Valorization of bioethanol by-products to produce unspecific peroxygenase with Agrocybe aegerita: Technological and proteomic perspectives.

Unspecific peroxygenase (UPO) presents a wide range of biotechnological applications. This study targets the use of by-products from bioethanol synthesis to produce UPO by Agrocybe aegerita. Solid-state and submerged fermentations (SSF and SmF) were evaluated, achieving the highest titers of UPO and laccase in SmF using vinasse as nutrients source. Optimized UPO production of 331 U/L was achieved in 50% (v:v) vinasse with an inoculum grown for 14 days. These conditions were scaled-up to a 4 L reactor, achieving a UPO activity of 265 U/L. Fungal proteome expression was analyzed before and after UPO activity appeared by shotgun mass spectrometry proteomics. Laccase, dye-decolorizing peroxidases (DyP), lectins and proteins involved in reactive oxygen species (ROS) production and control were detected (in addition to UPO). Interestingly, the metabolism of complex sugars and nitrogen sources had a different activity at the beginning and end of the submerged fermentation.

Polysaccharide from Agrocybe cylindracea prevents diet-induced obesity through inhibiting inflammation mediated by gut microbiota and associated metabolites.

Polysaccharide from Agrocybe cylindracea (ACP) has been demonstrated with various health benefits, but its anti-obesity effect and underlying mechanisms remain poorly understood. This study aimed to investigate the beneficial effects of ACP in high-fat diet (HFD)-induced obese mice by targeting gut microbiota and metabolites. 9-week ACP supplementation in HFD-fed mice reduced body weight, adipose accumulation, impaired insulin resistance, lipid levels, and liver injuries, which were negatively correlated to the pro-inflammatory factors, particularly tumor necrosis factor-alpha (TNF-α) and interleukin- 6 (IL-6). Moreover, ACP not only restored HFD-induced gut disorder, as indicated by the depletion of Desulfovibrio and Oscillibacter and the enrichment of the Bacteroides, Parabacteroides, Butyricimonas, and Dubosiella, but also positively regulated gut metabolites such as solavetivone and N-acetylneuraminic acid. Spearman's correlation analysis revealed that the ACP-altered microbes and metabolites were highly correlated with inflammation-related indexes. Notably, ACP greatly lowered the obesity-related TNF-α- and IL-6-levels partially by reducing Desulfovibrio and increasing Parabacteroides abundances, together with the associated decrease of solavetivone level. These findings suggest that ACP may be used as a prebiotic agent to prevent diet-induced obesity, and target-specific microbiota and metabolites may have unique therapeutic promise for inflammation-related diseases.

Optimization Extraction and Antioxidant Activity of Crude Polysaccharide from Chestnut Mushroom (Agrocybe aegerita) by Accelerated Solvent Extraction Combined with Response Surface Methodology (ASE-RSM).

The present work is conducted to investigate the optimal extraction technology of polysaccharide from chestnut mushroom (Agrocybe aegerita) using a new method based on accelerated solvent extraction combined with response surface methodology (ASE-RSM). The conventional reflux extraction (CRE) method and ultrasonic-assisted extraction (UAE) method were also carried out. Additionally, the in vitro antioxidant activities, including ABTS and DPPH assay, were evaluated. The RSM method, based on a three level and three variable Box-Behnken design (BBD), was developed to obtain the optimal combination of extraction conditions. In brief, the polysaccharide was optimally extracted with water as extraction solvent, extraction temperature of 71 °C, extraction time of 6.5 min, number of cycles of 3, and extraction pressure of 10 MPa. The 3D response surface plot and the contour plot derived from the mathematical models were applied to determine the optimal conditions. Under the above conditions, the experimental value of polysaccharide yield was 19.77 ± 0.12%, which is in close agreement with the value (19.81%) predicted by the model. These findings demonstrate that ASE-RSM produce much higher polysaccharide and consumed environmentally friendly extraction and solvent systems, have less extraction discrimination and shorter time and provide scientific basis for industrialization of polysaccharide extraction. Moreover, it was proved that the polysaccharide had the potential ability to scavenge ABTS and DPPH.

Transformation of Agrocybe cylindracea Galectin into αGalNAc-Specific Lectin.

A multi-specific fungal galectin from the mushroom Agrocybe cylindracea (ACG) binds a broad range of ß-galactosides, as well as their derivative GalNAcα1-3Gal. Site-directed mutagenesis of the hydrophilic residues His, Asn, Arg, and Glu, involved in carbohydrate recognition, abolished the binding affinity of the derived mutants to ß-galactosides, whereas only N46A caused increased affinity to GalNAcα1-3Gal-containing oligosaccharides and loss of ß-galactoside-binding activity. Detailed structural analysis revealed that Pro45, the preceding residue of Asn46 of the wild-type ACG, takes the cis imide conformation to tether Asn46 onto a loop region to make new hydrogen bonds with ß-galactosides and to compensate for the lack of evolutionarily conserved Asn. In contrast, in the N46A mutant, Pro45 takes the more stable trans conformation, resulting in "switched" specificity to αGalNAc. Such an altered recognition system in the binding specificity of galectins can be observed in other lectin molecules not only in nature but will also be observed in those engineered in the future.

