To become viable industrial catalysts, laccases need to be subjected to various treatments in order to make them robust, recyclable, or heterogeneous. One of the most studied treatments is immobilization, achieved by either chemical linkage or physical adsorption/entrapment in various carriers . Covalent coupling to a solid carrier (for example, celite, alumina, or nylon [7, 61, 120]) may allow multiple uses of the laccase (thus saving in enzyme cost or water usage) or increase its resilience against pH or thermal inactivation, although the immobilization may lead to a significant activity loss of the original enzyme. However, many targeted molecules or their products (particularly dyes) may also adsorb onto the carrier, resulting in either inactivated catalyst, inaccessible substrate, or inseparable product [57, 119]. Encapsulation in hydrated gels, for example Cu-
alginate , may better preserve laccase, although the hindered permeability or unproductive adsorption of a substrate or product may pose problems. Entrapment in micelles or immobilization on solid carriers may allow laccases to be applied in non-aqueous, novel solvents (organic solvents, ionic liquids, or supercritical CO2) or multiphasic systems.
Other kinds of modification have also been studied on laccases. Cross-linking laccases into enzyme crystals could enhance their stability . Wrapping laccases (via covalent surface linkage) with amphiphilic polymers , dendrimers , PEG [124, 125], or a dioleyl glucono glutamate surfactant  could protect them from proteolysis, facilitate their incorporation into micelles, solubilize them in organic solvents (particularly the non-aliphatic, reverse micelle-disrupting ones), or enhance their accessibility toward hydrophobic substrates. Non-ionic, cationic, and anionic polymers (including surfactants) tend to exhibit different effects on laccase catalysis, attributable to their different function in binding product or laccase [127-129, 129a].
Combination of laccase catalysis with other physical or chemical methods, such as electrochemistry and ultrasonics, has been studied [130, 131].
For applications that target large, insoluble, or non-substrate substances, small redox-active mediators have been found very useful in enhancing/extending laccase catalysis. This is exemplified by the laccase-mediator systems for pulp del-ignification (in which the targeted lignin is insoluble, hardly accessible, and mostly inactive towards laccase direct action) and for dyed fabric finishing (in which the targeted dye is adsorbed onto insoluble textile, making it mostly inaccessible and inactive for laccase). Numerous molecules have been studied as potential laccase mediators. Currently promising mediators include some types of phenol/aniline [7, 42, 132, 133], N-hydroxy/oxide/oximes [30, 31, 42, 54, 134136], phenazine/phenoxazine/phenothiazine [31, 42, 46, 134], and redox-active organics or metal complexes [29, 46, 64, 134, 137-139]. Mediated laccase catalysis has potential for the degradation or modification of lignin [39, 42, 47, 49, 51, 63, 84], delignification of pulp or bleaching of recycled paper [54, 88], degradation of xenobiotics and other pollutants [65a, 69, 71], bleaching of dyes [60, 64], and synthesis of fine or bioactive chemicals [108, 134, 141]. Table 2.4 shows some of the types of targets suitable for mediated laccase oxidation.
In general, any reducing substrate of laccase having an E ° close to ~1 V and a relatively stable oxidized state (or insignificant autooxidation) may serve as a potential mediator, capable of being oxidized/reoxidized by laccase and oxidizing target molecules (essentially shuttling oxidation equivalences between laccase and target). All the known mediators seem to be oxidized by laccase via "outer-sphere"-type electron transfer, whose kinetics is dominated by the AE° between the mediator and laccase (see Section 2.1.2). Based on the proposed mechanisms for oxidizing the target, known laccase mediators may be divided into three main groups [35, 142]. The first group, including ABTS, phenols, and phenothiazines (Table 2.3), seems to oxidize a target by electron transfer. For instance, laccase-generated ABTS radical and doubly oxidized form may extract electrons from a phenol and non-phenol target, respectively, with a rate/efficiency proportional to
2.2 Applications of Laccase for Industrial Oxidation Processes | 55 Table 2.4 Potential targets for mediated laccase biooxidation.
Representative mediator Representative target (targeted bond/atom in bold)
N-Hydroxybenzotriazole ^—^ r
Glucose oh oh err
Toluene thiophene o
Cyclohexene Linoleic acid oh
Cyclohexylmethanol r the AE° between the mediator and target [29, 140, 143, 144, 144a] or attributable to the Hammett correlation with regards to the electronic property of the target's substituents . The second group, including N-hydroxybenzotriazole (HBT), N-hydroxyacetanilide, and other N—OH compounds, seems to oxidize a target by H-abstraction. For instance, the laccase-generated N—OH+• cation radical in HBT may deprotonate into an N—O^ radical, which then abstracts an H from a benzylic target to form a benzylic C radical, with an efficiency dependent on the bond dissociation energy (BDE) difference [35, 135, 143-145]. The third group, represented by the radical 2,2,6,6-tetramethylpiperidin-1-yloxyl (TEMPO), seems to employ yet another mechanism (see Section 184.108.40.206). It is the second and third groups of mediators that have significantly extended laccase catalysis to nonsubstrate substances.
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