Synergistic enhancement of cellulase pairs linked by consensus ankyrin repeats: Determination of the roles of spacing, orientation, and enzyme identity: Inserting Multiple Cellulases into Ankyrin Scaffolds

Cunha, Eva S.; Hatem, Christine L.; Barrick, Doug

doi:10.1002/prot.25047

Title: Synergistic enhancement of cellulase pairs linked by consensus ankyrin repeats: Determination of the roles of spacing, orientation, and enzyme identity: Inserting Multiple Cellulases into Ankyrin Scaffolds

Journal Article · Tue May 03 00:00:00 EDT 2016 · Proteins

DOI:https://doi.org/10.1002/prot.25047· OSTI ID:1467438

Cunha, Eva S. ^[1]; Hatem, Christine L. ^[2]; Barrick, Doug ^[2]

Johns Hopkins Univ., Baltimore, MD (United States). T.C. Jenkins Dept. of Biophysics; Max Plank Inst. of Biophysics, Frankfurt am Main (Germany). Dept. of Structural Biology
Johns Hopkins Univ., Baltimore, MD (United States). T.C. Jenkins Dept. of Biophysics

Biomass deconstruction to small simple sugars is a potential approach to biofuels production, however the highly recalcitrant nature of biomass limits the economic viability of this approach. Thus, research on efficient biomass degradation is necessary to achieve large-scale production of biofuels. Enhancement of cellulolytic activity by increasing synergism between cellulase enzymes holds promise in achieving high-yield biofuels production. Here we have inserted cellulase pairs from extremophiles into hyper-stable α-helical consensus ankyrin repeat domain scaffolds. Such chimeric constructs allowed us to optimize arrays of enzyme pairs against a variety of cellulolytic substrates. We found that endocellulolytic domains CelA (CA) and Cel12A (C12A) act synergistically in the context of ankyrin repeats, with both three and four repeat spacing. The extent of synergy differs for different substrates. Also, having C12A N-terminal to CA provides greater synergy than the reverse construct, especially against filter paper. In contrast, we do not see synergy for these enzymes in tandem with CelK (CK) catalytic domain, a larger exocellulase, demonstrating the importance of enzyme identity in synergistic enhancement. Furthermore, we found endocellulases CelD and CA with three repeat spacing to act synergistically against filter paper. Importantly, connecting CA and C12A with a disordered linker of similar contour length, shows no synergistic enhancement, indicating that synergism results from connecting these domains with folded ankyrin repeats. These results show that ankyrin arrays can be used to vary spacing and orientation between enzymes, helping to design and optimize artificial cellulosomes, providing a novel architecture for synergistic enhancement of enzymatic cellulose degradation.

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Research Organization:: Johns Hopkins Univ., Baltimore, MD (United States)

Sponsoring Organization:: USDOE; National Institutes of Health (NIH); Portuguese Foundation for Science and Technology

Grant/Contract Number:: FG02-04ER25626; 1 R01-GM068462

OSTI ID:: 1467438

Journal Information:: Proteins, Vol. 84, Issue 8; ISSN 0887-3585

Publisher:: WileyCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 3 works

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Title: Synergistic enhancement of cellulase pairs linked by consensus ankyrin repeats: Determination of the roles of spacing, orientation, and enzyme identity: Inserting Multiple Cellulases into Ankyrin Scaffolds

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