Aryl-alcohol Oxidase Research Articles

Enzymatic Preparation of 2,5-Furandicarboxylic Acid (FDCA)-A Substitute of Terephthalic Acid-By the Joined Action of Three Fungal Enzymes.

Enzymatic oxidation of 5-hydroxymethylfurfural (HMF) and its oxidized derivatives was studied using three fungal enzymes: wild-type aryl alcohol oxidase (AAO) from three fungal species, wild-type peroxygenase from Agrocybe aegerita (AaeUPO), and recombinant galactose oxidase (GAO). The effect of pH on different reaction steps was evaluated and apparent kinetic data (Michaelis-Menten constants, turnover numbers, specific constants) were calculated for different enzyme-substrate ratios and enzyme combinations. Finally, the target product, 2,5-furandicarboxylic acid (FDCA), was prepared in a multi-enzyme cascade reaction combining three fungal oxidoreductases at micro-scale. Furthermore, an oxidase-like reaction is proposed for heme-containing peroxidases, such as UPO, horseradish peroxidase, or catalase, causing the conversion of 5-formyl-2-furancarboxylic acid into FDCA in the absence of exogenous hydrogen peroxide.

Functional and Transcriptomic Characterization of a Dye-decolorizing Fungus from Taxus Rhizosphere.

We isolated three laccase-producing fungus strains from Taxus rhizosphere. Myrotheium verrucaria strain DJTU-sh7 had the highest laccase activity of 216.2 U/ml, which was increased to above 300 U/ml after optimization. DJTU-sh7 had the best decolorizing effect for three classes of reactive dyes. The DJTU-sh7-containing fungal consortium displayed the robust decolorizing ability. Both color removal efficiency and chemical oxygen demand were increased in the consortium mediated biotransformation. Transcriptome changes of M. verrucaria elicited by azo dye and phenolic were quantified by the high throughput transcriptome sequencing, and the activities of the selected oxidases and reductases were determined. The possible involvement of oxidases and reductases, especially laccase, aryl alcohol oxidase, and ferric reductase in the biotransformation of dye and phenolic compounds was revealed at both transcriptomic and phenotypic levels. Revealing the transcriptomic mechanisms of fungi in dealing with organic pollutants facilitates the fine-tuned manipulation of strains in developing novel bioremediation and biodegradation strategies.

A role for small secreted proteins (SSPs) in a saprophytic fungal lifestyle: Ligninolytic enzyme regulation in Pleurotus ostreatus

Small secreted proteins (SSPs), along with lignocellulose degrading enzymes, are integral components of the secretome of Pleurotus ostreatus, a white rot fungus. In this study, we identified 3 genes (ssp1, 2 and 3) encoding proteins that are annotated as SSPs and that exhibited of ~4,500- fold expression, 24 hr following exposure to the toxic compound 5-hydroxymethylfurfural (HMF). Homologues to genes encoding these SSPs are present in the genomes of other basidiomycete fungi, however the role of SSPs is not yet understood. SSPs, aryl-alcohol oxidases (AAO) and the intracellular aryl-alcohol dehydrogenases (AAD) were also produced after exposure to other aryl-alcohols, known substrates and inducers of AAOs, and during idiophase (after the onset of secondary metabolism). A knockdown strain of ssp1 exhibited reduced production of AAO-and AAD-encoding genes after HMF exposure. Conversely, a strain overexpressing ssp1 exhibited elevated expression of genes encoding AAOs and ADD, resulting in a 3-fold increase in enzymatic activity of AAOs, as well as increased expression and protein abundance of versatile peroxidase 1, which directly degrades lignin. We propose that in addition to symbionts and pathogens, SSPs also have roles in saprophytes and function in P. ostreatus as components of the ligninolytic system.

Degradation of the herbicide paraquat by macromycetes isolated from southeastern Mexico.

