In vivo enzyme immobilization by use of engineered polyhydroxyalkanoate synthase

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 PRA 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 PRA-negative mutant of P. aeruginosa PAO1 showed a PHA synthase function of the fusion protein. PRA granules were isolated and showed beta-galactosidase activity. PRA 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 PRA granules. Localization of the beta-galactosidase at the PRA granule surface was confirmed by enzyme-linked immunosorbent assay using anti-beta-galactosidase antibodies. Treatment of these beta-galactosidase-PRA granules with urea suggested a covalent binding of the beta-galactosidase-PRA synthase to the PRA granule. The immobilized beta-galactosidase was enzymologically characterized, suggesting a Michaelis-Menten reaction kinetics. A K-m of 630 mu M and a V-max 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 PRA granules with covalently attached enzymes, suggesting an application in recycling of biocatalysts, such as in fine-chemical production.