported in the present paper have been submitted to the DDBJ, EMBL, and NCBI databases under the accession number AB45458. M. Ueda (*) : K. Ohata : T. Konishi : A. Sutrisno : H. Okada : M. Nakazawa : K. Miyatake Graduate School of Life Science and Environment, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan e-mail: mueda@biochem.osakafu-u.ac.jp