Bacterial inclusion bodies are aggregations of mostly inactive and misfolded proteins. However, previously the in vivo self-assembly of green fluorescent protein (GFP) fusions into fluorescent particles which displayed specific binding sites suitable for applications in bioseparation and diagnostics was demonstrated. Here, the suitability of GFP particles for enzyme immobilization was assessed. The enzymes tested were a thermostable ␣-amylase from Bacillus licheniformis, N-acetyl-D-neuraminic acid aldolase (NanA) from Escherichia coli, and organophosphohydrolase (OpdA) from Agrobacterium radiobacter. Respective GFP particles were isolated and could be stably maintained outside the cell. These enzyme-bearing GFP particles exhibited considerable stability across a range of temperature, pH, and storage conditions and could be recycled. The ␣-amylase-bearing particles retained activity after treatments at 4 to 85°C and at pHs 4 to 10, were stable for 3 months at 4°C, and could be recycled up to three times. OpdA-bearing particles retained degradation activity after treatments at 4 to 45°C and at pHs 5 to 10 and were able to be recycled up to four times. In contrast, the performance of NanA-bearing particles rapidly declined (>50% loss) after each recycling step and 3 months storage at 4°C. However, they were still able to convert N-acetylmannosamine and pyruvate to Nacetylneuraminic acid after treatment at 4 to 85°C and at pHs 4 to 11. Fluorescent GFP fusion particles represent a novel method for the immobilization and display of enzymes. Potential applications include diagnostic assays, biomass conversion, pharmaceutical production, and bioremediation.

Polyhydroxyalkanoate (PHA) is a carbon storage polymer produced by certain bacteria in unbalanced nutrient conditions. The PHA forms spherical inclusions surrounded by granule associate proteins including the PHA synthase (PhaC). Recently, the intracellular formation of PHA granules with covalently attached synthase from Ralstonia eutropha has been exploited as a novel strategy for oriented enzyme immobilisation. Fusing the enzyme of interest to PHA synthase results in a bifunctional protein able to produce PHA granules and immobilise the active enzyme of choice to the granule surface. Functionalised PHA granules can be isolated from the bacterial hosts, such as Escherichia coli, and maintain enzymatic activity in a wide variety of assay conditions. This approach to oriented enzyme immobilisation has produced higher enzyme activities and product levels than non-oriented immobilisation techniques such as protein inclusion based particles. Here, enzyme immobilisation via PHA synthase fusion is reviewed in terms of the genetic designs, the choices of enzymes, the control of enzyme orientations, as well as their current and potential applications.