Purification and characterization of an endogenous cellulase from the digestive system of grub of banana pseudostem weevil Odoiporus longicollis (Olivier)

Abstract

Insects have evolved with effectual strategies to utilise cellulose substrates for the energy source and can be used as an optimal resource in the field of bioenergy sector and insect-pest management by finding the novel cellulolytic enzymes from it. The present study was aimed at evaluating the endogenous cellulolytic system in the larval gut of banana pseudostem weevil Odoiporus longicollis. Initially, the cellulase activity was localized in the gut system in which the midgut showed highest activity of 2858 U min-1mg-1. The optimum temperature and thermal stability were found to be 60°C at the highest activity of 2712 U min-1mg-1. The enzyme was stable at a pH from 5 to 6. The effect of various divalent cations tested on the cellulase activity showed a differential enhancing and inhibitory activity upon varying concentrations. Purification of cellulase was carried out using anion exchange chromatography with the DEAE-Sepharose CL-6B matrix which was chosen according to the results of the batch assay. The total and specific activity of purified cellulase was 1166 U min-1mg-1 and 1190.46 U min-1mg-1, respectively, with 41.66 % recovery of activity. The molecular weight of the purified cellulase was determined as 47 kDa. The optimum temperature for the purified enzyme was 60°C with an activity of 1130 U min-1mg-1. The purified fraction had the highest activity between pH 5 and 6. Kinetic parameters of the purified cellulase enzyme were determined in which Km and Vmax values were 1.03 mg/ml and 343 U min-1mg-1, respectively. Mass spectrometry result identified the homology towards endoglucanase sequence belonging to the GHF5 family. The gut microbial cellulase activity showed no competence comparatively.