Fifty-four macromycetes, isolated from southeastern Mexico, were used in order to evaluate their capacity for degradation and tolerance to the herbicide paraquat. Ten of these strains were capable of growing in a solid culture medium in the presence of 200ppm paraquat. Subsequently, assays to evaluate the degradation of the xenobiotic in a liquid medium were carried out. Of the ten strains evaluated, three presented the highest levels of degradation of the compound, which were Trametes pavonia (54.2%), Trametes versicolor (54.1%) and Hypholoma dispersum. They presented the highest overall degradation percentage (70.7%) after 12days culture. The presence of ligninolytic enzymes in these strains was evaluated. H. dispersum only presented aryl alcohol oxidase activity; however, with the data obtained, it was not possible to conclude whether this specific enzyme is responsible for paraquat degradation. The level of degradation obtained is above the one reported for Pseudomonas putida, one of the few reports on paraquat degradation. This is the first report on the contaminant degradation capacity of H. dispersum.

Prevention of melanin formation during aryl alcohol oxidase production under growth-limited conditions using an Aspergillus nidulans cell factory

An Aspergillus nidulans cell factory was genetically engineered to produce an aryl alcohol oxidase (AAO). The cell factory initiated production of melanin when growth-limited conditions were established using stationary plates and shaken flasks. This phenomenon was more pronounced when the strain was cultured in a trickle bed reactor (TBR). This study investigated different approaches to reduce melanin formation in fungal mycelia and liquid medium in order to increase the enzyme production yield. Removal of copper from the medium recipe reduced melanin formation in agar cultures and increased enzyme activities by 48% in agitated liquid cultures. Copper has been reported as a key element for tyrosinase, an enzyme responsible for melanin production. Ascorbic acid (0.44g/L) stopped melanin accumulation, did not affect growth parameters and resulted in AAO activity that was more than two-fold greater than a control treatment with no ascorbic acid.

Peach and Cherry Agroindustrial Wastes: New and Economic Sources for the Production of Lignocellulolytic Enzymes.

Pleurotus eryngii was incubated on both peach and cherry agroindustrial wastes by solid state fermentation for 30 days without/with pretreatment conditions. The lignocellulosic substrates were pretreated with dilute acid and alkaline solutions, hot water before incubation. The maximum carboxymethyl cellulase and xylanase activities peaked on 3rd and 5th days under control conditions of both wastes, respectively. The highest laccase and manganese peroxidase activities reached to their maximum on 17th day as 2193.06 ± 50.4 UL-1 and 732.73 ± 19.8 UL-1, respectively. The highest aryl alcohol oxidase activity was obtained as 239.25 ± 7.3 UL-1 in control condition of peach cultures. The used pretreatment methods had generally negative effects on lignocellulolytic enzyme production. The highest lignocellulolytic activities were detected using peach wastes. To results, these wastes could.

Fungal pretreatment of sweet sorghum bagasse with supplements: improvement in lignin degradation, selectivity and enzymatic saccharification

Sweet sorghum bagasse (SSB) from food processing and agricultural industry has attracted the attention for uses in production of biofuel, enzymes and other products. The alteration in lignocellulolytic enzymes by use of supplements in fungal pretreatment of SSB to achieve higher lignin degradation, selectivity value and enzymatic hydrolysis to fermentable sugar was studied. Fungal strain Coriolus versicolor was selected for pretreatment due to high ligninolytic and low cellulolytic enzyme production resulting in high lignin degradation and selectivity value. SSB was pretreated with supplements of veratryl alcohol, syringic acid, catechol, gallic acid, vanillin, guaiacol, CuSO4 and MnSO4. The best results were obtained with CuSO4, gallic acid and syringic acid supplements. CuSO4 increased the activities of laccase (4.9-fold) and polyphenol oxidase (1.9-fold); gallic acid increased laccase (3.5-fold) and manganese peroxidase (2.5-fold); and syringic acid increased laccase (5.6-fold), lignin peroxidase (13-fold) and arylalcohol oxidase (2.8-fold) resulting in enhanced lignin degradations and selectivity values than the control. Reduced cellulolytic enzyme activities resulted in high cellulose recovery. Enzymatic hydrolysis of pretreated SSB yielded higher sugar due to degradation of lignin and reduced the crystallinity of cellulose. The study showed that supplements could be used to improve the pretreatment process. The results were confirmed by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy and thermogravimetric/differential thermogravimetric analysis of SSB.

