Directed Evolution of Fungal Laccases

Fungal laccases are generalists biocatalysts with potential applications that range from bioremediation to novel green processes. Fuelled by molecular oxygen, these enzymes can act on dozens of molecules of different chemical nature, and with the help of redox mediators, their spectrum of oxidizable substrates is further pushed towards xenobiotic compounds (pesticides, industrial dyes, PAHs), biopolymers (lignin, starch, cellulose) and other complex molecules. In recent years, extraordinary efforts have been made to engineer fungal laccases by directed evolution and semi-rational approaches to improve their functional expression or stability. All these studies have taken advantage of Saccharomyces cerevisiae as a heterologous host, not only to secrete the enzyme but also, to emulate the introduction of genetic diversity through in vivo DNA recombination. Here, we discuss all these endeavours to convert fungal laccases into valuable biomolecular platforms on which new functions can be tailored by directed evolution

Re-designing the substrate binding pocket of laccase for enhanced oxidation of sinapic acid

Iterative saturation mutagenesis was performed over six residues delimiting the substrate binding pocket of a high-redox potential chimeric laccase with the aim of enhancing its activity over sinapic acid, a lignin-related phenol of industrial interest. In total, more than 15000 clones were screened and two selected variants, together with the parent-type laccase, were purified and characterized. The new variants presented shifted pH activity profiles and enhanced turnover rates on sinapic acid and its methyl ester, whereas the oxidation of related phenols was not significantly enhanced. Neither the enzyme's redox potential nor the mechanism of the reaction were affected. Thus, quantum mechanics and molecular dynamics calculations were done to rationalize the effect of the selected mutations, revealing the critical role of the residues of the enzyme pocket to provide the precise binding of the substrate that enables an efficient electron transfer to the T1 copper. The results presented highlight the power of combining directed evolution and computational approaches to give novel solutions in enzyme engineering and to understand the mechanistic reasons behind them, offering new insights for further rational design towards specific targets

Engineering and Applications of fungal laccases for organic synthesis

Laccases are multi-copper containing oxidases (EC 1.10.3.2), widely distributed in fungi, higher plants and bacteria. Laccase catalyses the oxidation of phenols, polyphenols and anilines by one-electron abstraction, with the concomitant reduction of oxygen to water in a four-electron transfer process. In the presence of small redox mediators, laccase offers a broader repertory of oxidations including non-phenolic substrates. Hence, fungal laccases are considered as ideal green catalysts of great biotechnological impact due to their few requirements (they only require air, and they produce water as the only by-product) and their broad substrate specificity, including direct bioelectrocatalysis

Identification and characterization of laccase-type multicopper oxidases involved in dye-decolorization by the fungus Leptosphaerulina sp.

13 p.-4 fig.-4 tab.[Background] Fungal laccases are multicopper oxidases (MCOs) with high biotechnological potential due to their capability to oxidize a wide range of aromatic contaminants using oxygen from the air. Albeit the numerous laccase-like genes described in ascomycete fungi, ascomycete laccases have been less thoroughly studied than white-rot basidiomycetous laccases. A variety of MCO genes has recently been discovered in plant pathogenic ascomycete fungi, however little is known about the presence and function of laccases in these fungi or their potential use as biocatalysts. We aim here to identify the laccase-type oxidoreductases that might be involved in the decolorization of dyes by Leptosphaerulina sp. and to characterize them as potential biotechnological tools.[Results] A Leptosphaerulina fungal strain, isolated from lignocellulosic material in Colombia, produces laccase as the main ligninolytic oxidoreductase activity during decolorization of synthetic organic dyes. Four laccase-type MCO genes were partially amplified from the genomic DNA using degenerate primers based on laccase-specific signature sequences. The phylogenetic analysis showed the clustering of Lac1, Lac4 and Lac3 with ascomycete laccases, whereas Lac2 grouped with fungal ferroxidases (together with other hypothetical laccases). Lac3, the main laccase produced by Leptosphaerulina sp. in dye decolorizing and laccase-induced cultures (according to the shotgun analysis of both secretomes) was purified and characterized in this study. It is a sensu-stricto laccase able to decolorize synthetic organic dyes with high efficiency particularly in the presence of natural mediator compounds.[Conclusions] The searching for laccase-type MCOs in ascomycetous families where their presence is poorly known, might provide a source of biocatalysts with potential biotechnological interest and shed light on their role in the fungus. The information provided by the use of genomic and proteomic tools must be combined with the biochemical evaluation of the enzyme to prove its catalytic activity and applicability potential.This research was supported by the Program for Interuniversity Cooperation and Scientific Reasearch (PCI) from the Spanish Agency for International Cooperation and Development (AECID), Project AP/033932/11, and the Spanish Project NOESIS BIO2014-56388-R.Peer reviewe

