Purification and characterization of a viral chitinase active against plant pathogens and herbivores from transgenic tobacco
Antimo Di Maro, Irma Terracciano, Lucia Sticco, Luisa Fiandra, Michelina Ruocco, Giandomenico Corrado, Augusto Parente, Rosa Rao
Dipartimento di Scienze della Vita, Seconda UniversitÓ di Napoli, Via Vivaldi 43, I-81100 Caserta, Italy.
The Autographa californica nucleopolyhedrovirus chitinase A (AcMNPV ChiA) is a chitinolytic enzyme with fungicidal and insecticidal properties. Its expression in transgenic plants enhances resistance against pests and fungal pathogens. We exploited tobacco for the production of a biologically active recombinant AcMNPV ChiA (rChiA), as such species is an alternative to traditional biological systems for large-scale enzyme production. The protein was purified from leaves using ammonium sulfate precipitation followed by anion exchange and gel-filtration chromatography. Transgenic plants produced an estimated 14 mg kg−1 fresh leaf weight, which represents 0.2% of total soluble proteins. The yield of the purification was about 14% (2 mg kg−1 fresh leaf weight). The comparison between the biochemical and kinetic properties of the rChiA with those of a commercial Serratia marcescens chitinase A indicated that the rChiA was thermostable and more resistant at basic pH, two positive features for agricultural and industrial applications. Finally, we showed that the purified rChiA enhanced the permeability of the peritrophic membrane of larvae of two Lepidoptera (Bombyx mori and Heliothis virescens) and inhibited spore germination and growth of the phytopatogenic fungus Alternaria alternata. The data indicated that tobacco represents a suitable platform for the production of rChiA, an enzyme with interesting features for future applications as “eco-friendly” control agent in agriculture.