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dc.contributor.authorRasiah, Indira A
dc.contributor.authorRehm, Bernd HA
dc.date.accessioned2018-02-16T06:10:25Z
dc.date.available2018-02-16T06:10:25Z
dc.date.issued2009
dc.identifier.issn0099-2240
dc.identifier.doi10.1128/AEM.02782-08
dc.identifier.urihttp://hdl.handle.net/10072/369677
dc.description.abstractIndustrial 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.
dc.description.peerreviewedYes
dc.languageEnglish
dc.language.isoeng
dc.publisherAmerican Society for Microbiology
dc.relation.ispartofpagefrom2012
dc.relation.ispartofpageto2016
dc.relation.ispartofissue7
dc.relation.ispartofjournalApplied and Environmental Microbiology
dc.relation.ispartofvolume75
dc.subject.fieldofresearchBiochemistry and cell biology not elsewhere classified
dc.subject.fieldofresearchcode310199
dc.titleOne-Step Production of Immobilized α-Amylase in Recombinant Escherichia coli
dc.typeJournal article
dc.type.descriptionC1 - Articles
dc.type.codeC - Journal Articles
dc.description.versionVersion of Record (VoR)
gro.rights.copyright© 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.
gro.hasfulltextFull Text
gro.griffith.authorRehm, Bernd


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