title: In vivo enzyme immobilization by use of engineered polyhydroxyalkanoate synthase.
abstract: This study demonstrated that engineered polyhydroxyalkanoate (PHA) synthases can be employed as molecular tools to covalently immobilize enzymes at the PHA granule surface. The beta-galactosidase was fused to the N terminus of the class II PHA synthase from Pseudomonas aeruginosa. The open reading frame was confirmed to encode the complete fusion protein by T7 promoter-dependent overexpression. Restoration of PHA biosynthesis in the PHA-negative mutant of P. aeruginosa PAO1 showed a PHA synthase function of the fusion protein. PHA granules were isolated and showed beta-galactosidase activity. PHA granule attached proteins were analyzed and confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Surprisingly, the beta-galactosidase-PHA synthase fusion protein was detectable at a high copy number at the PHA granule, compared with PHA synthase alone, which was barely detectable at PHA granules. Localization of the beta-galactosidase at the PHA granule surface was confirmed by enzyme-linked immunosorbent assay using anti-beta-galactosidase antibodies. Treatment of these beta-galactosidase-PHA granules with urea suggested a covalent binding of the beta-galactosidase-PHA synthase to the PHA granule. The immobilized beta-galactosidase was enzymologically characterized, suggesting a Michaelis-Menten reaction kinetics. A Km of 630 microM and a Vmax of 17.6 nmol/min for orthonitrophenyl-beta-D-galactopyranoside as a substrate was obtained. The immobilized beta-galactosidase was stable for at least several months under various storage conditions. This study demonstrated that protein engineering of PHA synthase enables the manufacture of PHA granules with covalently attached enzymes, suggesting an application in recycling of biocatalysts, such as in fine-chemical production.
authors.affiliation: Institute of Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand.
authors.forename: Verena
authors.initials: V
authors.lastname: Peters
authors.forename: Bernd H A
authors.initials: BH
authors.lastname: Rehm
mesh_terms: D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism; D001709:Biotechnology / Q000379:methods; D004800:Enzymes, Immobilized; D010316:Particle Size; D011091:Polyesters / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
chemical_list: D004800:Enzymes, Immobilized; D011091:Polyesters; D011993:Recombinant Fusion Proteins; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; D001616:beta-Galactosidase