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  • One-Step Production of Immobilized α-Amylase in Recombinant Escherichia coli

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    Author(s)
    Rasiah, Indira A
    Rehm, Bernd HA
    Griffith University Author(s)
    Rehm, Bernd
    Year published
    2009
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    Abstract
    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 α-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 ...
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    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 α-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 α-amylase activity. The α-amylase beads were assessed with respect to α-amylase activity, which was demonstrated qualitatively and quantitatively. The immobilized α-amylase showed Michaelis-Menten enzyme kinetics exerting a Vmax of about 506 mU/mg of bead protein with a Km of about 5 μM, consistent with that of free α-amylase. The stability of the enzyme at 85°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 α-amylase in a single step by using the polyester granules forming protein PhaC as a fusion partner in engineered E. coli.
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    Journal Title
    Applied and Environmental Microbiology
    Volume
    75
    Issue
    7
    DOI
    https://doi.org/10.1128/AEM.02782-08
    Copyright Statement
    © 2009 American Society for Microbiology. The attached file is reproduced here in accordance with the copyright policy of the publisher. Please refer to the journal's website for access to the definitive, published version.
    Subject
    Biochemistry and cell biology not elsewhere classified
    Publication URI
    http://hdl.handle.net/10072/369677
    Collection
    • Journal articles

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