Optimization of chimeric chitinase42 prokaryotic expression and comparison of its chitinase activity with Chit42

Chitinases have the ability of chitin digestion that constitutes a main compound of the fungal cell wall, insect exoskeletons, and crustacean shells. Chitinase Chit42 from Trichoderma atroviride PTCC5220 is considered to play an important role in the biocontrol activity of this fungus against phytopathogenic fungi. The Chitin-Binding Domain (ChBD) of Serratia marcescens chitinase B was selected and fused to the fungal chitinase, T. atroviride Chit42 using SOEing PCR with overlapping primers. The chimeric fragment was cloned into prokaryotic expression vector (pET26b+) and transformed to E. coli BL21-DE3.Culture conditions for chimeric enzyme production by E. coli were optimized by Taguchi orthogonal array experimental design methodology. This approach facilitates the study of interaction of a large number of variables spanned by factors and their settings with a small number of experiments leading to considerable saving in time and cost for the process optimization. The objective of the current research was to determine the significant parameters on the production of chimeric chitinase enzyme in the culture. The process variables were IPTG concentration, incubation time, and temperature. The total protein extraction of all experiments were carried out and analyzed by SDS-PAGE, Total lab and Qualitek-4 software. The optimal levels of the different factors for chimeric chitinase production were 1mM IPTG, and 16 hours of incubation time at 28 °C. IPTG concentration was the most important factor in the enzyme production.