A novel goose-type lysozyme gene with chitinolytic activity from the moderately thermophilic bacterium Ralstonia sp. A-471: cloning, sequencing, and expression.
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 alpha-anomer by hydrolyzing beta-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.
Ueda
Mitsuhiro
M
Graduate School of Life Science and Environment, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan. mueda@biochem.osakafu-u.ac.jp
Ohata
Konomi
K
Konishi
Toshiaki
T
Sutrisno
Aji
A
Okada
Hitomi
H
Nakazawa
Masami
M
Miyatake
Kazutaka
K
D000595:Amino Acid Sequence / Q000235:genetics; D001483:Base Sequence; D002686:Chitin / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016366:Open Reading Frames; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D043367:Ralstonia / Q000201:enzymology*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D002686:Chitin; D009113:Muramidase