Electrical contacting of flavoenzymes and NAD(P)+-dependent enzymes by reconstitution and affinity interactions on phenylboronic acid monolayers associated with Au-electrodes.
The preparation of integrated, electrically contacted, flavoenzyme and NAD(P)(+)-dependent enzyme-electrodes is described. The reconstitution of apo-glucose oxidase, apo-GOx, on a FAD cofactor linked to a pyrroloquinoline quinone (PQQ) phenylboronic acid monolayer yields an electrically contacted enzyme monolayer (surface coverage 2.1 x 10(-)(12) mol cm(-)(2)) exhibiting a turnover rate of 700 s(-)(1) (at 22 +/- 2 degrees C). The system is characterized by microgravimetric quartz-crystal microbalance analyses, Faradaic impedance spectroscopy, rotating disk electrode experiments, and cyclic voltammetry. The performance of the enzyme-electrode for glucose sensing is described. Similarly, the electrically contacted enzyme-electrodes of NAD(P)(+)-dependent enzymes malate dehydrogenase, MalD, and lactate dehydrogenase, LDH, are prepared by the cross-linking of affinity complexes generated between the enzymes and the NADP(+) and NAD(+) cofactors linked to a pyrroloquinoline quinone phenylboronic acid monolayer, respectively. The MalD enzyme-electrode (surface coverage 1.2 x 10(-)(12) mol cm(-)(2)) exhibits a turnover rate of 190 s(-)(1), whereas the LDH enzyme-electrode (surface coverage 7.0 x 10(-)(12) mol cm(-)(2)) reveals a turnover rate of 2.5 s(-)(1). Chronoamperometric experiments reveal that the NAD(+) cofactor is linked to the PQQ-phenylboronic acid by two different binding modes. The integration of the LDH with the two NAD(+) cofactor configurations yields enzyme assemblies differing by 1 order of magnitude in their bioelectrocatalytic activities.
Zayats
Maya
M
Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Katz
Eugenii
E
Willner
Itamar
I
D000818:Animals; D001234:Aspergillus niger / Q000201:enzymology; D001897:Boronic Acids / Q000737:chemistry*; D002645:Chickens; D017097:Electric Impedance; D004563:Electrochemistry; D004566:Electrodes; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D005949:Glucose Oxidase / Q000737:chemistry*; D006046:Gold / Q000737:chemistry*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry*; D008099:Liver / Q000201:enzymology; D008291:Malate Dehydrogenase / Q000737:chemistry*; D009249:NADP / Q000737:chemistry*; D011817:Rabbits
D001897:Boronic Acids; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D006046:Gold; D007770:L-Lactate Dehydrogenase; D008291:Malate Dehydrogenase; D005949:Glucose Oxidase; C010686:benzeneboronic acid