Molecular characterization of an endochitinase from Bacillus thuringiensis subsp. konkukian

A chitinase gene from Bacillus thuringiensis serovar konkukian S4 was cloned, sequenced, and heterologously expressed in Escherichia coli M15. Recombinant enzyme (Chi74) was purified by Ni-NTA affinity column chromatography. The chi74 gene contains an open reading frame (ORF), with a capacity to encode an endochitinase with a deduced molecular weight 74 kDa and predicted isoelectric point of 5.67. Comparison of Chi74 with other chitinases has shown that it contains a modular structure with an N-terminal family 18 catalytic-domain, a Fibronectin-III like domain and a C-terminal carbohydrate binding module (CBM-II). Turn over rate (Kcat) of the enzyme was determined using colloidal chitin (28.3 ± 0.70 S−1) as substrate. The Purified enzyme was active at a broad range of pH (pH 3.5–7.5) and temperature (20–70°C) with a peak activity at pH 5.5 and 55°C. However, the enzyme was found to be stable up to 30°C for longer incubation periods. Moreover, the purified enzyme was shown to inhibit fungal spore germination and hyphal growth in the pathogenic fungi Fusarium oxysporum and Aspergillus niger. These studies will lead us to develop broad spectrum resistance in the crop plants via co-expression of the chitinases and the insecticidal proteins.