title: Molecular orientation of enzymes attached to surfaces through defined chemical linkages at the solid-liquid interface.
abstract: The immobilization of enzymes on solid supports is widely used in many applications, including biosensors, antifouling coatings, food packaging materials, and biofuel cells. Enzymes tend to lose their activity when in contact with a support surface, a phenomenon that has been attributed to unfavorable orientation and (partial) unfolding. In this work, specific immobilization of 6-phospho-β-galactosidase (β-Gal) on a self-assembled monolayer (SAM) containing maleimide end groups and oligo(ethylene glycol) spacer segments was achieved through a unique cysteinyl residue. A systematic means to characterize the interfacial orientation of immobilized enzymes has been developed using a combination of sum frequency generation vibrational spectroscopy and attenuated total reflectance FTIR-spectroscopy. The possible orientations of the immobilized β-Gal were determined and found to be well-correlated with the tested activity of β-Gal. This study will impact the development of an increasingly wide range of devices that use surface-immobilized enzymes as integral components with improved functions, better sensitivity, enhanced stability, and longer shelf life.
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Yuwei
authors.initials: Y
authors.lastname: Liu
authors.forename: Tadeusz L
authors.initials: TL
authors.lastname: Ogorzalek
authors.forename: Pei
authors.initials: P
authors.lastname: Yang
authors.forename: McKenna M
authors.initials: MM
authors.lastname: Schroeder
authors.forename: E Neil G
authors.initials: EN
authors.lastname: Marsh
authors.forename: Zhan
authors.initials: Z
authors.lastname: Chen
mesh_terms: D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D019855:Ethylene Glycol / Q000737:chemistry* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013294:Lactococcus lactis / Q000201:enzymology; D008301:Maleimides / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D013499:Surface Properties
chemical_list: D004800:Enzymes, Immobilized; D008301:Maleimides; C043592:maleimide; D006026:Glycoside Hydrolases; C022361:6-phospho-beta-galactosidase; D019855:Ethylene Glycol; D003545:Cysteine