Laccase Engineering

Limited protein engineering on laccase, by site-directed mutagenesis as well as directed evolution, has been previously made to probe the enzyme's structure-

function relationship [23-27, 151, 204]. Since this initial work, our knowledge on the atomic structure (see Section 2.1.1) and natural diversity (see Section 2.3.1.1) of laccase has been significantly expanded, which should enable more effective rational design and gene shuffling-based protein engineering of laccase. For instance, a recent site-directed engineering aimed at an aspartate important for the substrate binding/activation has led to significant shiftings of the optimal pH of a Trametes laccase [231].

With regard to its reactivity, laccase's T1 E° is probably the most important factor. It is known that both the high- and low-E° laccases can have the same amino acid residues to ligate the T1 Cu. Mutating the axial ligand has not been able to convert a low-E° laccase into a high-E° laccase [23, 25-27]. A random mutagenesis or gene shuffling approach may be needed to alter the global conformation of the protein backbone to perturb the T1 Cu ligation geometry enough to significantly raise the E°.

As discussed in Section 2.3.1.1, some bacterial or archaeon laccases have very high alkaline pH or thermal stability. Understanding the structural basis of the property may guide us to render fungal laccases, which generally are more active at ambient temperature or neutral pH than bacterial ones, more alkaline or thermal activity/stability desirable for many applications. Further studying the lac-cases from marine microorganisms or extremophiles may help us to enhance the salinity tolerance or other robustnesses of the biocatalyst [223]. Directed evolution may be used to engineer laccases active in organic solvents [24, 224].

Target-specific/binding molecules may be fused with or linked to laccase to create "smart" biocatalysts. For example, llama heavy-chain antibody may enhance the specificity of laccase towards a target that is immunogenic to the antibody [224a]. Carbohydrate-binding motifs may bring laccase to close vicinity of cellulose-adsorbed targets for better action [224b]. Peroxisome-targeting signal may insert laccase to a fatty particulate for better oxidation [225]. Artificial binding peptides, constructed by either organic synthesis (such as combinatorial chemistry) or genetic engineering (such as phage display), may also enhance laccase's specificity, such as that towards carotenoid [73].

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