Production of ligninolytic enzymes by solid state fermentation using Pleurotus ostreatus

Abstract Solid state fermentation (SSF) stands out in the production of lignocellulolytic and other industrially important enzymes. SSF, an alternative culture method, has several advantages over the conventional submerged ones, like higher yields of enzymes. The production of ligninolytic enzymes, such as laccase (Lac), manganese peroxidase (MnP), lignin peroxidase (LiP) and aryl alcohol oxidase (AAO) by Pleurotus ostreatus (Jacq.) Pleurotus Kumm. (MCC16) was investigated under SSF, which was performed using a support-substrate from potato peel waste (PPW). PPW as dry and fresh was pretreated with base to neutralize organic acids and distilled water. Chemical content of PPW was investigated. Ligninolytic enzyme production patterns were investigated during the growth of the organism for a period of 23 days at 25 °C in the stationary SSF using pretreated PPW. The highest Lac and MnP activities were determined in dry PPW, pretreated with distilled water as 6708.3 ± 75 U/L and 2503.6 ± 50 U/L on day 17, respectively. In addition, amylase and protease enzyme activities were detected under same conditions. According to the results, PPW has a potential as supports for ligninolytic enzyme production by P. ostreatus under SSF.

Fungal Pretreatment of Sweet Sorghum Bagasse with Combined CuSO4-Gallic Acid Supplement for Improvement in Lignin Degradation, Selectivity, and Enzymatic Saccharification.

Sweet sorghum (Sorghum sp.) has high biomass yield. Hydrolysis of lignocellulosic sweet sorghum bagasse (SSB) to fermentable sugar could be useful for manufacture of biofuel or other fermentation products. Pretreatment of lignocellulosic biomass to degrade lignin before enzymatic hydrolysis is a key step. Fungal pretreatment of SSB with combined CuSO4-gallic acid supplements in solid-state fermentation (SSF) to achieve higher lignin degradation, selectivity value (SV), and enzymatic hydrolysis to sugar was studied. Coriolus versicolor was selected due to high activities of ligninolytic enzymes laccase, lignin peroxidase (LiP), manganese peroxidase (MnP), polyphenol oxidase (PPO), and arylalcohol oxidase (AAO) and low activities of cellulolytic enzymes CMCase, FPase, and β-glucosidase with high lignin degradation and SV in 20days. CuSO4/gallic acid increased the activities of ligninolytic enzymes resulting in enhanced lignin degradations and SVs. Cumulative/synergistic effect of combined supplements further increased the activities of laccase, LiP, MnP, PPO, and AAO by 7.6, 14.6, 2.67, 2.06, and 2.15-folds, respectively (than control), resulting in highest lignin degradation 31.1±1.4% w/w (1.56-fold) and SV 2.33 (3.58-fold). Enzymatic hydrolysis of pretreated SSB yielded higher (~2.2 times) fermentable sugar. The study showed combined supplements can improve fungal pretreatment of lignocellulosic biomass. XRD, SEM, FTIR, and TGA/DTG of SSB confirmed the results.

Degradation of hardwoods by treatment of white rot fungi and its pyrolysis kinetics studies

The present work investigated the degradation of two Indian hardwoods (Pithecellobium dulce and Tamarindus indica) and the activities of lignocellulolytic enzymes by Pseudolagarobasidium acaciicola AGST3 and Tricholoma giganteum AGDR1. Laccase exhibited the highest ligninolytic activity, followed by manganese peroxidase and lignin peroxidase in both white rot fungi. Accessory enzymes such as aryl alcohol oxidase and cellobiose dehydrogenase showed higher activities till 21 days after incubation and then declined till 30 days. Xylanase and β-glucosidase were also found to be stable in both fungi during the degradation of hardwoods. The cultures, P. acaciicola AGST3 and T. giganteum AGDR1 accumulated high amounts of oxalic acid and produced weddellite (0.2–9 μm) and whewellite (5–21 μm) crystals, respectively. Comparative decay studies revealed that both the fungi were able to degrade lignin, cellulose and hemicelluloses of two hardwoods efficiently. During the pyrolysis reaction, decrease in onset temperature and activation energy was observed with the fungal treated wood as compared to the untreated wood. The basidiomycete cultures used in the study are efficient lignin degraders and can be applied as a pretreatment for second generation biofuels.

