Diseño de nuevos sistemas lacasa-mediadorempleo de mediadores de origen natural y mejora de lacasas por evolución dirigida

Abstract

The use of environmentally‐friendly redox mediators easily available at low cost together with the design of tailor‐made laccases, more stable or with improved catalytic efficiencies, could smooth the progress of application of laccase‐mediator systems in decontaminating or white‐biotechnological industrial processes. This thesis comprises both, searching of alternative laccase mediators of natural origin and directed evolution of the high‐redox potential laccase from the fungus Pycnoporus cinnabarinus. The mediating capabilities of naturally‐occurring phenolic compounds, related to the lignin polymer, were demonstrated during oxidation of polycyclic aromatic hydrocarbons (PAH) and dyes, by P. cinnabarinus laccase. Moreover, relevant pieces of evidence on the mechanisms followed by these laccase‐natural mediator systems were also given. p‐Hydroxycinnamic acids posses significantly higher antioxidant activity than benzylic phenols, as showed by the TEAC assay. However, they are also capable to act as efficient laccase redox mediators once oxidized by the enzyme, promoting the oxidation of recalcitrant aromatic compounds. Vanillin, acetovanillone, ferulic acid and p‐coumaric acid significantly promoted oxidative transformation of PAH by laccase. The outstanding efficiency of p‐coumaric acid (PCA) as laccase mediator resulted close similar to that of HBT. Complete removal of anthracene and benzo[a]pyrene and 50 % transformation of pyrene were attained with laccase‐PCA (laccase alone only oxidized anthracene). Quinones were detected as oxidation products of anthracene and benzo[a]pyrene by laccase and natural mediators (and HBT or ABTS). However, 6‐benzo[a]pyrenyl acetate accumulated during transformation of benzo[a]pyrene by laccase‐ABTS but was not detected with HBT or PCA. The absence of this intermediate upholds the dissimilarity of the oxidation mechanisms follow by laccase‐PCA and laccase‐HBT (HAT) vs laccase‐ABTS (ET). The recalcitrance of phenanthrene hinders its oxidation by the enzyme even in the presence of PCA or HBT. However, in the presence of unsaturated fatty acids phenanthrene was efficiently transformed by laccase‐PCA (and laccase‐HBT). The involvement of lipid peroxidation reactions, forming peroxyl radicals that acted as strong oxidizers, was confirmed by the TBARS assay. Among the three p‐hydroxycinnamic acids, sinapic acid (SA) was the best substrate of P. cinnabarinus laccase (specificity constant 400‐fold PCA) and caused the fastest dye decolorization. The strong mediating capacity of SA might be explained by its fast oxidation by the enzyme, producing high concentration of phenoxyl radicals. Their high tendency for β‐βʹ coupling generates phenolic dimers that would act as laccase mediators, as demonstrated here with syringaresinol. The design of enzymes by directed evolution consists of generating genetic diversity by induced mutagenesis or recombination of the gene(s) under study, expression of this diversity and selection of the best mutants at the conditions the enzyme is wanted to be improved. Successive evolution rounds will provide the accumulation of beneficial mutations and the desired feature. Directed evolution of P. cinnabarinus laccase (PcL) requires its expression in S. cerevisiae, the heterologous host used in the evolution assays. Unfortunately, expression of basidiomycete laccases in S. cerevisiae is a hard task. In order to achieve the functional expression of the enzyme, the signal sequence of the protein was replaced by the sequence of the S. cerevisiae α factor pre‐proleader (α‐PcL). Optimization of the expression conditions was also accomplished by adding ethanol and mM concentrations of CuSO4 to the medium, and lowering the incubation temperature. The complete α‐PcL sequence was submitted to the evolution process in order to improve both laccase expression in yeast and catalytic activity. The use of two polymerases with different mutagenic biases enhanced the genetic variability generated by random mutagenesis. The in vivo recombination of the resulting DNA enhanced this diversity, and promoted the accumulation of beneficial mutations incorporated in successive evolution rounds. High‐throughput screening of the libraries was based in two different colorimetric assays with ABTS and 2,6‐dimethoxyphenol. After three evolution rounds, laccase activity in S. cerevisiae microcultures was enhanced 800 fold. The selected mutant (7A9) possessed seven beneficial mutations. Four of them are located in the α factor pre‐proleader and would be responsible for increasing protein secretion rates. The other three are located in the mature protein; two of them in the CuT1 environment would be related to the improvement of enzymatic features. The evolved PcL variant (7A9) presented 6‐fold improvement of catalytic efficiency for ABTS oxidation, was rather more thermostable and more active at neutral pH than wild PcL.