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Applied and Environmental Microbiology, March 2006, p. 1777-1783, Vol. 72, No. 3
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.3.1777-1783.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

In Vivo Enzyme Immobilization by Use of Engineered Polyhydroxyalkanoate Synthase

Verena Peters and Bernd H. A. Rehm*

Institute of Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand

Received 3 November 2005/ Accepted 20 December 2005

This study demonstrated that engineered polyhydroxyalkanoate (PHA) synthases can be employed as molecular tools to covalently immobilize enzymes at the PHA granule surface. The ß-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 ß-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 ß-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 ß-galactosidase at the PHA granule surface was confirmed by enzyme-linked immunosorbent assay using anti-ß-galactosidase antibodies. Treatment of these ß-galactosidase-PHA granules with urea suggested a covalent binding of the ß-galactosidase-PHA synthase to the PHA granule. The immobilized ß-galactosidase was enzymologically characterized, suggesting a Michaelis-Menten reaction kinetics. A Km of 630 µM and a Vmax of 17.6 nmol/min for orthonitrophenyl-ß-D-galactopyranoside as a substrate was obtained. The immobilized ß-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.

* Corresponding author. Mailing address: Institute of Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand. Phone: 64 6 350 5515, ext. 7890. Fax: 64 6 350 5688. E-mail: B.Rehm{at}massey.ac.nz.

Applied and Environmental Microbiology, March 2006, p. 1777-1783, Vol. 72, No. 3
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.3.1777-1783.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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