Identification and characterization of a chitinase antigen from Pseudomonas aeruginosa strain 385.
A chitinase antigen has been identified in Pseudomonas aeruginosa strain 385 using sera from animals immunized with a whole-cell vaccine. The majority of the activity was shown to be in the cytoplasm, with some activity in the membrane fraction. The chitinase was not secreted into the culture medium. Purification of the enzyme was achieved by exploiting its binding to crab shell chitin. The purified enzyme had a molecular mass of 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 5.2. NH2-terminal amino acid sequencing revealed two sequences of M(I/L)RID and (Q/M/V)AREDAAAAM that gave an exact match to sequences in a translated putative open reading frame from the P. aeruginosa genome. The chitinase was active against chitin azure, ethylene glycol chitin, and colloidal chitin. It did not display any lysozyme activity. Using synthetic 4-methylumbelliferyl chitin substrates, it was shown to be an endochitinase. The Km and kcat for 4-nitrophenyl-beta-D-N,N'-diacetylchitobiose were 4.28 mM and 1.7 s(-1) respectively, and for 4-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose, they were 0.48 mM and 0.16 s(-1) respectively. The pH optimum was determined to be pH 6.75, and 90% activity was maintained over the pH range 6.5 to 7.1. The enzyme was stable over the pH range 5 to 10 for 3 h and to temperatures up to 50 degrees C for 30 min. The chitinase bound strongly to chitin, chitin azure, colloidal chitin, lichenan, and cellulose but poorly to chitosan, xylan, and heparin. It is suggested that the chitinase functions primarily as a chitobiosidase, removing chitobiose from the nonreducing ends of chitin and chitin oligosaccharides.
Thompson
S E
SE
School of Biological Sciences, Liverpool University, Liverpool, Merseyside L69 72B, United Kingdom.
Smith
M
M
Wilkinson
M C
MC
Peek
K
K
D000907:Antibodies, Bacterial / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology; D001428:Bacterial Vaccines / Q000276:immunology; D015153:Blotting, Western; D002688:Chitinases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000276:immunology; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001428:Bacterial Vaccines; D002688:Chitinases