A novel goose-type lysozyme gene with chitinolytic activity from the moderately thermophilic bacterium <Emphasis Type="Italic">Ralstonia</Emphasis> sp. A-471: cloning,sequencing, and expression |
| |
Authors: | Mitsuhiro Ueda Konomi Ohata Toshiaki Konishi Aji Sutrisno Hitomi Okada Masami Nakazawa Kazutaka Miyatake |
| |
Institution: | (1) Graduate School of Life Science and Environment, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan |
| |
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.
The sequence data reported in the present paper have been submitted to the DDBJ, EMBL, and NCBI databases under the accession
number AB45458. |
| |
Keywords: | Goose-type lysozyme Ralstonia Chitinase Oligosaccharides Molecular cloning |
本文献已被 PubMed SpringerLink 等数据库收录! |
|