In vivo immobilization of an organophosphorus hydrolyzing enzyme on bacterial polyhydroxyalkanoate nano-granules.

Background: Polyhydroxyalkanoate (PHA) are nano-granules naturally produced by bacteria. Two types of proteins, PHA synthase (PhaC) and phasins (PhaPs), are attached to the PHA surface by covalent and hydrophobic interactions. Utilizing these anchored proteins, functionalized PHA nano-granules displaying proteins of interest can be easily prepared by fermentation.

Results: In this study, a one-step fabrication method was developed for stable and efficient immobilization of an organophosphorus degrading enzyme on PHA nano-granules. The nano-biocatalysts were produced in recombinant Escherichia coli cells into which the polyhydroxyalkanoate synthesis pathway from Cupriavidus necator had been introduced. Two different strategies, covalent attachment and hydrophobic binding, were investigated by fusing bacterial organophosphorus anhydride hydrolase (OPAA4301) with PhaC and PhaP, respectively. Using both methods, the tetrameric enzyme successfully self-assembled and was displayed on the PHA surface. The display density of the target fused enzyme was enhanced to 6.8% of total protein on decorated PHA by combination of covalent and non-covalent binding modes. Immobilization of the enzyme on PHA granules resulted in higher catalytic efficiency, increased stability and excellent reusability. The k values of the immobilized enzymes increased by threefold compared to that of the free enzyme. The pH stability under acidic conditions was significantly enhanced, and the immobilized enzyme was stable at pH 3.0-11.0. Furthermore, more than 80% of the initial enzyme activity retained after recycling ten times.

Conclusions: This study provides a promising approach for cost-efficient in vivo immobilization of a tetrameric organophosphorus degrading enzyme. The immobilization process expands the utility of the enzyme, and may inspire further commercial developments of PHA nano-biocatalysts. As revealed by our results, combination of covalent and non-covalent binding is recommended for display of enzymes on PHA granules.