Abstract
Recalcitrance of biomass feedstock remains a challenge for microbial conversion of lignocellulose into biofuel and biochemicals. Clostridium cellulosi, one thermophilic bacterial strain dominated in compost, could hydrolyze lignocellulose at elevated temperature by secreting more than 38 glycoside hydrolases belong to 15 different families. Though one multi-modular endoglucanase CcCel9A has been identified from C. cellulosi CS-4-4, mechanism of synergistic degradation of cellulose by various cellulases from strain CS-4-4 remains elusive. In this study, CcCel9A, CcCel9B, and CcCel48A were characterized as processive endoglucanase, non-processive endoglucanase, and exoglucanase, respectively. To understand how they cooperate with each other, we estimated the approximate concentration ratio on the zymogram and optimized it using purified enzymes in vitro. Synergism between individual glycoside hydrolase during cellulose hydrolysis in the mixture was observed. CcCel9A and CcCel48A could degrade cellulose chain from non-reducing ends and reducing ends, respectively, to cello-oligosaccharide. CcCel9B could cut cellulose chain randomly and cello-oligosaccharides with varied length were released. In addition, a β-glucosidase BlgA from Caldicellulosiruptor sp. F32 which could cleave cello-oligosaccharides including G2-G6 to glucose was added to the enzyme mixture to remove the product inhibition of its partners. The combination and ratios of these cellulases were optimized based on the release rate of glucose. Hydrolysis of corn stalk was conducted by a four-component cocktail (CcCel9A:CcCel9B:CcCel48A:BlgA = 25:25:10:18), and only glucose was detected as main production by using high-performance anion-exchange chromatography. Processive endoglucanase CcCel9A, dominated in secretome of C. cellulosi, showed good potential in developing cellulase cocktail due to its exquisite cooperation with various cellulases.
Highlights
Lignocellulose is the most abundant renewable biomass for the production of biofuels and biochemicals
Due to the recalcitrance and complexity of lignocellulose, the cost of enzymatic hydrolysis is very high, which is the bottleneck of this industry
The specific activity of CcCel48A on filter paper was poor, it was the only active band identified on the exoglucanase-zymogram, suggesting that CcCel48A was the most significant exoglucanase in C. cellulosi CS-4-4 (Zhang et al, 2014)
Summary
Lignocellulose is the most abundant renewable biomass for the production of biofuels and biochemicals. The bioconversion of lignocellulose to fuel and chemical is carried out via three processes: pretreatment, enzymatic hydrolysis, and fermentation (Lynd et al, 2005). Due to the recalcitrance and complexity of lignocellulose, the cost of enzymatic hydrolysis is very high, which is the bottleneck of this industry. Development of cellulose cocktails became a mainstream strategy to enhance the process. The components of the cocktails could cooperate with each other in modifying the structure of crystalline cellulose, making cellulose more accessible for cellulases (Jeoh et al, 2006; Arantes and Saddler, 2010). Mining cellulases candidates which can synergize in nature was imperative
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