title: Physisorption of enzymatically active chymotrypsin on titania colloidal particles.
abstract: In this study we use a straightforward experimental method to probe the presence and activity of the proteolytic enzyme α-chymotrypsin adsorbed on titania colloidal particles. We show that the adsorption of α-chymotrypsin on the particles is irreversible and pH-dependent. At pH 8 the amount of adsorbed chymotrypsin is threefold higher compared to the adsorption at pH 5. However, we observe that the adsorption is accompanied by a substantial loss of enzymatic activity, and only around 6-9% of the initial enzyme activity is retained. A Michaelis-Menten kinetics analysis of both unbound and TiO2-bound chymotrypsin shows that the K(M) value is increased from ∼10 μM for free chymotrypsin to ∼40 μM for the particle bound enzyme. Such activity decrease could be related by the hindered accessibility of substrate to the active site of adsorbed chymotrypsin, or by adsorption-induced structural changes. Our simple experimental method does not require any complex technical equipment, can be applied to a broad range of hydrolytic enzymes and to various types of colloidal materials. Our approach allows an easy, fast and reliable determination of particle surface-bound enzyme activity and has high potential for development of future enzyme-based biotechnological and industrial processes.
authors.affiliation: Advanced Ceramics, Faculty of Production Engineering, University of Bremen, Germany.
authors.forename: Ludmilla
authors.initials: L
authors.lastname: Derr
authors.affiliation: Center for Environmental Research and Sustainable Technology, University of Bremen, Germany; Center for Biomolecular Interactions Bremen, Faculty 2 (Biology/Chemistry), University of Bremen, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Dringen
authors.affiliation: Advanced Ceramics, Faculty of Production Engineering, University of Bremen, Germany. Electronic address: treccani@uni-bremen.de.
authors.forename: Laura
authors.initials: L
authors.lastname: Treccani
authors.affiliation: Hybrid Materials Interfaces Group, Faculty of Production Engineering and Bremen Center for Computational Materials Science, University of Bremen, Germany.
authors.forename: Nils
authors.initials: N
authors.lastname: Hildebrand
authors.affiliation: Center for Environmental Research and Sustainable Technology, University of Bremen, Germany; Hybrid Materials Interfaces Group, Faculty of Production Engineering and Bremen Center for Computational Materials Science, University of Bremen, Germany; MAPEX Center for Materials and Processes, University of Bremen, Germany.
authors.forename: Lucio Colombi
authors.initials: LC
authors.lastname: Ciacchi
authors.affiliation: Advanced Ceramics, Faculty of Production Engineering, University of Bremen, Germany; MAPEX Center for Materials and Processes, University of Bremen, Germany.
authors.forename: Kurosch
authors.initials: K
authors.lastname: Rezwan
mesh_terms: D000327:Adsorption; D020134:Catalytic Domain; D002918:Chymotrypsin / Q000737:chemistry*; D003102:Colloids; D057075:Enzyme Assays; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D009596:Nitrophenols / Q000737:chemistry; D014025:Titanium / Q000737:chemistry*
chemical_list: D003102:Colloids; D004800:Enzymes, Immobilized; D009596:Nitrophenols; C009495:titanium dioxide; C008642:4-nitrophenyl acetate; D014025:Titanium; D002918:Chymotrypsin; C117608:alpha-chymotrypsin
keywords: Chymotrypsin; Colloidal titania particles; Enzymatic activity; Enzyme adsorption; Physisorption; TiO(2)