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Electronic Journal of Biotechnology
Universidad Católica de Valparaíso
ISSN: 0717-3458
Vol. 8, No. 2, 2005, pp. 134-145
Bioline Code: ej05016
Full paper language: English
Document type: Research Article
Document available free of charge

Electronic Journal of Biotechnology, Vol. 8, No. 2, 2005, pp. 134-145

 en Chitinase from Enterobacter sp. NRG4: Its purification, characterization and reaction pattern
Dahiya, Neetu; Tewari, Rupinder; Tiwari, Ram P. & Hoondal, Gurinder Singh

Abstract

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 Km and Vmax values of 1.43 mg ml-1 and 83.33 μM μg-1 h-1 for swollen chitin, 1.41 mg ml-1 and 74.07 μM μg-1 h-1 for colloidal chitin, 1.8 mg ml-1 and 40 M μg-1 h-1 for regenerated chitin and 2.0 mg ml-1 and 33.33 μM μg-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.

Keywords
chemical modification, chitinase, Enterobacter sp. NRG4, purification, substrate binding.

 
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