title: Insertion of endocellulase catalytic domains into thermostable consensus ankyrin scaffolds: effects on stability and cellulolytic activity.
abstract: Degradation of cellulose for biofuels production holds promise in solving important environmental and economic problems. However, the low activities (and thus high enzyme-to-substrate ratios needed) of hydrolytic cellulase enzymes, which convert cellulose into simple sugars, remain a major barrier. As a potential strategy to stabilize cellulases and enhance their activities, we have embedded cellulases of extremophiles into hyperstable α-helical consensus ankyrin domain scaffolds. We found the catalytic domains CelA (CA, GH8; Clostridium thermocellum) and Cel12A (C12A, GH12; Thermotoga maritima) to be stable in the context of the ankyrin scaffold and to be active against both soluble and insoluble substrates. The ankyrin repeats in each fusion are folded, although it appears that for the C12A catalytic domain (CD; where the N and C termini are distant in the crystal structure), the two flanking ankyrin domains are independent, whereas for CA (where termini are close), the flanking ankyrin domains stabilize each other. Although the activity of CA is unchanged in the context of the ankyrin scaffold, the activity of C12A is increased between 2- and 6-fold (for regenerated amorphous cellulose and carboxymethyl cellulose substrates) at high temperatures. For C12A, activity increases with the number of flanking ankyrin repeats. These results showed ankyrin arrays to be a promising scaffold for constructing designer cellulosomes, preserving or enhancing enzymatic activity and retaining thermostability. This modular architecture will make it possible to arrange multiple cellulase domains at a precise spacing within a single polypeptide, allowing us to search for spacings that may optimize reactivity toward the repetitive cellulose lattice.
authors: Institute for Multiscale Modeling of Biological Interactions, Johns Hopkins University, Baltimore, Maryland, USA.
authors: Eva S
authors: ES
authors: Cunha
authors: Christine L
authors: CL
authors: Hatem
authors: Doug
authors: D
authors: Barrick
mesh_terms: D000595:Amino Acid Sequence; D017487:Ankyrins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D056804:Biofuels; D001709:Biotechnology / Q000379:methods; D044602:Cellulases / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D048013:Acetivibrio thermocellus / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D045170:Matrix Attachment Regions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D020124:Thermotoga maritima / Q000235:genetics*
chemical_list: D017487:Ankyrins; D056804:Biofuels; D017931:DNA Primers; D044602:Cellulases
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