title: Forizymes - functionalised artificial forisomes as a platform for the production and immobilisation of single enzymes and multi-enzyme complexes.
abstract: The immobilisation of enzymes plays an important role in many applications, including biosensors that require enzyme activity, stability and recyclability in order to function efficiently. Here we show that forisomes (plant-derived mechanoproteins) can be functionalised with enzymes by translational fusion, leading to the assembly of structures designated as forizymes. When forizymes are expressed in the yeast Saccharomyces cerevisiae, the enzymes are immobilised by the self-assembly of forisome subunits to form well-structured protein bodies. We used glucose-6-phosphate dehydrogenase (G6PDH) and hexokinase 2 (HXK2) as model enzymes for the one-step production and purification of catalytically active forizymes. These structures retain the typical stimulus-response reaction of the forisome and the enzyme remains active even after multiple assay cycles, which we demonstrated using G6PDH forizymes as an example. We also achieved the co-incorporation of both HXK2 and G6PDH in a single forizyme, facilitating a two-step reaction cascade that was 30% faster than the coupled reaction using the corresponding enzymes on different forizymes or in solution. Our novel forizyme immobilisation technique therefore not only combines the sensory properties of forisome proteins with the catalytic properties of enzymes but also allows the development of multi-enzyme complexes for incorporation into technical devices.
authors: University of Münster, Institute of Plant Biology and Biotechnology, Münster, 48143, Germany.
authors: Franziska
authors: F
authors: Visser
authors: University of Münster, Institute of Plant Biology and Biotechnology, Münster, 48143, Germany.
authors: Boje
authors: B
authors: Müller
authors: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Münster, 48143, Germany.
authors: Judith
authors: J
authors: Rose
authors: University of Münster, Institute of Plant Biology and Biotechnology, Münster, 48143, Germany.|Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Münster, 48143, Germany.
authors: Dirk
authors: D
authors: Prüfer
authors: University of Münster, Institute of Plant Biology and Biotechnology, Münster, 48143, Germany.
authors: Gundula A
authors: GA
authors: Noll
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006593:Hexokinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D005954:Glucosephosphate Dehydrogenase; D006593:Hexokinase