Polyhydroyxalkanoate Synthase Fusions as a Strategy for Oriented Enzyme Immobilisation

Abstract

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.

Insights into the surface topology of polyhydroxyalkanoate synthase: self-assembly of functionalized inclusions

“…This approach of using a single engineered PhaC as the sole protein anchor on PHA beads will maintain the simplicity of the PHA bead overexpression system and purification processes while allowing more complex applications, such as novel multivalent particulate vaccines, to be explored (Hooks et al 2014). Fig.…”

“…Until now, limited understanding of the structure of PhaC has confined Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6719-6) contains supplementary material, which is available to authorized users. protein engineering to fusions at the PhaC C-terminus and/or N-terminus (Brockelbank et al 2006;Parlane et al 2011;Blatchford et al 2012;Hooks et al 2013Hooks et al , 2014Chen et al 2014).…”

Polyhydroxyalkanoates, a family of natural polymers, and their applications in drug delivery

“…Similarly, during polymerisation, PhaC remains covalently linked to PHA granules via the active site cysteine residue . The insertion of desired gene sequences to the phaP or phaC genes can produce fusion proteins . Owing to the high affinity of PhaP for PHAs, these fusion proteins can be linked to PHA particles or to PHA granules within the bacteria.…”

“…Formation of these fusion proteins can mostly maintain enzyme activity . Research is therefore being directed towards functionalising PHA nanoparticles using this approach …”

“…111 Research is therefore being directed towards functionalising PHA nanoparticles using this approach. 104,109,110,112 Yao et al 112 fused PhaP with recombinant human epidermal growth factor (rhEGF) in order to specifically target its receptor. Up-regulation of the EGF receptor (EGFR) is a feature of many cancers, including hepatocellular carcinoma.…”