Purification, characterization, and gene analysis of a chitosanase (ChoA) from Matsuebacter chitosanotabidus 3001.
The extracellular chitosanase (34,000 M(r)) produced by a novel gram-negative bacterium Matsuebacter chitosanotabidus 3001 was purified. The optimal pH of this chitosanase was 4.0, and the optimal temperature was between 30 and 40 degrees C. The purified chitosanase was most active on 90% deacetylated colloidal chitosan and glycol chitosan, both of which were hydrolyzed in an endosplitting manner, but this did not hydrolyze chitin, cellulose, or their derivatives. Among potential inhibitors, the purified chitosanase was only inhibited by Ag(+). Internal amino acid sequences of the purified chitosanase were obtained. A PCR fragment corresponding to one of these amino acid sequences was then used to screen a genomic library for the entire choA gene encoding chitosanase. Sequencing of the choA gene revealed an open reading frame encoding a 391-amino-acid protein. The N-terminal amino acid sequence had an excretion signal, but the sequence did not show any significant homology to other proteins, including known chitosanases. The 80-amino-acid excretion signal of ChoA fused to green fluorescent protein was functional in Escherichia coli. Taken together, these results suggest that we have identified a novel, previously unreported chitosanase.
Park
J K
JK
Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan.
Shimono
K
K
Ochiai
N
N
Shigeru
K
K
Kurita
M
M
Ohta
Y
Y
Tanaka
K
K
Matsuda
H
H
Kawamukai
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D020562:Betaproteobacteria / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D002686:Chitin / Q000031:analogs & derivatives / Q000378:metabolism; D048271:Chitosan; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D015698:Genomic Library; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D002686:Chitin; D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase