Abstract
BackgroundPolyhydroxyalkanoate (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.ResultsIn 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 kcat 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.ConclusionsThis 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.
Highlights
Many microorganisms naturally produce nanometer scale particles
Display of organophosphorus hydrolase on PHA granules via anchored proteins To enable production of PHA granules in E. coli BL21 (DE3), the hydroxyalkanoate biosynthetic pathway from C. necator ATCC 17699 was first introduced to the BL21(DE3) strain using pACYC-phaAB transforming plasmid encoding β-ketothiolase and acetoacetyl-CoA reductase (Fig. 2a)
This study demonstrated the functional display of a bacterial tetrameric organophosphorus hydrolase on the surface of PHA granules, which was achieved by coexpression of target enzyme and surface anchoring proteins in E. coli
Summary
Many microorganisms naturally produce nanometer scale particles. These biologically produced nanoparticles (BNPs) exist in a wide range of forms including polyhydroxyalkanoates, endospores, exosomes, and magnetosomes [1]. BNPs are composed of a spherical core–shell structure, where the organic or inorganic cores are associated with a number of proteins on their surfaces [2,3,4]. These surface proteins have attracted much attention and inspired investigations to explore them as anchoring tags to attach proteins and peptides to the surface of BNPs. The engineered BNPs are exciting prospects for drug targeting [5], bio-catalysis [6], biochemical separation [7] and bioremediation [8]. 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 prepared by fermentation
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