Enhancing enzyme activity of heterologously expressed Chit42 in Nicotiana benthamiana for chitin degradation

This study focused on enhancing the activity of the chitinase42 enzyme from Trichoderma atroviride . A Chitin-Binding Domain (ChBD) from T. atroviride chitinase18.10 was incorporated into the genomic DNA of the chitinase42 at the N-terminal using SOEing PCR. The engineered chimeric chitinase42 and the native chitinase42 were cloned into the expression vector pGDDEE, under the control of the synthetic inducible promoter SP-DDEE, resulting in constructs pGDDEENM1 and pGDDEEJN1, respectively. These were introduced into Nicotiana benthamiana using Agrobacterium tumefaciens strain 3101. Enzyme activity was optimized via colorimetric methods. Findings revealed optimal transient expression conditions: expression induction by spraying methyl jasmonate and sampling five days post- induction (5 DPI). The chimeric chitinase42 exhibited its highest enzymatic activity, surpassing chitinase42, at 40 °C and pH 4 with 1.55-fold higher than the native chitinase42 (lacking ChBD). Crucially, the chimeric chitinase42 demonstrated superior activity to the native enzyme, underscoring the potential for enhanced chitin degradation.