Purification and Characterization of Cellulases and Xylanases from Bacterial Isolates for Hydrolysis of Pretreated Sugarcane Bagasse

Abstract

Enzymatic saccharification of organo-solvent pretreated sugarcane bagasse (SCB) by cellulases and hemicellulases (xylanase) is a widely used technique for the extraction of reducing sugar (cellulose and xylose). Sugarcane bagasse contains 25-30% hemicellulose and 35-50% cellulose which act as major substrates for these enzymes to obtain valuable products. In current study bacterial strains CC16 and CC4 have been screened to produce cellulase and xylanase enzyme respectively. Different culture parameters were optimized for CC16 and CC4 for maximum production of cellulase and xylanase. Cellulase and xylanase produced by CC16 and CC4 were further purified using column chromatography with Sephadex G-100 gel resin, and all proteins were eluted according to their molecular size. The maximum specific activity of purified cellulase from bacterial strain CC16 was 114.2 U/mg, 2.03 purification fold, and 55.6% yield and for purified xylanase from bacterial strain CC4 specific activity was 232.0U/mg, 1.74 purification fold, and 55.3% yield. Purified cellulase shows the highest activity and stability at pH 7.0 and temperature 45°C while purified xylanase revealed the highest activity and stability at pH 9.0 and 50°C temperature. The enzyme cellulase and xylanase were active in the presence of different metal ions like Cu2+, Fe2+, Ca2+, and Mg2+ whereas Pb strongly inhibited its activity. Surfactants such as Tween 20, Tween 80, CTAB, and Triton X 100 potentially enhanced activity of both enzymes while SDS significantly decreased their activity at both 1% and 10% concentrations in case of both enzymes. The enzyme cellulase from CC16 remained stable in the presence of 10% organic solvents such as Methanol, Acetone, Ethyl acetate, N-Hexane DMSO, and glycerol while xylanase from CC4 was found to be stable in the presence of Methanol, Acetonitrile, Acetone, DMSO and glycerol for 120 minutes. Enzymatic hydrolysis of pretreated sugarcane bagasse by cellulase and xylanase resulted in 33.2 g/L and 0.91 g/g of total reducing sugars (TRS). The significance of the current study is that all the selected parameters possess a substantial effect on the production of both cellulase and xylanase enzymes, which upon the optimization and purification process showed stability at a wide range of temperatures, pH, various concentrations of metal ions, organic solvents and surfactants leading to increased hydrolytic proficiency of cellulase and xylanase for saccharification of pre treated sugarcane bagasse that can be used on industrial scale for the production of bioethanol and other valuable products.