Bacterial polyhydroxyalkanoate granules: biogenesis, structure, and potential use as nano-/micro-beads in biotechnological and biomedical applications.

Polyhydroxyalkanoates (PHAs) are naturally occurring organic polyesters that are of interest for industrial and biomedical applications. These polymers are synthesized by most bacteria in times of unbalanced nutrient availability from a variety of substrates and they are deposited intracellularly as insoluble spherical inclusions or PHA granules. The granules consist of a polyester core, surrounded by a boundary layer with embedded or attached proteins that include the PHA synthase, phasins, depolymerizing enzymes, and regulatory proteins. Apart from ongoing industrial interest in the material PHA, more recently there has also been increasing interest in applications of the PHA granules as nano-/micro-beads after it was conceived that fusions to the granule associated proteins (GAPs) provide a way to immobilize target proteins at the granule surface. This review gives an overview of PHA granules in general, including biogenesis and GAPs, and focuses on their potential use as nano-/micro-beads in biotechnological and biomedical applications.

One-step production of immobilized alpha-amylase in recombinant Escherichia coli.

Industrial enzymes are often immobilized via chemical cross-linking onto solid supports to enhance stability and facilitate repeated use in bioreactors. For starch-degrading enzymes, immobilization usually places constraints on enzymatic conversion due to the limited diffusion of the macromolecular substrate through available supports. This study describes the one-step immobilization of a highly thermostable alpha-amylase (BLA) from Bacillus licheniformis and its functional display on the surface of polyester beads inside engineered Escherichia coli. An optimized BLA variant (Termamyl) was N-terminally fused to the polyester granule-forming enzyme PhaC of Cupriavidus necator. The fusion protein lacking the signal sequence mediated formation of stable polyester beads exhibiting alpha-amylase activity. The alpha-amylase beads were assessed with respect to alpha-amylase activity, which was demonstrated qualitatively and quantitatively. The immobilized alpha-amylase showed Michaelis-Menten enzyme kinetics exerting a V(max) of about 506 mU/mg of bead protein with a K(m) of about 5 microM, consistent with that of free alpha-amylase. The stability of the enzyme at 85 degrees C and the capacity for repeated usage in a starch liquefaction process were also demonstrated. In addition, structural integrity and functionality of the beads at extremes of pH and temperature, demonstrating their suitability for industrial use, were confirmed by electron microscopy and protein/enzyme analysis. This study proposes a novel, cost-effective method for the production of immobilized alpha-amylase in a single step by using the polyester granules forming protein PhaC as a fusion partner in engineered E. coli.

Variation in bacterial DGGE patterns from human saliva: over time, between individuals and in corresponding dental plaque microcosms.

OBJECTIVE: Eubacterial 16S rDNA fingerprints of human saliva and dental plaque microcosm biofilms grown in the multi-plaque artificial mouth (MAM) were characterised using denaturing gradient gel electrophoresis (DGGE). DESIGN: The stability of the bacterial community in the saliva of one individual collected over 7 years was assessed and compared with bacterial patterns in the saliva of 10 different individuals. DGGE was also used to assess changes in bacterial composition between saliva and mature plaque microcosms developed in the MAM from these 10 individual saliva samples. RESULTS: A relatively stable bacterial community (>87% concordance) was maintained within the individual oral environment of the standard donor over 7 years of monitoring. By comparison, DGGE fingerprint patterns of saliva from 10 different donors displayed greater variability (66% concordance). Variability between individual DGGE profiles increased further in mature plaque microcosms grown from the saliva of the 10 donors (52% concordance) with an increase in detected species diversity and evidence for conserved similarity and hence the maintenance of organisation during community development. CONCLUSIONS: These results suggest that stable ecological conditions were maintained long-term within the oral environment of the individual saliva donor but that transient fluctuations also occurred. The ecology and predominating microbiota in different individuals was host-specific and these differences were maintained to a degree during development into mature plaque microcosms. These findings also demonstrate the potential usefulness of applying DGGE to monitor temporal and developmental changes and possibly pathogenic patterns in oral bacterial communities from saliva and plaque.