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Applied and Environmental Microbiology, April 2009, p. 2012-2016, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.02782-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

One-Step Production of Immobilized {alpha}-Amylase in Recombinant Escherichia coli{triangledown} ,{dagger}

Indira A. Rasiah and Bernd H. A. Rehm*

Institute of Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand

Received 7 December 2008/ Accepted 26 January 2009

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 Vmax of about 506 mU/mg of bead protein with a Km of about 5 µM, consistent with that of free {alpha}-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 {alpha}-amylase in a single step by using the polyester granules forming protein PhaC as a fusion partner in engineered E. coli.

* Corresponding author. Mailing address: Institute of Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand. Phone: 64 6 350 5515, ext. 7890. Fax: 64 6 350 2267. E-mail: b.rehm{at}massey.ac.nz

{triangledown} Published ahead of print on 5 February 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, April 2009, p. 2012-2016, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.02782-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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