Modification of galactitol dehydrogenase from Rhodobacter sphaeroides D for immobilization on polycrystalline gold surfaces.
Galactitol dehydrogenase (GatDH) from Rhodobacter sphaeroides is a multifunctional enzyme that catalyzes in the presence of oxidized beta-nicotinamide adenine dinucleotide (NAD(+)) the interconversion of various multivalent aliphatic alcohols to the corresponding ketones. The recombinant GatDH was provided with an N-terminal His(6)-tag to which distally up to three cysteine residues were attached. This protein construct maintained nearly full enzymatic activity, and it could be covalently immobilized via thiol bonds onto the surface of a gold electrode. Binding of GatDH onto the gold electrode was verified by SPR measurements, and residual enzyme activity was measured by cyclic voltammetry using 1,2-hexanediol as substrate, the cofactor NAD(+) and the redox mediator CTFM (4-carboxy-2,5,7-trinitrofluorenyliden-malonnitrile) in solute form. The results demonstrate the possibility of a directed functional immobilization of proteins on gold surfaces, which represents a proof-of-concept for the development of reactors for electrochemical synthon preparation using dehydrogenases.
Applied Microbiology, Campus A1.5., Saarland University, D-66123 Saarbruecken, Germany.
P
P
Kornberger
J
J
Gajdzik
H
H
Natter
G
G
Wenz
F
F
Giffhorn
G W
GW
Kohring
R
R
Hempelmann
D002122:Calcium Chloride / Q000378:metabolism; D020134:Catalytic Domain; D003545:Cysteine / Q000378:metabolism; D004563:Electrochemistry; D004566:Electrodes; D004579:Electron Transport; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006046:Gold / Q000737:chemistry*; D009154:Mutation; D009243:NAD / Q000378:metabolism; D011108:Polymers / Q000737:chemistry*; D015202:Protein Engineering; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013499:Surface Properties
D004800:Enzymes, Immobilized; D011108:Polymers; D013438:Sulfhydryl Compounds; D009243:NAD; D006046:Gold; D013401:Sugar Alcohol Dehydrogenases; C095946:galactitol 2-dehydrogenase; D003545:Cysteine; D002122:Calcium Chloride