Purification and characterization of endo β-1,4-D-glucanase from Trichoderma harzianum strain HZN11 and its application in production of bioethanol from sweet sorghum bagasse

An acidophilic-solvent-thermostable endo beta-1,4-D-glucanase produced from a potential Trichoderma harzianum strain HZN11 was purified to homogeneity by DEAE-Sepharose and Sephadex G-100 chromatography with 33.12 fold purification with specific activity of 66.25 U/mg and molecular mass of similar to 55 kDa. The optimum temperature and pH were 60 degrees C and 5.5 retaining 76 and 85 % of activity after 3 h, respectively. It showed stability between pH 4.5-6.0 and temperature between 50-70 degrees C indicating thermostability. Endo beta-1,4-D-glucanase was activated by Ca2+ and Mg2+ but inhibited by Hg2+, Pb2+ and Cd2+. The effect of thiol reagents, metal chelators, oxidizing agents and surfactants on enzyme activity has been studied. Purified endo beta-1,4-D-glucanase exhibited highest specificity towards carboxymethyl cellulose. Kinetic analysis showed the K-m, V-max and K-i (cellobiose inhibitor) of 2.5 mg/mL, 83.75 U/mg and 0.066 M, respectively. The storage stability of purified endo beta-1,4-D-glucanase showed a loss of mere 13 % over a period of 60 days. The hydrolysis efficiency of purified endo beta-1,4-D-glucanase mixed with cocktail was demonstrated over commercial enzyme. Optimized enzymatic hydrolysis of sweet sorghum and sugarcane bagasse released 5.2 g/g (36 h) and 6.8 g/g (48 h) of reducing sugars, respectively. Separate hydrolysis and fermentation of sweet sorghum bagasse yielded 4.3 g/L bioethanol (16 h) confirmed by gas chromatography-mass spectrometry (GC-MS). Morphological and structural changes were assessed by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy. Elemental analysis was carried out by SEM equipped with energy dispersive X-ray technique. These unique properties prove the potentiality of enzyme for biomass conversion to biofuel and other industrial applications.