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 β-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 ~55 kDa. The optimum temperature and pH were 60 °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 °C indicating thermostability. Endo β-1,4-D-glucanase was activated by Ca and Mg but inhibited by Hg, Pb and Cd. The effect of thiol reagents, metal chelators, oxidizing agents and surfactants on enzyme activity has been studied. Purified endo β-1,4-D-glucanase exhibited highest specificity towards carboxymethyl cellulose. Kinetic analysis showed the K , V and K (cellobiose inhibitor) of 2.5 mg/mL, 83.75 U/mg and 0.066 M, respectively. The storage stability of purified endo β-1,4-D-glucanase showed a loss of mere 13 % over a period of 60 days. The hydrolysis efficiency of purified endo β-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.