In Vivo Enzyme Immobilization by Use of Engineered Polyhydroxyalkanoate Synthase
Open Access
- 1 March 2006
- journal article
- research article
- Published by American Society for Microbiology in Applied and Environmental Microbiology
- Vol. 72 (3), 1777-1783
- https://doi.org/10.1128/aem.72.3.1777-1783.2006
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 β-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 β-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 β-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 β-galactosidase at the PHA granule surface was confirmed by enzyme-linked immunosorbent assay using anti-β-galactosidase antibodies. Treatment of these β-galactosidase-PHA granules with urea suggested a covalent binding of the β-galactosidase-PHA synthase to the PHA granule. The immobilized β-galactosidase was enzymologically characterized, suggesting a Michaelis-Menten reaction kinetics. A K m of 630 μM and a V max of 17.6 nmol/min for orthonitrophenyl-β- d -galactopyranoside as a substrate was obtained. The immobilized β-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.Keywords
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