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 <i>Trichoderma atroviride</i>. A Chitin-Binding Domain (ChBD) from <i>T. atroviride</i> 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 <i>Nicotiana benthamiana</i> using <i>Agrobacterium tumefaciens</i> 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.