Characterization of cheesy odor formed during fermentation of soy drink with Agrocybe aegerita.

The market for plant protein-based substitutes for cheeses is growing, but the sensory properties are distinctively different from the original products. Hence, natural and vegan cheesy flavors are needed to aromatize the products. A cheesy, sweaty and parmesan-like aroma was produced by fermentation of soy drink with Agrocybe aegerita. Aroma dilution analysis revealed short-chain fatty acids (SCFAs) as main influencing cheesy odorants analyzed by gas chromatography-mass spectrometry-olfactometry. In comparison to the five cheese varieties, the SCFA profile of the fermented soy drink revealed similarities with Parmesan and Emmental cheese. Meanwhile, principal component analysis showed an approximation of the aroma profile after fermentation with A. aegerita to those of cheeses. 3-Methylbutanoic acid was synthesized from the protein fraction, while the oil fraction contributed to the formation of unbranched SCFAs like butanoic acid. Accordingly, the production of these compounds can be increased by addition of the fractions.

Prebiotic Agrocybe cylindracea crude polysaccharides combined with Lactobacillus rhamnosus GG postpone aging-related oxidative stress in mice.

This study aimed to investigate the potential anti-aging mechanisms of Agrocybe cylindracea crude polysaccharides (APS), when used synergistically with Lactobacillus rhamnosus GG (APS + LGG) in a D-galactose-induced aging mouse model. In the Morris water maze test, APS + LGG showed a significantly higher memory and learning capacity compared to untreated, APS only treated and LGG treated mice. This was thought to be mediated by increased levels of brain-derived neurotrophic factor, which decreased escape latency. In addition to this, in the aging mouse model, APS + LGG co-treatment markedly alleviated liver oxidation and metabolism by enhancing the antioxidant activity of enzymes; this decreased the lipid metabolism and peroxidation levels. Furthermore, high throughput sequencing analysis revealed that an APS + LGG supplemented feed increased the relative abundance of positive bacteria in the gut microbiota such as Alloprevotella and Parvibacter. Importantly, Alloprevotella and Parvibacter showed a negative relationship with low density lipoprotein-cholesterol in the Spearman correlation analysis. These results illustrate that APS, in combination with LGG, postponed aging related oxidative stress when used as a prebiotic. The proposed mechanism for this is the reduction in liver oxidation and lipid metabolism, as well as the regulation of gut microbiota.

Whole Agrocybe cylindracea Prevented Obesity Linking with Modification of Gut Microbiota and Associated Fecal Metabolites in High-Fat Diet-Fed Mice.

SCOPE: Whole-food-based strategies to prevent metabolic diseases are growing interests. Agrocybe cylindracea (AC) is a major edible mushroom with high values of nutrition, but little is known about its health benefits as a portion of whole food. METHODS AND RESULTS: Diet-induced obese, C57BL/6J mice are fed an high-fat diet (HFD) with or without AC (3% or 5%, w/w in the diet) for 9 weeks. The results show that dietary AC reduced body weight, adipose accumulation, impairment of glucose tolerance, lipid levels, and liver injury in HFD-fed mice. Moreover, AC not only prevents HFD-induced gut disorder, as indicates by the enriched probiotic Bifidobacterium and reduced endotoxin-bearing Proteobacteria, but also improve the lipopolysaccharide (LPS) level and gut tissue structure. Fecal metabolites such as harmine and harmanine are also remarkably altered by AC. Spearman's correlation analysis reveals that the AC-altered microbes and metabolites are strongly correlated with obesity-related indexes. CONCLUSION: These findings suggest that dietary AC prevents HFD-induced obesity and its complications in association with modulating gut microbiota and associated fecal metabolites.

The protective effects of the Lactarius deliciosus and Agrocybe cylindracea mushrooms on histopathology of carbon tetrachloride induced oxidative stress in rats.

We investigated the protective effects of L. deliciosus and A. cylindracea supplementation against carbon tetrachloride (CCI4) induced oxidative stress by measuring levels of adenosine deaminase (ADA) and myeloperoxidase (MPO), and by observing histopathological changes in liver and kidney tissues of rats. We divided 36 rats into six groups: control, CCl4, L. deliciosus, A. cylindracea, CCl4 + L. deliciosus, and CCl4 + A. cylindracea. We found that administration of CCI4, A. cylindracea, and CCl4 + A. cylindracea increased MPO and ADA levels. We observed severe hepato-renal degenerative and necrotic lesions in the CCI4, A. cylindracea and CCl4 + A. cylindracea groups. Severe lesions of the liver and kidney were not observed with A. cylindracea administration. CCI4 induced hepato-renal lesions were ameliorated by L. deliciosus extract supplementation. L. deliciosus could be an important dietary antioxidant for preventing histologic lesions in liver and kidney due to CCI4 induced oxidative stress in rats.

Magnaporthe oryzae as an expression host for the production of the unspecific peroxygenase AaeUPO from the basidiomycete Agrocybe aegerita.