High-yield production of aryl alcohol oxidase under limited growth conditions in small-scale systems using a mutant Aspergillus nidulans strain.

Aryl alcohol oxidase (MtGloA) is an enzyme that belongs to the ligninolytic consortium and can play an important role in the bioenergy industry. This study investigated production of an MtGloA client enzyme by a mutant strain of Aspergillus nidulans unable to synthesize its own pyridoxine. Pyridoxine limitation can be used to control cell growth, diverting substrate to protein production. In agitated culture, enzyme production was similar when using media with 1mg/L and without pyridoxine (26.64±6.14U/mg mycelia and 26.14±8.39 U/mg mycelia using media with and without pyridoxine, respectively). However, the treatment lacking pyridoxine had to be supplemented with pyridoxine after 156h of fermentation to sustain continued enzyme production. Use of extremely diluted pyridoxine levels allowed reduced fungal growth while maintaining steady enzyme production. Concentrations of 9 and 13.5µg/L pyridoxine allowed MtGloA production with a growth rate of only 5% of that observed when using the standard 1mg/L pyridoxine media.

Protein dynamics promote hydride tunnelling in substrate oxidation by aryl-alcohol oxidase.

The temperature dependence of hydride transfer from the substrate to the N5 of the FAD cofactor during the reductive half-reaction of Pleurotus eryngii aryl-alcohol oxidase (AAO) is assessed here. Kinetic isotope effects on both the pre-steady state reduction of the enzyme and its steady-state kinetics, with differently deuterated substrates, suggest an environmentally-coupled quantum-mechanical tunnelling process. Moreover, those kinetic data, along with the crystallographic structure of the enzyme in complex with a substrate analogue, indicate that AAO shows a pre-organized active site that would only require the approaching of the hydride donor and acceptor for the tunnelled transfer to take place. Modification of the enzyme's active-site architecture by replacement of Tyr92, a residue establishing hydrophobic interactions with the substrate analogue in the crystal structure, in the Y92F, Y92L and Y92W variants resulted in different temperature dependence patterns that indicated a role of this residue in modulating the transfer reaction.

Extracellular proteins of Trametes hirsuta st. 072 induced by copper ions and a lignocellulose substrate.

BackgroundFungi are organisms with the highest natural capacity to degrade lignocellulose substrates, which is enabled by complex systems of extracellular enzymes, whose expression and secretion depend on the characteristics of substrates and the environment.ResultsThis study reports a secretome analysis for white-rot basidiomycete Trametes hirsuta cultivated on a synthetic media and a lignocellulose substrate. We demonstrate that T. hirsuta st. 072 produces multiple extracellular ligninolytic, cellulolytic, hemicellulolytic, peroxide generating, and proteolytic enzymes, as well as cerato-platanins. In contrast to other white rot species described earlier, which mostly secreted glucanases and mannosidases in response to the presence of the lignocellulose substrate, T. hirsuta expressed a spectrum of extracellular cellulolytic enzymes containing predominantly cellobiases and xylanases. As proteomic analysis could not detect lignin peroxidase (LiP) among the secreted lignin degrading enzymes, we attributed the observed extracellular LiP - like activity to the expressed versatile peroxidase (VP). An accessory enzyme, glyoxal oxidase, was found among the proteins secreted in the media during submerged cultivation of T. hirsuta both in the presence and in the absence of copper. However, aryl-alcohol oxidase (AAO) was not identified, despite the presence of AAO enzymatic activity secreted by the fungus.The spectra of the expressed enzymes dramatically changed depending on the growth conditions. Transfer from submerged cultivation to surface cultivation with the lignocellulose substrate switched off expression of exo-β-1,3-glucanase and α-amylase and turned on secretion of endo-β-1,3-glucanase and a range of glycosidases. In addition, an aspartic peptidase started being expressed instead of family S53 protease. For the first time, we report production of cerato-platanin proteins by Trametes species. The secretion of cerato-platanins was observed only in response to contact with lignocellulose, thus indicating a specific role of these proteins in degradation of the lignocellulose substrates.ConclusionsOur results suggest a sequential mechanism of natural substrate degradation by T. hirsuta, in which the fungus produces different sets of enzymes to digest all main components of the substrate during cultivation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0729-0) contains supplementary material, which is available to authorized users.

