title: In vivo self-assembly of stable green fluorescent protein fusion particles and their uses in enzyme immobilization.
abstract: Bacterial inclusion bodies are aggregations of mostly inactive and misfolded proteins. However, previously the in vivo self-assembly of green fluorescent protein (GFP) fusions into fluorescent particles which displayed specific binding sites suitable for applications in bioseparation and diagnostics was demonstrated. Here, the suitability of GFP particles for enzyme immobilization was assessed. The enzymes tested were a thermostable α-amylase from Bacillus licheniformis, N-acetyl-d-neuraminic acid aldolase (NanA) from Escherichia coli, and organophosphohydrolase (OpdA) from Agrobacterium radiobacter. Respective GFP particles were isolated and could be stably maintained outside the cell. These enzyme-bearing GFP particles exhibited considerable stability across a range of temperature, pH, and storage conditions and could be recycled. The α-amylase-bearing particles retained activity after treatments at 4 to 85°C and at pHs 4 to 10, were stable for 3 months at 4°C, and could be recycled up to three times. OpdA-bearing particles retained degradation activity after treatments at 4 to 45°C and at pHs 5 to 10 and were able to be recycled up to four times. In contrast, the performance of NanA-bearing particles rapidly declined (>50% loss) after each recycling step and 3 months storage at 4°C. However, they were still able to convert N-acetylmannosamine and pyruvate to N-acetylneuraminic acid after treatment at 4 to 85°C and at pHs 4 to 11. Fluorescent GFP fusion particles represent a novel method for the immobilization and display of enzymes. Potential applications include diagnostic assays, biomass conversion, pharmaceutical production, and bioremediation.
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.
authors.forename: Mark
authors.initials: M
authors.lastname: Venning-Slater
authors.forename: David O
authors.initials: DO
authors.lastname: Hooks
authors.forename: Bernd H A
authors.initials: BH
authors.lastname: Rehm
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology / Q000235:genetics; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007652:Oxo-Acid-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C045290:phosphorylphosphatase; D010744:Phosphoric Monoester Hydrolases; D000516:alpha-Amylases; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase