Efficient biomass saccharification using a novel cellobiohydrolase from Clostridium clariflavum for utilization in biofuel industry†
The present study describes the cloning of the cellobiohydrolase gene from a thermophilic bacterium Clostridium clariflavum and its expression in Escherichia coli BL21(DE3) utilizing the expression vector pET-21a(+). The optimization of various parameters (pH, temperature, isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, time of induction) was carried out to obtain the maximum enzyme activity (2.78 ± 0.145 U ml−1) of recombinant enzyme. The maximum expression of recombinant cellobiohydrolase was obtained at pH 6.0 and 70 °C respectively. Enzyme purification was performed by heat treatment and immobilized metal anionic chromatography. The specific activity of the purified enzyme was 57.4 U mg−1 with 35.17% recovery and 3.90 purification fold. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the molecular weight of cellobiohydrolase was 78 kDa. Among metal ions, Ca2+ showed a positive impact on the cellobiohydrolase enzyme with increased activity by 115%. Recombinant purified cellobiohydrolase enzyme remained stable and exhibited 77% and 63% residual activity in comparison to control in the presence of n-butanol and after incubation at 80 °C for 1 h, respectively. Our results indicate that our purified recombinant cellobiohydrolase can be used in the biofuel industry.