An aryl-alcohol oxidase of Pleurotus sapidus: heterologous expression, characterization, and application in a 2-enzyme system

Aryl-alcohol oxidases (AAOs) are enzymes supporting the degradation of lignin by fungal derived class II peroxidases produced by white-rot fungi. AAOs are able to generate H2O2 as a by-product via oxidation of an aryl-alcohol into its correspondent aldehyde. In this study, an AAO was heterologously expressed in a basidiomycete host for the first time. The gene for an AAO of the white-rot fungus Pleurotus sapidus, a close relative to the oyster mushroom Pleurotus ostreatus, was cloned into an expression vector and put under control of the promotor of the glyceraldehyde-3-phosphate dehydrogenase gene 2 (gpdII) of the button mushroom Agaricus bisporus. The expression vector was transformed into the model basidiomycete Coprinopsis cinerea, and several positive transformants were obtained. The best producing transformants were grown in shake-flasks and in a stirred tank reactor reaching enzymatic activities of up to 125UL(-1) using veratryl alcohol as a substrate. The purified AAO was biochemically characterized and compared to the previously described native and recombinant AAOs from other Pleurotus species. In addition, a two-enzyme system comprising a dye-decolorizing peroxidase (DyP) from Mycetinis scorodonius and the P. sapidus AAO was successfully employed to bleach the anthraquinone dye Reactive Blue 5.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening.

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in <em>Saccharomyces cerevisiae</em>: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Activities of Secreted Aryl Alcohol Quinone Oxidoreductases from Pycnoporus cinnabarinus Provide Insights into Fungal Degradation of Plant Biomass.

Auxiliary activities family 3 subfamily 2 (AA3_2) from the CAZy database comprises various functions related to ligninolytic enzymes, such as fungal aryl alcohol oxidases (AAO) and glucose oxidases, both of which are flavoenzymes. The recent study of the Pycnoporus cinnabarinus CIRM BRFM 137 genome combined with its secretome revealed that four AA3_2 enzymes are secreted during biomass degradation. One of these AA3_2 enzymes, scf184803.g17, has recently been produced heterologously in Aspergillus niger Based on the enzyme's activity and specificity, it was assigned to the glucose dehydrogenases (PcinnabarinusGDH [PcGDH]). Here, we analyze the distribution of the other three AA3_2 enzymes (scf185002.g8, scf184611.g7, and scf184746.g13) to assess their putative functions. These proteins showed the highest homology with aryl alcohol oxidase from Pleurotus eryngii Biochemical characterization demonstrated that they were also flavoenzymes harboring flavin adenine dinucleotide (FAD) as a cofactor and able to oxidize a wide variety of phenolic and nonphenolic aryl alcohols and one aliphatic polyunsaturated primary alcohol. Though presenting homology with fungal AAOs, these enzymes exhibited greater efficiency in reducing electron acceptors (quinones and one artificial acceptor) than molecular oxygen and so were defined as aryl-alcohol:quinone oxidoreductases (AAQOs) with two enzymes possessing residual oxidase activity (PcAAQO2 and PcAAQO3). Structural comparison of PcAAQO homology models with P. eryngii AAO demonstrated a wider substrate access channel connecting the active-site cavity to the solvent, explaining the absence of activity with molecular oxygen. Finally, the ability of PcAAQOs to reduce radical intermediates generated by laccase from P. cinnabarinus was demonstrated, shedding light on the ligninolytic system of this fungus.

Characterization of a new aryl-alcohol oxidase secreted by the phytopathogenic fungus Ustilago maydis.

