Characterization and Catalytic Property of Surfactant‐Laccase Complex in Organic Media

Abstract

The oxidation of o-phenylenediamine catalyzed in anhydrous organic solvents by surfactant-laccase complex was investigated. The complex was prepared by 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 aqueous buffer solution in which its activity was optimized, exhibited no catalytic activity in nonaqueous media. To optimize the preparation and reaction conditions for the surfactant-enzyme complexes, we examined the effects of pH in the water pool of W/O emulsions, the concentration of enzyme and surfactant at the preparation stage, and the nature of organic solvents at the reaction stage on the laccase activity in organic media. Surfactant-laccase complex showed a strong pH-dependent catalytic activity in organic media. Its optimum activity was obtained when the complex was prepared at a pH of about 3. Interestingly, native laccase in an aqueous buffer solution exhibited an optimum activity at the same pH of 3. The optimum preparation conditions of surfactant-laccase complex were [laccase] = 0.8 mg/mL and [surfactant] = 10 mM, and the complex showed the highest catalytic activity in toluene among nine anhydrous organic solvents. The effect of a cosolubilized mediator (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 that without HBT.

Enzymatic catalysis of 2,6-dimethoxyphenol by laccases and products characterization in organic solutions

“…Due to the complete symmetry of carbon atoms at positions 3 and 5, their chemical shifts should be at 147.2 ppm, and the peaks at 133.5 ppm and 134.5 ppm belong to carbon atoms at positions 1 and 4. The calculation indicated that the deshielding effect of carbon (1) is larger than carbon (4) and should be at the lower magnetic field which is proven by the COLOC analysis ( Figure 5). The methoxyl groups or hydrogen atoms clearly interact with the carbon atoms at positions 3 and 5 of the benzene ring through 3 J coupling.…”

“…Simply based on the function of catalyzing the oxidation of substituted phenols and/or anilines-type substrates through four-electron reduction of O 2 to H 2 O [3][4][5][6][7] , these enzymes constitute a very prospective class of enzymes for physiological development and industrial utility, such as bioremediation, organic catalyzed synthesis, pulp and paper biobleaching, and analytic uses [4,[8][9][10] .…”

Micellar Enzymology- Chemistry and Applications

“…Very low concentration of surfactant < 150 m. M increases the water pool of reverse micelles which reduces an overall affinity of enzyme towards its substrate by increasing the distance between them [18]. On the other hand, a very high concentration leads to electrostatic repulsion and leads to denaturation of the protein.…”

“…Activity of enzymes/proteins at higher pH values in the AOT-enclosed water droplets could be due to the presence of the negatively charged surfactant, which leads to attraction of protons from the interior of the droplet and shift optimum pH values in reversed micelles [9,18].…”

“…The Km value is influenced by the distribution coefficient of the substrates and the volume ratio of the phases. Different types of multi-phasic theoretical models and experimental approaches are considered for the interpretation of results for reverse micelles [18,20,28,31,32]. The first order rate constant kcat [Sec] -1 is reflecting a true reactivity of enzyme in the reverse micelles because this kinetic parameter is not complicated by the distribution effects of the substances and may be regarded as an objective parameter.…”