title: Stable and Simple Immobilization of Proteinase K Inside Glass Tubes and Microfluidic Channels.
abstract: Engyodontium album proteinase K (proK) is widely used for degrading proteinaceous impurities during the isolation of nucleic acids from biological samples, or in proteomics and prion research. Toward applications of proK in flow reactors, a simple method for the stable immobilization of proK inside glass micropipette tubes was developed. The immobilization of the enzyme was achieved by adsorption of a dendronized polymer-enzyme conjugate from aqueous solution. This conjugate was first synthesized from a polycationic dendronized polymer (denpol) and proK and consisted, on average, of 2000 denpol repeating units and 140 proK molecules, which were attached along the denpol chain via stable bis-aryl hydrazone bonds. Although the immobilization of proK inside the tube was based on nonspecific, noncovalent interactions only, the immobilized proK did not leak from the tube and remained active during prolonged storage at 4 °C and during continuous operation at 25 °C and pH = 7.0. The procedure developed was successfully applied for the immobilization of proK on a glass/PDMS (polydimethylsiloxane) microchip, which is a requirement for applications in the field of proK-based protein analysis with such type of microfluidic devices.
authors.affiliation: Polymer Chemistry Group, Department of Materials (D-MATL), ETH Zürich , Vladimir-Prelog-Weg 5, 8093 Zürich, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Küchler
authors.affiliation: Polymer Chemistry Group, Department of Materials (D-MATL), ETH Zürich , Vladimir-Prelog-Weg 5, 8093 Zürich, Switzerland.
authors.forename: Julian N
authors.initials: JN
authors.lastname: Bleich
authors.affiliation: Bioanalytics Group, Department of Biosystems Science and Engineering (D-BSSE), ETH Zürich , Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Sebastian
authors.affiliation: Bioanalytics Group, Department of Biosystems Science and Engineering (D-BSSE), ETH Zürich , Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland.
authors.forename: Petra S
authors.initials: PS
authors.lastname: Dittrich
authors.affiliation: Polymer Chemistry Group, Department of Materials (D-MATL), ETH Zürich , Vladimir-Prelog-Weg 5, 8093 Zürich, Switzerland.
authors.forename: Peter
authors.initials: P
authors.lastname: Walde
mesh_terms: D000327:Adsorption; D019286:Endopeptidase K / Q000378:metabolism*; D004795:Enzyme Stability; D005898:Glass / Q000737:chemistry*; D006868:Hydrolysis; D055767:Immobilized Proteins / Q000378:metabolism*; D007700:Kinetics; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D017711:Nonlinear Dynamics; D059748:Proteolysis; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet
chemical_list: D055767:Immobilized Proteins; D019286:Endopeptidase K
keywords: adsorption; conjugate; dendronized polymer; enzyme; flow reactor; immobilization; microchip; protease