Abstract
In recent work, we reported the self-assembly of a comprehensive set of defined "bifunctional" chimeric cellulosomes. Each complex contained the following: (i) a chimeric scaffoldin possessing a cellulose-binding module and two cohesins of divergent specificity and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. This approach allowed the controlled integration of desired enzymes into a multiprotein complex of predetermined stoichiometry and topology. The observed enhanced synergy on recalcitrant substrates by the bifunctional designer cellulosomes was ascribed to two major factors: substrate targeting and proximity of the two catalytic components. In the present work, the capacity of the previously described chimeric cellulosomes was amplified by developing a third divergent cohesin-dockerin device. The resultant trifunctional designer cellulosomes were assayed on homogeneous and complex substrates (microcrystalline cellulose and straw, respectively) and found to be considerably more active than the corresponding free enzyme or bifunctional systems. The results indicate that the synergy between two prominent cellulosomal enzymes (from the family-48 and -9 glycoside hydrolases) plays a crucial role during the degradation of cellulose by cellulosomes and that one dominant family-48 processive endoglucanase per complex is sufficient to achieve optimal levels of synergistic activity. Furthermore cooperation within a cellulosome chimera between cellulases and a hemicellulase from different microorganisms was achieved, leading to a trifunctional complex with enhanced activity on a complex substrate.
Highlights
Typical cellulosomes, such as those produced by Clostridium cellulolyticum and Clostridium thermocellum contain a scaffolding protein devoid of enzymatic activity that displays a powerful substratetargeting family-3a carbohydrate-binding module (CBM)1 and numerous cohesin modules [2, 3]
A series of trifunctional designer cellulosomes was self-assembled by combining the new chimeric scaffoldin with three different enzymes
Selection and Characterization of a Third Divergent CohesinDockerin Device—Previous studies have shown that complexation of cellulase pairs, appended with suitable dockerins, into hybrid miniscaffoldins containing two divergent cohesins served to induce significant increases in cellulolytic activity, especially toward recalcitrant cellulose such as microcrystalline cellulose [6, 11]
Summary
CONTROLLED INCORPORATION OF THREE DISTINCT ENZYMES INTO A DEFINED TRIFUNCTIONAL SCAFFOLDIN*. Each complex contained the following: (i) a chimeric scaffoldin possessing a cellulose-binding module and two cohesins of divergent specificity and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins This approach allowed the controlled integration of desired enzymes into a multiprotein complex of predetermined stoichiometry and topology. Cellulosomes are extracellular macromolecular complexes produced by cellulolytic anaerobic bacteria that efficiently degrade cellulose (the most abundant biopolymer on earth) and related polysaccharides of the plant cell wall [1] Typical cellulosomes, such as those produced by Clostridium cellulolyticum and Clostridium thermocellum contain a scaffolding protein devoid of enzymatic activity that displays a powerful substratetargeting family-3a carbohydrate-binding module (CBM) and numerous cohesin modules (eight and nine for the two respective species) [2, 3]. A library of 75 different bifunctional designer cellulosomes was constructed by combining one of the five chimeric scaffoldins with one of the
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