title: Coated-wall microreactor for continuous biocatalytic transformations using immobilized enzymes.
abstract: Microstructured flow reactors are emerging tools for biocatalytic process development. A compelling design is that of the coated-wall reactor where enzyme is present as a surface layer attached to microchannel walls. However, preparation of a highly active wall biocatalyst remains a problem. Here, a stainless steel microreactor was developed where covalent immobilization of the enzyme in multiple linear flow channels of the reaction plate was supported by a macroporous wash-coat layer of gamma-aluminum oxide. Using surface functionalization with aminopropyl triethoxysilane followed by activation with glutardialdehyde, the thermophilic beta-glycosidase CelB from Pyrococcus furiosus was bound with retention of half of the specific activity of the free enzyme (800 U/mg), yielding a high catalyst loading of about 500 U/mL. This microreactor was employed for the continuous hydrolysis of lactose (100 mM) at 80 degrees C, providing a space-time yield of 500 mg glucose/(mL h) at a stable conversion of > or =70%. The immobilized enzyme displayed a half-life of 15 days under the operational conditions. Due to the absence of hydrophobic solute-material interactions, which limit the scope of microstructures fabricated from poly(dimethylsiloxane) for biocatalytic applications, the new microreactor was fully compatible with the alternate enzyme substrate 2-nitro-phenyl-beta-D-galactoside and the 2-nitro-phenol product resulting from its hydrolysis catalyzed by CelB.
authors.affiliation: Research Center Applied Biocatalysis, Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Graz, Austria.
authors.forename: Malene S
authors.initials: MS
authors.lastname: Thomsen
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D019149:Bioreactors; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013499:Surface Properties; D013696:Temperature; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; C406923:CelB protein, Pyrococcus furiosus; D001617:beta-Glucosidase; D007785:Lactose