Abstract
Microbial production of cellulose-degrading enzymes could be significantly improved using traditional mutagenesis treatment. Development of high-titre cellulase producing mutants drastically reduces the costs involved in cellulase production and downstream processing in commercial-scale enzyme production. Here, we have evaluated the efficacy of different Aspergillus terreus D34 mutants for hyper-production of improved cellulase enzymes utilizing locally available lignocellulosic biomass residues as growth substrates in solid state fermentation conditions. Further, enzymatic hydrolysis of mild-alkali pre-treated rice straw was performed using the improved cellulases. A 4.9-fold higher β-glucosidase activity was obtained from ethyl methyl sulphonate (EMS) treated mutant strain (EMS2) when grown on mixed rice straw/sugarcane bagasse (RSBG) biomass growth substrate. Similarly with the EMS2 mutant and BG-grown culture extract a 1.1-fold higher xylanase activity was observed. Irrespective of the growth substrates and the mutant strains, the maximum cellulase (FPase, carboxymethyl cellulase, avicelase, β-glucosidase) and xylanase activities (U mL−1) were 2.34, 39.8, 2.46, 19.9 and 655, respectively. Further, external supplementation of 20% bovine serum albumin (BSA), 3% tween 80 and 20% polyethylene glycol (PEG) 6000 to the crude enzyme extract increased the FPase activity nearly 4.0-, 2.8- and 2.2-fold. Addition of 0.05% sodium benzoate marginally increased the stability of cellulase enzyme and retained more than 60% of the initial activity after 96 h incubation at 37°C. While at 4°C, no loss in enzyme activity was observed even after prolonged incubation period (up to 90 days). Further, maximum reducing sugars of 0.842 g g−1 at a rate of 0.25 mM g−1 h−1 at 10% biomass loading of mild-alkali pretreated rice straw was produced using the BG-grown culture extract of EMS2 mutant strain. The extracellular protein production and corresponding cellulase activities of A. terreus D34 were significantly enhanced after combined UV and chemical mutagenesis treatments. In the present study, besides accelerating the rate of cellulase production, we have also demonstrated production of high reducing sugars by enzymatic saccharification of pretreated lignocellulosic biomass using hyper-produced cellulase enzymes. Due to high enzyme activity of the cellulase enzymes produced from the mutant strains, the volume of enzyme loadings in enzymatic hydrolysis could be reduced up to 7-fold. These studies clearly show the potential of the developed hyper-cellulase producing mutants in decreasing the overall process economics of cellulosic ethanol technology.
Highlights
Microbial production of cellulose-degrading enzymes could be significantly improved using traditional mutagenesis treatment
UV and chemical mutagenesis of A. terreus Recently, we have reported the growth of A. terreus D34 on different lignocellulosic biomass residues and biochemical studies of crude cellulose-degrading enzymes (Kumar and Parikh 2015; Narra et al 2012)
Significant improvement in extracellular protein production and cellulase activities was observed when the filamentous fungus was subjected to conventional mutagenesis by UV and chemical treatments
Summary
Microbial production of cellulose-degrading enzymes could be significantly improved using traditional mutagenesis treatment. We have evaluated the efficacy of different Aspergillus terreus D34 mutants for hyper-production of improved cellulase enzymes utilizing locally available lignocellulosic biomass residues as growth substrates in solid state fermentation conditions. Lignocellulosic biomass is a renewable, abundant and inexpensive natural bioresource and is preferably used in the bioconversion processes for production of biofuels and other value added products. This bioconversion is quite challenging owing to the complex structural integrity of plant cell wall materials which is inherently designed to resist microbial degradation. Is well known for commercial cellulase production but lacks in secreting sufficient β-glucosidase, while on the contrary, majority of the Aspergillus sp. Trichoderma sp. is well known for commercial cellulase production but lacks in secreting sufficient β-glucosidase, while on the contrary, majority of the Aspergillus sp. produce high β-glucosidase enzymes
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