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.

FIG. 1.

Construction of plasmid pBHR80AlacZ.

FIG. 2.

SDS-PAGE analysis of overproduced PhaC1 and LacZ-PhaC1 in E. coli BL21(DE3). Lane 1, molecular mass standard (New England Biolabs, United Kingdom); lane 2, whole-cell lysate of E. coli BL21(DE3) harboring pET14b-lacZphaC1; lane 3, whole-cell lysate of E. coli BL21(DE3) harboring pET14b-phaC1; lane 4, whole-cell lysate of E. coli XL1-Blue harboring pBHR80ALacZ; lane 5, molecular mass standard (New England Biolabs, United Kingdom). The arrows indicate proteins confirmed by peptide mass fingerprinting using MALDI-TOF MS (Table 3).

FIG. 3.

SDS-PAGE analysis of PHA granules. Lane 1, molecular mass standard (New England Biolabs, United Kingdom); lane 2, PHA granules from wild-type P. aeruginosa PAO1; lane 3, PHA granules from P. aeruginosa ΔphaC1-Z-C2(pBBR1JO5-lacZphaC1). The arrows indicate proteins confirmed by peptide mass fingerprinting using MALDI-TOF MS (Table 3).

FIG. 4.

SDS-PAGE analysis of PHA granules before and after treatment with 8 M urea. Lane 1, PHA granules from P. aeruginosa ΔphaC1-Z-C2(pBBR1JO5-lacZphaC1) before treatment with 8 M urea; lane 2, PHA granules (insoluble fraction) after treatment with urea; lane 3, proteins (soluble fraction) released from PHA granules after treatment with 8 M urea; lane 4, molecular mass standard (New England Biolabs, United Kingdom). The arrows indicate proteins confirmed by peptide mass fingerprinting using MALDI-TOF MS (Table 3).

FIG. 5.

Model of in vivo enzyme immobilization using engineered PHA synthase. CoA, coenzyme A.