Poly(hydroxyalkanoate) Generation from Nonchiral Substrates Using Multiple Enzyme Immobilizations on Peptide Nanofibers.

ABSTRACT

We developed a method for the immobilization of multiple active enzymes, allowing the production of chiral products from nonchiral substrates with recycling of expensive cofactors. Using a rapid, two-step process under nondenaturing conditions, we could preserve enzyme activity by separating the production of an immobilization scaffold from the attachment of the enzymes. The technique is applicable to a wide range of enzymes and will facilitate simple, cost-effective enzyme immobilization for research and industrial purposes. An (R)-specific poly(hydroxyalkanoate) synthase (PhaCRe from Ralstonia eutropha), an (S)-specific dehydrogenase (FadB from Pseudomonas putida), and an (R)-specific hydratase (PhaJ4Pa from P. aeruginosa) were immobilized by affinity tag-assisted binding to self-assembled antiparallel type ß-sheets with a coiled fiber structure formed from a decapeptide (P-K-F-K-I-I-E-F-E-P). The functionalized scaffolds were capable of producing poly(3-hydroxybutyrate) from ß-butyrolactone with the recycling of coenzyme A. Enzyme immobilization was confirmed by fluorescence microscopy using fusion proteins of the enzymes with fluorescent marker proteins, and activity was confirmed by spectroscopic activity assays.