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

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.

Two Distinct α- l -Arabinofuranosidases in Caldicellulosiruptor Species Drive Degradation of Arabinose-Based Polysaccharides

“…The samples were eluted at the speed of 1 ml/min via the use of an eluent consisting of 200 mM NaOH. In order to assess the synergy of each mixture, the degree of synergy (DOS) (23,39) was calculated using the following formula: DOS ϭ (total sugars released from enzyme mixture)/(total sugars released from incubation with individual enzymes). 1 H-NMR analysis of products.…”

Synergistic Enzyme Cocktail to Enhance Hydrolysis of Steam Exploded Wheat Straw at Pilot Scale

“…Recently, Oliva-Taravilla et al (2014) reported that the relative glucose recovery in presence of laccase with cellulosic substrate (Sigmacell and steam exploded wheat straw) was almost 10% lower than in the absence of laccase. Yang et al (2016) reported that the ratios and cooperation of cellulases are crucial in the hydrolysis and the efficiency could be further improved after adding accessory enzymes such as oxidoreductases, expansions, for which the bacterial enzyme pool is so abundant to excavate in the future (Agrawal et al, 2015d).…”

Artificially selecting microbial communities using propagule strategies

“…In particular, the hydrolysis of complex polymers into smaller molecular weight molecules (which can be then metabolized into useful byproducts by other community members) is often a limiting step in bioconversion (Minty et al 2013;Kim et al 2014;Yang et al 2016) . As a proof of concept, we set out to apply artificial selection to find a community with an enhanced ability to hydrolyze starch.…”