Induction and Expression of Chitinases from Four Sub Species of Bacillus thuringiensis

Chitin is a naturally occurring linear polymer of N-acetylglucosamine and the major structural component of fungal cell walls and exoskeletons of insects and arthropods. Chitinases are the enzymes that breakdown chitin to economically important derivatives and found in a range of organisms including bacteria, insects, crustaceans, invertebrates, some vertebrates and higher plants. In the present study, four Bacillus thuringiensis (Bt) isolates were grown in media supplemented with 0.1% (w/v) regenerated chitin and chitinase inducing medium and screened for chitinolytic activity. All isolates showed notable extracellular chitinase activity with very low levels in cell bound fractions. When the isolates were screened with fluorogenic substrates, Bt HD133, Bt T7002 and Bt IPS78 showed a similar pattern of distribution with the highest production from Bt IPS 78. The lowest activity was detected from the isolate Bt 36-3. Similar to the results obtained with fluorogenic substrates, Bt 36-3 and Bt IPS 78 expressed the lowest and the highest activities with the substrate Carboxymethyl-Chitin Remazol Brilliant Violet 5R (CM-chitin RBV 5R) respectively. When the culture supernatants of isolates grown in nutrient broth supplemented with regenerated chitin were electrophoresed on activity gels, one chitinase band of approximate molecular mass of 36 kDa was obtained from all four sub species. Future experiments can be carried out to find out whether these chitinases have any insecticidal activity aiming, to develop environmental-friendly biopesticides, and in vivo antifungal activities to test against plant pathogenic fungi in growth chambers and under glasshouse condition.