Purification and molecular characterization of chitinases from soil actinomycetes

Microbial extracellular chitinases are used in agriculture as effective biocontrol agents and in waste degradation, pharmaceutical and food industry. Actinomycetes are widely tapped group for production of extracellular chitinases. In the present study, approximately 260 actinomycetes were isolated from various ecological habitats was subjected to primary analyses and screened for production of chitinase by plate assay method. Diameter of zones of hydrolysis ranged from 8 to 16 mm. Based on the results, isolates 130, 194, 184, NRRLB 24916 (Streptomyces mexicanus) and NRRLB 16746 (Streptomyces albidoflavus, positive control) were selected for secondary screening and purification. Enzyme activity was estimated in crude cell free extract and partially purified samples. Activity ranged from 7.16 to 14.12 IU/ml (in crude extracts) and 12.1 to 23.10 IU/ml (in partially purified samples). In case of highest chitinase producing isolate 130, effect of various fermentation conditions (pH, temperature and substrate concentration) was studied in crude extract. Furthermore, complete purification of isolate 130 was done by column chromatography and the activity in purified fraction was found to be 32.12 IU/ml. The Km and Vmax values of the purified fractions for isolate 130 were 2.11 µg/ml/min and 53.11mg/ml respectively. This shows that the enzyme has high affinity for the substrate. SDS gel electrophoresis of the purified fraction showed presence of single band of approximately 65 to 70 kDa. Analyses of purified chitinase were done using MS/MS technique. N-terminal sequence corresponded to chitinase, the gene encodes a protein of 453 amino acid residues. Comparison of deduced amino acid sequence to other chitinases in the database indicated that enzyme showed 70% similarity with chitinase from Streptomyces plicatus and belongs to glycoside hydrolase family 18. Homology modeling showed that the enzyme was folded into a domain of (α/β)8 barrel structure. Identification of secondary structure was done by CD spectroscopy. Isolate 130 was capable of degrading biodegradable wastes such as crustacean shells. Key words: Actinomycetes, extracellular chitinase, primary screening, secondary screening, purification, MS/MS analyses, homology modelling, protein structure, biodegradation.