Research Article: Biosynthesis and characterization of a thermostable, alkali-tolerant chitinase from Bacillus pumilus JUBCH08 displaying antagonism against phytopathogenic Fusariumoxysporum

Date Published: March 7, 2016

Publisher: Springer Berlin Heidelberg

Author(s): Sourav Bhattacharya, Arijit Das, Saikat Samadder, Subbaramiah Sundara Rajan.


The present investigation highlights the process parameters influencing the submerged fermentation of chitinase by Bacillus pumilus JUBCH08, purification and characterization of the enzyme and determination of antagonistic activity of the bacterium against Fusarium oxysporum. Medium supplemented with 0.5 % chitin and peptone, at initial pH 8.0, when incubated at 35 °C for 72 h favored highest chitinase production. The enzyme was purified 25.1-fold to homogeneity. The chitinase was found to be thermostable and alkali-tolerant with maximum activity at pH 8.0 and 70 °C for 1 h. The molecular weight of chitinase was found to be 64 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Mg2+, Co2+, Ca2+ and Mn2+ improved the chitinase activity. The Km and Vmax values of the enzyme were 0.13 mg/ml and 38.23 U/ml, respectively. When subjected to dual plate assay, the bacterium showed 45 % antagonism against F. oxysporum. Thus, it could be inferred that cultural conditions strongly affected the chitinase production by B. pumilus JUBCH08. The enzyme being thermostable and best functional under alkaline conditions could be useful for the feed industry and related biotechnological applications. Inhibition of F. oxysporum by the culture through lytic mechanism indicates its potentiality as a biocontrol agent.

Partial Text

Chitin, an insoluble polysaccharide, is one of the most abundant biopolymers on earth and is composed of linear chains of β-1, 4-N–acetyl glucosamine residues that are highly cross-linked by hydrogen bonds. Chitin as a structural polysaccharide is present in the fungal cell wall, nematodes, exoskeleton of insects and outer shell of crustaceans. Chitin forms a significant proportion of dry weight of the waste generated from shellfish industry (Wang and Chang 1997). Annually, shellfish production and processing industry contribute to the accumulation of a substantial amount of chitin rich waste that may prove to be an environmental hazard (Wang et al. 2002).

The present investigation revealed that B. pumilus JUBCH08 is a potent chitinase producer. Medium supplemented with chitin and peptone, at initial pH 8.0, when incubated at 35 °C for 72 h favored highest chitinase production. The chitinase was unique in some properties, being functional under alkaline conditions and displaying thermostability at 70 °C for 1 h. The results of the dual plate assay emphasized that the chitinolytic bacterium greatly inhibited the fungal pathogen through lytic mechanism, a characteristic that may be utilized for the formulation of a biocontrol agent as an alternative to harmful chemical pesticides.




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