Chitinase from Enterobacter sp NRG4:: Its purification, characterization and reaction pattern

Enterobacter sp. NRG4 was shown to excrete chitinase into the culture supernatant when cultivated in medium containing chitin. A 60 kDa extracellular chitinase was purified to homogeneity and characterized. The enzyme hydrolyzed swollen chitin, colloidal chitin, regenerated chitin and glycol chitin but did not hydrolyze chitosan. The chitinase exhibited K-m and V-max values of 1.43 mg ml(-1) and 83.33 mu M mu g(-1) h(-1) for swollen chitin, 1.41 mg ml(-1) and 74.07 mu M mu g(-1) h(-1) for colloidal chitin, 1.8 mg ml(-1) and 40 mu M mu g(-1) h(-1) for regenerated chitin and 2.0 mg ml(-1) and 33.33 mu M mu g(-1) h(-1) for glycol chitin, respectively. The optimal temperature and pH for activity were 45 C and pH 5.5, respectively. Mg2+, K+ and Ca2+ stimulated chitinase activity by 13, 16 and 18%, respectively whereas Cu2+, Co2+, Ag+ and Hg2+ inhibited chitinase activity by 9.7, 15, 22 and 72.2%, respectively at 1 mM concentration. N-bromosuccinamide (NBS) at 1 mM and iodoacetamide at 10 mM concentration completely inhibited the enzyme activity. Dithiobisnitrobenzoic acid ( DTNB) at 10 mM concentration inhibited chitinase activity by 97.2%. Chitin was hydrolyzed to chitobiose and N-acetyl D-glucosamine when incubated with the purified enzyme. The hydrolysis pattern of the purified enzyme indicated that the chitinase was an endochitinase.