Production of chitinolytic enzymes from a novel species of Aeromonas

A bacterial strain secreting potent chitinolytic activity was isolated from shrimp-pond water by enrichment culture using colloidal crab-shell chitin as the major carbon source, The isolated bacterium, designated as Aeromonas sp No. 16, exhibited a rod-like morphology with a polar flagellum. Under optimal culture conditions in 500-ml shaker flasks, it produced a chitinolytic activity of 1.4 U ml(-1). A slightly higher enzymatic activity of 1.5 U ml(-1) was obtained when cultivation was carried out in a 5-liter jar fermenter using a medium containing crystalline chitin as the carbon source. The secretion of the enzyme(s) was stimulated by several organic nitrogenous supplements. Most carbon sources tested (glucose, maltose, N-acetylglucosamine, etc) enhanced cell growth, but they slightly inhibited enzyme secretion. Glucosamine (0.5% w/v) severely inhibited cell growth (16% of the control), but it did not significantly affect enzyme secretion. The production of chitinolytic enzymes was pH sensitive and was enhanced by increasing the concentration of colloidal chitin to 1.5%. The observed chitinolytic activity could be attributed to the presence of beta-N-acetylglucosaminidase and chitinase. Chitinase was purified by ammonium sulfate fractionation and preparative gel electrophoresis to three major bands on SDS-PAGE. An in-gel enzymatic activity assay indicated that all three bands possessed chitinase activity. Analysis of the enzymatic products indicated that the purified enzyme(s) hydrolyzed colloidal chitin predominantly to N,N-diacetyl-chitobiose and, to a much lesser extent, the mono-, tri-, and tetramer of N-acetylglucosamine, suggesting that they are mainly endochitinases.