Optimizing the Mass-Specific Activity of Bilirubin Oxidase Adlayers through Combined Electrochemical Quartz Crystal Microbalance and Dual Polarization Interferometry Analyses.
Two surface analysis techniques, dual polarization interferometry (DPI) and analysis by an electrochemical quartz crystal microbalance with dissipation capability (E-QCM-D), were paired to find the deposition conditions that give the highest and most stable electrocatalytic activity per adsorbed mass of enzyme. Layers were formed by adsorption from buffered solutions of bilirubin oxidase from Myrothecium verrucaria at pH 6.0 to planar surfaces, under high enzyme loading (≥1 mg mL(-1)) for contact periods of up to 2 min. Both unmodified and carboxylate-functionalized gold-coated sensors showed that a deposition solution concentration of 10-25 mg mL(-1) gave the highest activity per mass of adsorbed enzyme with an effective catalytic rate constant (k(cat)) of about 60 s(-1). The densification of adsorbed layers observed by DPI correlated with reduced bioactivity observed by parallel E-QCM-D measurements. Postadsorption changes in thickness and density observed by DPI were incorporated into Kelvin-Voigt models of the QCM-D response. The modeled response matched experimental observations when the adlayer viscosity tripled after adsorption.
School of Materials and Manchester Institute of Biotechnology, University of Manchaster , 131 Princess Street, Manchester M1 7DN, United Kingdom.
Trevor
T
McArdle
School of Materials and Manchester Institute of Biotechnology, University of Manchaster , 131 Princess Street, Manchester M1 7DN, United Kingdom.
Thomas P
TP
McNamara
School of Materials and Manchester Institute of Biotechnology, University of Manchaster , 131 Princess Street, Manchester M1 7DN, United Kingdom.
Fan
F
Fei
School of Materials and Manchester Institute of Biotechnology, University of Manchaster , 131 Princess Street, Manchester M1 7DN, United Kingdom.|Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford , South Parks Road, Oxford OX1 3QR, United Kingdom.
Kulveer
K
Singh
School of Materials and Manchester Institute of Biotechnology, University of Manchaster , 131 Princess Street, Manchester M1 7DN, United Kingdom.
Christopher F
CF
Blanford
D015097:3-Mercaptopropionic Acid / Q000737:chemistry; D000327:Adsorption; D002384:Catalysis; D004560:Electricity; D004563:Electrochemistry / Q000379:methods*; D005658:Fungi / Q000201:enzymology; D007368:Interferometry / Q000379:methods*; D008958:Models, Molecular; D008970:Molecular Weight; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000378:metabolism*; D010100:Oxygen / Q000737:chemistry; D058525:Quartz Crystal Microbalance Techniques / Q000379:methods*
D015097:3-Mercaptopropionic Acid; D044925:Oxidoreductases Acting on CH-CH Group Donors; C042401:bilirubin oxidase; D010100:Oxygen
enzymatic biofuel cell; industrial biotechnology; laccase; multicopper oxidase; protein immobilization; protein−surface interactions