The discovery of novel fungal lignocellulolytic enzymes is essential to improve the breakdown of plant biomass for the production of second-generation biofuels or biobased materials in green biorefineries. We previously reported that Ustilago maydis grown on maize secreted a diverse set of lignocellulose-acting enzymes including hemicellulases and putative oxidoreductases. One of the most abundant proteins of the secretome was a putative glucose-methanol-choline (GMC) oxidoreductase. The phylogenetic prediction of its function was hampered by the few characterized members within its clade. Therefore, we cloned the gene and produced the recombinant protein to high yield in Pichia pastoris. Functional screening using a library of substrates revealed that this enzyme was able to oxidize several aromatic alcohols. Of the tested aryl-alcohols, the highest oxidation rate was obtained with 4-anisyl alcohol. Oxygen, 1,4-benzoquinone, and 2,6-dichloroindophenol can serve as electron acceptors. This GMC oxidoreductase displays the characteristics of an aryl-alcohol oxidase (E.C.1.1.3.7), which is suggested to act on the lignin fraction in biomass.

A survey of genes encoding H2O2-producing GMC oxidoreductases in 10 Polyporales genomes

The genomes of three representative Polyporales (Bjerkandera adusta, Phlebia brevispora and a member of the Ganoderma lucidum complex) recently were sequenced to expand our knowledge on the diversity and distribution of genes involved in degradation of plant polymers in this Basidiomycota order, which includes most wood-rotting fungi. Oxidases, including members of the glucose-methanol-choline (GMC) oxidoreductase superfamily, play a central role in the above degradative process because they generate extracellular H2O2 acting as the ultimate oxidizer in both white-rot and brown-rot decay. The survey was completed by analyzing the GMC genes in the available genomes of seven more species to cover the four Polyporales clades. First, an in silico search for sequences encoding members of the aryl-alcohol oxidase, glucose oxidase, methanol oxidase, pyranose oxidase, cellobiose dehydrogenase and pyranose dehydrogenase families was performed. The curated sequences were subjected to an analysis of their evolutionary relationships, followed by estimation of gene duplication/reduction history during fungal evolution. Second, the molecular structures of the near one hundred GMC oxidoreductases identified were modeled to gain insight into their structural variation and expected catalytic properties. In contrast to ligninolytic peroxidases, whose genes are present in all white-rot Polyporales genomes and absent from those of brown-rot species, the H2O2-generating oxidases are widely distributed in both fungal types. This indicates that the GMC oxidases provide H2O2 for both ligninolytic peroxidase activity (in white-rot decay) and Fenton attack on cellulose (in brown-rot decay), after the transition between both decay patterns in Polyporales occurred.

Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides.

Aryl-alcohol oxidase (AAO) is an extracellular flavoprotein that supplies ligninolytic peroxidases with H2O2 during natural wood decay. With a broad substrate specificity and highly stereoselective reaction mechanism, AAO is an attractive candidate for studies into organic synthesis and synthetic biology, and yet the lack of suitable heterologous expression systems has precluded its engineering by directed evolution. In this study, the native signal sequence of AAO from Pleurotus eryngii was replaced by those of the mating α-factor and the K1 killer toxin, as well as different chimeras of both prepro-leaders in order to drive secretion in Saccharomyces cerevisiae. The secretion of these AAO constructs increased in the following order: preproα-AAO > preαproK-AAO > preKproα-AAO > preproK-AAO. The chimeric preαproK-AAO was subjected to focused-directed evolution with the aid of a dual screening assay based on the Fenton reaction. Random mutagenesis and DNA recombination was concentrated on two protein segments (Met[α1]-Val109 and Phe392-Gln566), and an array of improved variants was identified, among which the FX7 mutant (harboring the H91N mutation) showed a dramatic 96-fold improvement in total activity with secretion levels of 2 mg/liter. Analysis of the N-terminal sequence of the FX7 variant confirmed the correct processing of the preαproK hybrid peptide by the KEX2 protease. FX7 showed higher stability in terms of pH and temperature, whereas the pH activity profiles and the kinetic parameters were maintained. The Asn91 lies in the flavin attachment loop motif, and it is a highly conserved residue in all members of the GMC superfamily, except for P. eryngii and P. pulmonarius AAO. The in vitro involution of the enzyme by restoring the consensus ancestor Asn91 promoted AAO expression and stability.