Biochemical and antifungal characteristics of recombinant class I chitinase from Drosera rotundifolia.

DrChit is class I chitinase involved in the digestion of insect prey of Drosera rotundifolia plants. Herein, we cloned the DrChit open reading frame lacking the 5'- sequence coding signal peptide into the pET32a vector and its derivate lacking the thioredoxin tag. After DrChit + Trx and DrChit-Trx overexpression in Escherichia coli cells and purification on Ni-NTA agarose, both enzymes exhibited maximum activity at pH 6.0 and 38 °C. Surprisingly, the DrChit  + Trx exerted double enzyme activity and improved all kinetic parameters for FITC-chitin substrate degradation resulting in catalytic efficiency three times higher (46.2 mM. s) than DrChit-Trx (13.63 mM. s). The 3D-structure of DrChit + Trx revealed different spatial arrangement of the three tyrosine residues in chitin-binding site, while their aromatic rings showed better stacking geometry for CH/π interactions with the carbohydrate substrate. In contrast, there were no significant differences between both enzymes when the effect of metal ions and their antifungal potential were tested. Quantitative in vitro assays showed growth suppression of Fusarium poae (40%), Trichoderma viride (43.8%), and Alternaria solani (52.6%) but not Rhizoctonia solani (sp.). Our study indicates that sundew chitinase has potential in biotechnology either for degradation of chitin to oligomers applicable in medicine or for plant defense fortification.