A chitinase with high activity toward partially N-acetylated chitosan from a new, moderately thermophilic, chitin-degrading bacterium, Ralstonia sp. A-471.
A moderately thermophilic bacterium, strain A-471, capable of degrading chitin was isolated from a composting system of chitin-containing waste. Analysis of the 16S rDNA sequence revealed that the bacterium belongs to the genus Ralstonia. A thermostable chitinase A ( Ra-ChiA) was purified from culture fluid of the bacterium grown in colloidal chitin medium. Purification of the enzyme was achieved mainly by exploiting its binding to the colloidal chitin. The molecular mass of the enzyme was estimated to be 70 kDa and the isoelectric point approximately 4.7. N-terminal amino acid sequencing revealed a sequence of ADPYLKVAYYP, which had high homology (66% identity) with that of chitinase A1 from Bacillus circulans WL-12. The pH and temperature optima were determined to be 5.0 and 70 degrees C, respectively. The enzyme was classified as a retaining glycosyl hydrolase and was most active against partially N-acetylated chitosans. Its activities towards the partially N-acetylated chitosans, i.e. chitosan 7B, chitosan 8B, and chitosan 9B, were about 11-fold, 9-fold, and 5-fold higher than towards colloidal chitin, respectively. Ra-ChiA cleaved (GlcNAc)6 almost exclusively into (GlcNAc)2. Activation of Ra-ChiA was observed by the addition of 1 mM Cu2+, Mn2+, Ca2+, or Mg2+. Degradation of the partially N-acetylated chitosan produced oligosaccharides with a degree of polymerization ranging from 1-8; these are products that offer potential application for functional oligosaccharide production.
Sutrisno
A
A
Laboratory of Biocycle Engineering, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Sakai, 599-8531 Osaka, Japan.
Ueda
M
M
Abe
Y
Y
Nakazawa
M
M
Miyatake
K
K
D002686:Chitin / Q000031:analogs & derivatives* / Q000378:metabolism*; D002688:Chitinases / Q000302:isolation & purification* / Q000378:metabolism*; D048271:Chitosan; D004275:DNA, Ribosomal / Q000737:chemistry; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000032:analysis; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D043367:Ralstonia / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature
D004275:DNA, Ribosomal; D020536:Enzyme Activators; D008670:Metals; D012336:RNA, Ribosomal, 16S; D002686:Chitin; D048271:Chitosan; D002688:Chitinases