Gene cloning, expression, purification and characterization of rice (Oryza sativa L.) class II chitinase CHT11

Class II chitinase CHT11 is one of the 12 chitinases identified in rice (Oryza sativa L.). In order to elucidate its enzymatic properties, the Cht11 gene was cloned and overexpressed in Escherichia coli BL21 cells as a glutathione-S-transferase (GST) fusion protein. The optimal culture temperature and the optimal time after induction were found to be 16 °C and 12 h, respectively. In the process of purification, a non-specific 70-kDa protein bound to the fusion protein was successfully removed from recombinant CHT11 by adding 3 mM ATP-Mg in wash buffer. In the culture conditions and purification procedures presented in this study, the final yield of recombinant CHT11 from 800 ml of the culture supernatant was 4.1 mg with 11.5-fold purification. The optimal temperature and pH of recombinant CHT11 were determined to be 35–40 °C and 5.5–6.5, respectively. It inhibited the hyphal growth of both avirulent and virulent strains of Trichoderma viride at 100 and 300 μg in medium, but the growth inhibition zone was subtle even when 300 μg of protein was applied. This indicates that the antifungal activity of recombinant CHT11 is considerably lower than other chitinases.