The oxidation of o-phenylenediamine catalyzed in anhydrous organic solvents
by surfactant-laccase complex was investigated. The complex was prepared b
y utilizing a novel preparation technique in water-in-oil (W/O) emulsions.
The surfactant-laccase complex effectively catalyzed the oxidation reaction
in various dry organic solvents, while laccase, lyophilized from an aqueou
s buffer solution in which its activity was optimized, exhibited no catalyt
ic activity in nonaqueous media. To optimize the preparation and reaction c
onditions for the surfactant-enzyme complexes, we examined the effects of p
H in the water pool of W/O-emulsions, the concentration of enzyme and surfa
ctant at the preparation stage, and the nature of organic solvents at the r
eaction stage on the laccase activity in organic media. Surfactant-laccase
complex showed a strong pH-dependent catalytic activity in organic media. I
ts optimum activity was obtained when the complex was prepared at a pH of a
bout 3. Interestingly, native laccase in an aqueous buffer solution exhibit
ed an optimum activity at the same pH of 3. The optimum preparation conditi
ons of surfactant-laccase complex were [laccase] = 0.8 mg/mL and [surfactan
t] = 10 mM, and the complex showed the highest catalytic activity in toluen
e among nine anhydrous organic solvents. The effect of a cosolubilized medi
ator (1-hydroxybenzotriazole (HBT)) on the reaction was also investigated.
The addition of HBT at the preparation stage of the enzyme complex did not
accelerate the catalytic reaction because HBT was converted to an inactive
benzotriazole (BT) by laccase. However, the addition of HBT at the reaction
stage enhanced the catalytic performance by a factor of five compared to t
hat without HBT.