Optimization of a laccase-mediator stage for TCF bleaching of flax pulp

7 p.-3 fig.-3 tab.Flax pulp obtained by anthraquinone-soda pulping, resulting in a kappa number of 11.1, a viscosity of 950 ml/g and 36.7% ISO brightness, was bleached in a totally chlorine-free sequence using the enzyme laccase from the fungus Pycnoporus cinnabarinus and 1-hydroxybenzotriazole (HBT) as redox mediator (stage L), followed by a hydrogen peroxide stage (P). The laccase treatment was optimized using a three-variable sequential statistical plan over the following ranges: 1–20 U/g o.d.p. (oven-dried pulp) laccase dose, 0.5–7.5% o.d.p. HBT dose and 1–24 h reaction time. The influence of these variables on several pulp properties after the P stage of the LP sequence was examined.The models defined from the results obtained predicted variations in ISO brightness, viscosity and kappa number of 57.6–74.8%, 590–955 ml/g and 0–6.2, respectively. The variables most strongly influencing these pulp properties were found to be the reaction time and the enzyme dose. A compromise was adopted as regards the operating conditions in order to ensure optimum results. The study was completed by conducting a biobleaching assay in a pressurized reactor (590 kPa) to assess the effect of oxygen pressure. The high pressure level resulted in improved pulp properties by the laccase-mediator system.This work was supported by Spanish CICyT (Project PPQ2000–1068-C02-02, and FEDER Project 2FD97-0896-C02) and Comunidad Autónoma de Madrid, the EU Commission (QLK3-99-590) and the Spanish grant 2002FI 00556.Peer reviewe

Paper pulp delignification using laccase and natural mediators

aCentro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain; bCentro de Investigación y Tecnología, ENCE, Carretera de Campañó, Ribeiro Vao, E-36157 Pontevedra, Spain; cInstituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, PO Box 1052,E-41080, Seville, Spain; E-mail address: [email protected] (A.T. Martínez)Three plant phenols, namely acetosyringone syringaldehyde and p-coumaric acid, were selected as laccase redox mediators to investigate the enzymatic delignification of paper pulp (obtained from kraft cooking of eucalypt wood) in combination with peroxide bleaching. The effects of these natural mediators were compared with those obtained using the synthetic mediator 1-hydroxybenzotriazole. p-Coumaric acid only caused minor increase of pulp brightness and did not lowered its kappa number (a rough estimation of the lignin content), whereas the use of acetosyringone or syringaldehyde as laccase mediators enabled over 15 % increase of final brightness and a similar decrease of final kappa number. Pulp delignification by laccase in the presence of the two latter natural mediators was demonstrated by analytical pyrolysis, which does not suffer from interferences by other pulp constituents as kappa number does, showing a preferential removal of lignin marker compounds compared with carbohydrate markers (up to 25% decrease of the corresponding ratio). This technique also revealed a modification of the residual lignin composition in terms of phenylpropane units after the laccase-mediator treatment. The use of laccase in combination with natural mediators, widely available from plant materials and pulping liquors, represents a promising alternative for environmentally-friendly delignification of paper pulp.This study has been funded by two ENCE-CSIC contracts, the Spanish projects AGL2005-01748, CTQ2005-8925-CO2-02/PPQ and BIO2005-2224, and the CSIC project 2006-4-0I-039. Marcel Asther from INRA (Marseille, France) is acknowledged for the P. cinnabarinus strain, and Beldem (Andenne, Belgium) is acknowledged for laccase production. S.C. thanks a R&C contract of the Spanish MEC, and D.I. thanks a I3P Fellowship of the Spanish CSIC.Peer reviewe

Computer-aided laccase engineering: toward biological oxidation of arylamines

Oxidation of arylamines, such as aniline, is of high industrial interest and laccases have been proposed as biocata-lysts to replace harsh chemical oxidants. However, the reaction is hampered by the redox potential of the substrate at acid pH and enzyme engineering is required to improve the oxidation. In this work, instead of trying to improve the redox potential of the en-zyme, we aim towards the (transient) substrate’s one and propose this as a more reliable strategy. We have successfully combined a computational approach with experimental validation to rationally design an improved biocatalyst. The in silico protocol combines classical and quantum mechanics to deliver atomic and electronic level detail on the two main processes involved: substrate binding and electron transfer. After mutant expression and comparison to the parent type, kinetic results show that the protocol accurately predicts aniline’s improved oxidation (2-fold kcat increase) in the engineered variant for biocatalyzed polyaniline production.This study was supported by the INDOX (KBBE-2013-7-613549) EU-project, and the NOESIS (BI0201456388-R) and OxiDesign (CTQ2013-48287-R) Spanish project. GS thanks an FPI grant of the Spanish Ministry of Competitiveness.Peer ReviewedPostprint (author's final draft

