High Temperature Glycoside Hydrolase Enzymes Synergistically Degrade Lignocellulose

Lignocellulose is the most valuable renewable material for producing biofuels and biochemicals after treatment with different process. Lignocellulose mainly consists of cellulose, hemicellulose and lignin. Due to the recalcitrance and complication o f lignocellulose, the cost of enzymatic hydrolysis is very high, which is the bottleneck for industrial applications of this material. Development of cellulase cocktails became a mainstream strategy for enhancing this process. Mixtures of thermostable bacterial cellulases have an important significance for the theoretical research and practical application of the synergistic degradation of cellulose leading to form a basic foundation for the subsequent biofuel production by monosaccharide.In this paper, four different genes including CcCel9 A, CcCel9 B, CcCel48 A, and ?-glucosidase Blg A were cloned, expressed in E.coli, and purified. CcCel9 A was a processive endoglucanase identified from a cellulolytic thermophile Clostridium cellulosi CS-4-4, adding a nonprocessive endoglucanase CcCel9 B, an exoglucanase CcCel48 A from the same strain and a ?-glucosidase Blg A from Caldicellulosiruptor sp. F32. Different types of enzyme cocktails were evaluated and optimized. The best enzyme cocktail(CcCel9 A & CcCel9 B & CcCel48 A & BlgA=25:25:10:18) were calculated by reducing sugar concentration and DOS. The cocktail can hydrolysis 33.5% of the substrate(filter paper) to soluble sugars. Fluorophore-assisted carbohydrate electrophoresis and high-performance anion-exchange chromatography analysis showed that glucose was the only end product.The optimum cocktail was applied in the degradation of corn straw and it could convert 15.8% of the substrate to soluble sugars, and the glucose was the only end product. This is the first report of using these four enzymes for an enzyme cocktail. The ratios and interactions of cellulases were the key factors in the hydrolysis process.