P17: Synthesis and characterization of a thermostable fusion protein based on endoglucanase and β-glucosidase for efficient cellulose degradation

Biofuels are drawing increasing attention worldwide as substitutes for petroleum-derived transportation fuels to help address energy cost, energy security and global warming concerns associated with liquid fossil fuels. Identification and design of new cellulolytic enzymes with higher catalytic efficiency is a key in reducing the production cost of lignocellulosic biofuel. We report here synthesis and characterization of cellulolytic chimeric protein which will help reduce biofuel production cost. We isolated a Paenibacillus strain, with MTCC deposit no. MTCC5639, from the gut of a cotton bollworm, on the basis of its ability to secrete a variety of plant-hydrolyzing enzymes. In this study, we cloned, expressed and characterized two enzymes, β-1,4-endoglucanase, and β-1,4-glucosidase from MTCC5639 strain and synthesized recombinant bifunctional enzymes based on β-1,4-endoglucanase, and β-1,4-glucosidase. The β-1,4-glucosidase hydrolyzed cellodextrin up to five chain length, with highest efficiency towards cellobiose. Thus, addition of glucosidase improved the ability of endoglucanase in releasing the reducing sugars from cellulose. Also, both enzymes exhibited maximum specific activity at similar pH and temperature. Therefore, we constructed series of bifunctional chimeric models by fusing the encoding genes of these two enzymes either end to end or through a linker. One of the chimeric construct showed much higher catalytic efficiency as compared to individual recombinant enzymes. The thermal denaturation monitored by CD demonstrated increased thermostability of both the domains in the fusion protein. Together with the channelling effect in the fusion protein, the chimeric protein could be effective for hydrolyzing plant biomass.