title: Entrapment in E. coli improves the operational stability of recombinant beta-glycosidase CelB from Pyrococcus furiosus and facilitates biocatalyst recovery.
abstract: beta-Glycosidase CelB from the hyperthermophilic archaeon Pyrococcus furiosus is a versatile biocatalyst that has been used for the hydrolysis and synthesis of beta-d-glycosidic compounds at high temperatures and in non-conventional solvents. In spite of its outstanding thermal stability, CelB is prone to inactivation in the presence of reducing sugars and through recirculation in loop enzyme reactors. Entrapment into E. coli cells was used here to improve the stability of recombinant CelB under conditions promoting strong inactivation. Glutardialdehyde-mediated protein cross-linking or rigidification of the cell membrane by adding magnesium ions was required to prevent release of CelB from within the cell into the bulk solution. In the presence of 1M glucose or when applying recirculation rates of 2.6 min(-1), the entrapped enzyme was around two-fold more stable at 80 degrees C than free CelB. The significance of the stabilisation was attenuated by the decrease in CelB initial activity which was due to cross-linking and glutardialdehyde concentration-dependent. Entrapment facilitated downstream processing of CelB and biocatalyst recovery in repeated batchwise conversions of lactose at elevated temperatures.
authors.affiliation: Research Centre Applied Biocatalysis, Petersgasse 14, c/o Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kamrat
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D019149:Bioreactors; D002384:Catalysis / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D007785:Lactose / Q000378:metabolism; D020027:Pyrococcus furiosus / Q000187:drug effects / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism*; D013696:Temperature; D001617:beta-Glucosidase / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; C406923:CelB protein, Pyrococcus furiosus; D001617:beta-Glucosidase; D005947:Glucose; D007785:Lactose