AUTHOR=Yang Min , Zhang Kun-Di , Zhang Pei-Yu , Zhou Xia , Ma Xiao-Qing , Li Fu-Li 

TITLE=Synergistic Cellulose Hydrolysis Dominated by a Multi-Modular Processive Endoglucanase from Clostridium cellulosi

JOURNAL=Frontiers in Microbiology

VOLUME=7

YEAR=2016

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2016.00932

DOI=10.3389/fmicb.2016.00932

ISSN=1664-302X

ABSTRACT=<p>Recalcitrance of biomass feedstock remains a challenge for microbial conversion of lignocellulose into biofuel and biochemicals. <italic>Clostridium cellulosi</italic>, 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 <italic>Cc</italic>Cel9A has been identified from <italic>C. cellulosi</italic> CS-4-4, mechanism of synergistic degradation of cellulose by various cellulases from strain CS-4-4 remains elusive. In this study, <italic>Cc</italic>Cel9A, <italic>Cc</italic>Cel9B, and <italic>Cc</italic>Cel48A 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 <italic>in vitro</italic>. Synergism between individual glycoside hydrolase during cellulose hydrolysis in the mixture was observed. <italic>Cc</italic>Cel9A and <italic>Cc</italic>Cel48A could degrade cellulose chain from non-reducing ends and reducing ends, respectively, to cello-oligosaccharide. <italic>Cc</italic>Cel9B could cut cellulose chain randomly and cello-oligosaccharides with varied length were released. In addition, a β-glucosidase BlgA from <italic>Caldicellulosiruptor</italic> 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 (<italic>Cc</italic>Cel9A:<italic>Cc</italic>Cel9B:<italic>Cc</italic>Cel48A:BlgA = 25:25:10:18), and only glucose was detected as main production by using high-performance anion-exchange chromatography. Processive endoglucanase <italic>Cc</italic>Cel9A, dominated in secretome of <italic>C. cellulosi</italic>, showed good potential in developing cellulase cocktail due to its exquisite cooperation with various cellulases.</p>