James P Eberhardt , Hugh ONeill , Patrick Coleman, and Jonathan Woodward

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Characterization of Glucose Dehydrogenase Immobili
zed on Acrylic Beads
James P Eberhardt , Hugh ONeill , Patrick
Coleman, and Jonathan Woodward Albion
College, Albion, Michigan 49224 3M
Corporation, Biomaterials Research Center
Bldg. 209-1W-24 3M Center, St. Paul, Minnesota
55144 Chemical Technology Division
Oak Ridge National Laboratory Oak Ridge,
Tennessee 37831
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Introduction
If a fuel cell can be constructed which uses
hydrogen produced by an enzymatic pathway, the
use of carbohydrates as an alternative fuel is
possible. In order to construct an enzymatic fuel
cell, all of the enzymes need to be immobilized.
Immobilization techniques for invertase from
Candida utilis and b-glucosidase from Pyrococcus
furiosus have been shown previously. An
immobilization procedure for glucose
dehydrogenase from Thermosplasma acidophilum is
the focus of this study. Initial results indicate
that immobilization of the active enzyme is
possible on azlactone beads. This study is a
characterization of the immobilized glucose
dehydrogenase.
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Fuel Cell Basics
http//www.fuelcells.org/
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Hydrogen Production from Sucrose
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Coupling Reaction

• It was previously shown that an azlactone bead
  sample provided by the 3M Corporation was
  suitable for enzyme immobilization studies. These
  beads varied in their vinyl-dimethyl azlactone
  content.
• R. Sims, Unpublished

Azlactone Group On Support
Formation of Amide Bond With Ring Opening
Amine On Enzyme
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Immobilization Method
1 U Enzyme 10 mg Azlactone Beads 10
?l 50 mM NADP 25 ?l 10 mg/ml BSA 25
?l 0.5M Glucose 430 ?l 0.1M Sodium
Phosphate 0 .6M Sodium Citrate
0.1 Triton X100 pH 7.5
Stir 2.5h _at_ 25 0 C
Wash 400 ?l 50 mM Sodium Phosphate pH 6.5
400 ?l Quenching Buffer 20 min
Resuspend Store 400 ?l 0.1M Tris pH 8.0
Wash 400 ?l 50 mM Sodium Phosphate pH 6.5
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Immobilization Yields

• n4
• One unit of enzyme is defined as the amount of
  enzyme that is required to produce 1 ?mol NADPH
  per min at 26C. The variation in yield can be
  contributed in part to changing reaction
  conditions such as pH and temperature.

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Values are expressed
as a percent of the activity of the immobilized
enzyme assayed immediately after coupling.
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Immobilization Yields55540 Beads

• Stored at 25C, All Assays Conducted at 45C

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Km 0.20 mM NADP
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Km 7.58 mM Glucose
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Conclusion

• It is possible to immobilize GDH on 55540
  azlactone beads while retaining the native
  enzymes activity and stability.
• The kinetic parameters of the immobilized GDH
  differ slightly from the native enzyme.
• This is a viable method of immobilizing glucose
  dehydrogenase.

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