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Research article - Peer-reviewed, 2019

Coupled chemistry kinetics demonstrate the utility of functionalized Sup35 amyloid nanofibrils in biocatalytic cascades

Schmuck, Benjamin; Gudmundsson, Mikael; Härd, Torleif; Sandgren, Mats

Abstract

Concerns over the environment are a central driver for designing cell-free enzymatic cascade reactions that synthesize non?petrol-based commodity compounds. An often-suggested strategy that would demonstrate the economic competitiveness of this technology is recycling of valuable enzymes through their immobilization. For this purpose, amyloid nanofibrils are an ideal scaffold to realize chemistry-free covalent enzyme immobilization on a material that offers a large surface area. However, in most instances, only single enzyme?functionalized amyloid fibrils have so far been studied. To embark on the next stage, here we displayed xylanase A, ?-xylosidase, and an aldose sugar dehydrogenase on Sup35(1?61) nanofibrils to convert beechwood xylan to xylonolactone. We characterized this enzymatic cascade by measuring the time-dependent accumulation of xylose, xylooligomers, and xylonolactone. Furthermore, we studied the effects of relative enzyme concentrations, pH, temperature, and agitation on product formation. Our investigations revealed that a modular cascade with a mixture of xylanase and ?-xylosidase, followed by product removal and separate oxidation of xylose with the aldose sugar dehydrogenase, is more productive than an enzyme mix containing all of these enzymes together. Moreover, we found that the nanofibril-coupled enzymes do not lose activity compared with their native state. These findings provide proof of concept of the feasibility of functionalized Sup35(1?61) fibrils as a molecular scaffold for biocatalytic cascades consisting of reusable enzymes that can be used in biotechnology.

Keywords

amyloid; enzyme kinetics; fusion protein; protein aggregation; protein chimera; protein engineering; aldose sugar dehydrogenase; beta-xylosidase; protein nanofibrils; xylan; xylanase

Published in

Journal of Biological Chemistry
2019, volume: 294, number: 41, pages: 14966–14977

Authors' information

Swedish University of Agricultural Sciences, Department of Molecular Sciences
Gudmundsson, Mikael
Swedish University of Agricultural Sciences, Department of Molecular Sciences
Swedish University of Agricultural Sciences, Department of Molecular Sciences
Swedish University of Agricultural Sciences, Department of Molecular Sciences

Sustainable Development Goals

SDG12 Responsible consumption and production

UKÄ Subject classification

Biochemistry and Molecular Biology

Publication Identifiers

DOI: https://doi.org/10.1074/jbc.RA119.008455

URI (permanent link to this page)

https://res.slu.se/id/publ/101591