Fishing of beta-amylase with a SAM of alpha-cyclodextrin-poly- (ethylene glycol) conjugate.
Alpha-cyclodextrin (alpha-CD) with an amino group was conjugated to an alpha, omega-dicarboxylated poly(ethylene glycol) (PEG). The inhibition constant (Ki) of the alpha-CD-PEG conjugate for the catalysis by beta-amylase was larger than that of alpha-CD, due to a steric obstruction of the PEG moiety to the binding of alpha-CD moiety to beta-amylase. alpha-CD-PEG was further modified with cystamine (CD-PEG-Cys) or cysteamine methyl disulfide (CD-PEG-MDS), and the disulfide-carrying alpha-CD-PEG was accumulated on a gold surface as a self-assembled monolayer (SAM). The binding of beta-amylase to the alpha-CD-PEG SAM was followed by a decrease in cathodic peak current in the voltammogram of hydroquinone as a probe using a cyclic voltammetry (CV). The beta-amylase bound to the alpha-CD-PEG SAM was desorbed by the addition of free alpha-CD, and the ratio of desorbed beta-amylase from the SAM of alpha-CD-PEG-Cys to the total amount of the enzyme bound to the SAM was 40% whereas that from the alpha-CD-PEG-MDS SAM was 83-85%. The percentage of desorption was increased to 100% by the treatment of the alpha-CD-PEG-MDS SAM-carrying electrode with 2-hydroxyethyldisulfide prior to the immersion in the enzyme solution. Adsorption and desorption processes of beta-amylase to the surface of alpha-CD-PEG-MDS SAM were clearly observed using localized surface plasmon resonance absorption spectroscopy. The binding constant of the enzyme to the surface-confined alpha-CD-PEG was much larger than that to free alpha-CD, probably due to a large local concentration of the alpha-CD moiety on the gold surface.
Kitano
Hiromi
H
Department of Chemical and Biochemical Engineering, Toyama University, Toyama 930-8555, Japan. kitano@eng.toyama-u.ac.jp
Miyamoto
Takashi
T
Kawasaki
Hideaki
H
D002384:Catalysis; D004563:Electrochemistry; D004566:Electrodes; D005898:Glass; D006046:Gold / Q000737:chemistry; D015394:Molecular Structure; D049329:Nanostructures / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D020349:Surface Plasmon Resonance; D013499:Surface Properties; D047391:alpha-Cyclodextrins / Q000737:chemistry*; D001614:beta-Amylase / Q000737:chemistry*
D047391:alpha-Cyclodextrins; D011092:Polyethylene Glycols; D006046:Gold; D001614:beta-Amylase; C032613:alpha-cyclodextrin