Advanced synthesis of conductive polyaniline using laccase as biocatalyst

18 p.-7 fig.-1 tab.Polyaniline is a conductive polymer with distinctive optical and electrical properties. Its enzymatic synthesis is an environmentally friendly alternative to the use of harsh oxidants and extremely acidic conditions. 7D5L, a high-redox potential laccase developed in our lab, is the biocatalyst of choice for the synthesis of green polyaniline (emeraldine salt) due to its superior ability to oxidize aniline and kinetic stability at the required polymerization conditions (pH 3 and presence of anionic surfactants) as compared with other fungal laccases.Doses as low as 7.6 nM of 7D5L catalyze the polymerization of 15 mM aniline (in 24 h, room temperature, 7% yield) in the presence of different anionic surfactants used as doping templates to provide linear and water-soluble polymers. Aniline polymerization was monitored by the increase of the polaron absorption band at 800 nm (typical for emeraldine salt). Best polymerization results were obtained with 5 mM sodium dodecylbenzenesulfonate (SDBS) as template. At fixed conditions (15 mM aniline and 5mM SDBS), polymerization rates obtained with 7D5L were 2.5-fold the rates obtained with commercial Trametes villosa laccase. Moreover, polyaniline yield was notably boosted to 75% by rising 7D5L amount to 0.15 μM, obtaining 1g of green polyaniline in 1L-reaction volume. The green polymer obtained with the selected system (7D5L/SDBS) holds excellent electrochemical and electro-conductive properties displayed in water-dispersible nanofibers,which is advantageous for the nanomaterial to be readily cast into uniform films for different applications.This work was funded by INDOX (KBBE-2013-7-613549) European project and NOESIS (BIO2014-56388-R) Spanish national project.Peer reviewe

Biodegradation of lignocellulosics: microbial, chemical, and enzymatic aspects of the fungal attack of lignin

Wood is the main renewable material on Earth and is largely used as building material and in paper-pulp manufacturing. This review describes the composition of lignocellulosic materials, the different processes by which fungi are able to alter wood, including decay patterns caused by white, brown, and soft-rot fungi, and fungal staining of wood. The chemical, enzymatic, and molecular aspects of the fungal attack of lignin, which represents the key step in wood decay, are also discussed. Modern analytical techniques to investigate fungal degradation and modification of the lignin polymer are reviewed, as are the different oxidative enzymes (oxidoreductases) involved in lignin degradation. These include laccases, high redox potential ligninolytic peroxidases (lignin peroxidase, manganese peroxidase, and versatile peroxidase), and oxidases. Special emphasis is given to the reactions catalyzed, their synergistic action on lignin, and the structural bases for their unique catalytic properties. Broadening our knowledge of lignocellulose biodegradation processes should contribute to better control of wood-decaying fungi, as well as to the development of new biocatalysts of industrial interest based on these organisms and their enzymes. [Int Microbiol 2005; 8(3):195-204

Depolymerisation of Kraft Lignin by Tailor-Made Alkaliphilic Fungal Laccases

Lignins released in the black liquors of kraft pulp mills are an underutilised source of aromatics. Due to their phenol oxidase activity, laccases from ligninolytic fungi are suitable biocatalysts to depolymerise kraft lignins, which are characterised by their elevated phenolic content. However, the alkaline conditions necessary to solubilise kraft lignins make it difficult to use fungal laccases whose activity is inherently acidic. We recently developed through enzyme-directed evolution high-redox potential laccases active and stable at pH 10. Here, the ability of these tailor-made alkaliphilic fungal laccases to oxidise, demethylate, and depolymerise eucalyptus kraft lignin at pH 10 is evidenced by the increment in the content of phenolic hydroxyl and carbonyl groups, the methanol released, and the appearance of lower molecular weight moieties after laccase treatment. Nonetheless, in a second assay carried out with higher enzyme and lignin concentrations, these changes were accompanied by a strong increase in the molecular weight and content of β–O–4 and β–5 linkages of the main lignin fraction, indicating that repolymerisation of the oxidised products prevails in one-pot reactions. To prevent it, we finally conducted the enzymatic reaction in a bench-scale reactor coupled to a membrane separation system and were able to prove the depolymerisation of kraft lignin by high-redox alkaliphilic laccase