Abstract
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 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 β-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 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-016-0421-y) contains supplementary material, which is available to authorized users.
Highlights
In the current scenario, the major concerns are towards the diminishing of fossil fuels which have forced the energy industries and researchers to develop alternatives to the existing fuels (Bentsen and Felby 2012)
The endo b-1,4-D-glucanase produced by Trichoderma harzianum strain HZN11 was purified to homogeneity by (NH4)2SO4 precipitation, DEAE-Sepharose and Sephadex G-100 chromatography
The elution profiles of DEAE-Sepharose and Sephadex G-100 chromatography are shown in Fig. S1 (Supplementary Information, SI) which did not show any multiple isoforms of enzyme
Summary
The major concerns are towards the diminishing of fossil fuels which have forced the energy industries and researchers to develop alternatives to the existing fuels (Bentsen and Felby 2012). One of the attractive sustainable substitutes is the microbial production of bioethanol from lignocellulosic wastes as it is costeffective and renewable (Ren et al 2009). Plant biomass constitute of cellulose which is the major organic polysaccharide found in the biosphere (Bhat and Bhat 1997) and is renewable. Biodegradation of plant based biomass requires cellulose and hemicellulose saccharifying enzymes. Cellulases participate in saccharification of biomass for bioethanol production (Dhillon et al 2011), by mainly acting on b-1,4-glycosidic bonds of cellulose. Cellulolytic enzymes have been classified as: endoglucanase (endo-1,4-D-glucanase, EG), cellobiohydrolase (exo-1,4-D-glucanase, CBH) and glucosidase (1,4D-glucosidase, BG) (Saha 2004), which have been shown to act synergistically for effective degradation
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