Chitinolytic activity of an estuarine bacterium
An estuarine isolate, identified as Alteromonas haloplanktis was isolated from surface sediment of the Ythan estuary, Aberdeenshire, by its ability to degrade chitin. The isolate could employ a number of carbon sources but required seawater (40-60% ) for maximum growth. When grown in the presence of chitin or its breakdown product N-acteylglucosamine, chitinolytic enzymes were secreted into the medium (both N-acetylglucosaminidase and chitinase) and were detectd by a highly sensitive fluorogenic assay based on 4-methylumbelliferyl glycosides of N-acetylglucosamine oligosaccharides. Specific activity was greatest at the onset of stationary phase. Enzyme production was inducible and subject to catabolic repression. Anion-exchange chromatography revealed the presence of at least three N-acetylglucosaminidase enzymes and one chitinase. The latter was purified to homogeneity and was detected as a single band, by SDS-PAGE, of molecular weight 38kDa. Kinetic studies with chitinolytic preparations demonstrated that activity could be strikingly stimulated by the presence of salts (both NaCl and KCl). Chitinase activity could be inhibited with the specific inhibitor allosamidin, in a competitive manner (IC 50 of 2μM). N-acetylglucosaminidase activity could be inhibited with the lactone 2-acetamido-1,5-imino-1,2,5-trideoxy-D-glucitol, in a competitive manner (IC 50 of 0.5mM).