The filamentous fungus Magnaporthe oryzae has the potential to be developed as an alternative platform organism for the heterologous production of industrially important enzymes. M. oryzae is easy to handle, fast-growing and unlike yeast, posttranslational modifications like N-glycosylations are similar to the human organism. Here, we established M. oryzae as a host for the expression of the unspecific peroxygenase from the basidiomycete Agrocybe aegerita (AaeUPO). Note, UPOs are attractive biocatalysts for selective oxyfunctionalization of non-activated carbon-hydrogen bonds. To improve and simplify the isolation of AaeUPO in M. oryzae, we fused a Magnaporthe signal peptide for protein secretion and set it under control of the strong EF1α-promoter. The success of the heterologous production of full-length AaeUPO in M. oryzae and the secretion of the functional enzyme was confirmed by a peroxygenase-specific enzyme assay. These results offer the possibility to establish the filamentous ascomycete M. oryzae as a broad applicable alternative expression system.

Toxicity and membrane perturbation properties of the ribotoxin-like protein Ageritin.

Ageritin is the prototype of a new ribotoxin-like protein family, which has been recently identified also in basidiomycetes. The protein exhibits specific RNase activity through the cleavage of a single phosphodiester bond located at sarcin/ricin loop of the large rRNA, thus inhibiting protein biosynthesis at early stages. Conversely to other ribotoxins, its activity requires the presence of divalent cations. In the present study, we report the activity of Ageritin on both prokaryotic and eukaryotic cells showing that the protein has a prominent effect on cancer cells viability and no effects on eukaryotic and bacterial cells. In order to rationalize these findings, the ability of the protein to interact with various liposomes mimicking normal, cancer and bacterial cell membranes was explored. The collected results indicate that Ageritin can interact with DPPC/DPPS/Chol vesicles, used as a model of cancer cell membranes, and with DPPC/DPPG vesicles, used as a model of bacterial cell membranes, suggesting a selective interaction with anionic lipids. However, a different perturbation of the two model membranes, mediated by cholesterol redistribution, was observed and this might be at the basis of Ageritin selective toxicity towards cancer cells.

Directed evolution of unspecific peroxygenase in organic solvents.

Fungal unspecific peroxygenases (UPOs) are efficient biocatalysts that insert oxygen atoms into nonactivated C-H bonds with high selectivity. Many oxyfunctionalization reactions catalyzed by UPOs are favored in organic solvents, a milieu in which their enzymatic activity is drastically reduced. Using as departure point the UPO secretion mutant from Agrocybe aegerita (PaDa-I variant), in the current study we have improved its activity in organic solvents by directed evolution. Mutant libraries constructed by random mutagenesis and in vivo DNA shuffling were screened in the presence of increasing concentrations of organic solvents that differed both in regard to their chemical nature and polarity. In addition, a palette of neutral mutations generated by genetic drift that improved activity in organic solvents was evaluated by site directed recombination in vivo. The final UPO variant of this evolutionary campaign carried nine mutations that enhanced its activity in the presence of 30% acetonitrile (vol/vol) up to 23-fold over PaDa-I parental type, and it was also active and stable in aqueous acetone, methanol and dimethyl sulfoxide mixtures. These mutations, which are located at the surface of the protein and in the heme channel, seemingly helped to protect UPO from harmful effects of cosolvents by modifying interactions with surrounding residues and influencing critical loops.

Transcriptome of different fruiting stages in the cultivated mushroom Cyclocybe aegerita suggests a complex regulation of fruiting and reveals enzymes putatively involved in fungal oxylipin biosynthesis.

BACKGROUND: Cyclocybe aegerita (syn. Agrocybe aegerita) is a commercially cultivated mushroom. Its archetypal agaric morphology and its ability to undergo its whole life cycle under laboratory conditions makes this fungus a well-suited model for studying fruiting body (basidiome, basidiocarp) development. To elucidate the so far barely understood biosynthesis of fungal volatiles, alterations in the transcriptome during different developmental stages of C. aegerita were analyzed and combined with changes in the volatile profile during its different fruiting stages. RESULTS: A transcriptomic study at seven points in time during fruiting body development of C. aegerita with seven mycelial and five fruiting body stages was conducted. Differential gene expression was observed for genes involved in fungal fruiting body formation showing interesting transcriptional patterns and correlations of these fruiting-related genes with the developmental stages. Combining transcriptome and volatilome data, enzymes putatively involved in the biosynthesis of C8 oxylipins in C. aegerita including lipoxygenases (LOXs), dioxygenases (DOXs), hydroperoxide lyases (HPLs), alcohol dehydrogenases (ADHs) and ene-reductases could be identified. Furthermore, we were able to localize the mycelium as the main source for sesquiterpenes predominant during sporulation in the headspace of C. aegerita cultures. In contrast, changes in the C8 profile detected in late stages of development are probably due to the activity of enzymes located in the fruiting bodies. CONCLUSIONS: In this study, the combination of volatilome and transcriptome data of C. aegerita revealed interesting candidates both for functional genetics-based analysis of fruiting-related genes and for prospective enzyme characterization studies to further elucidate the so far barely understood biosynthesis of fungal C8 oxylipins.

Ligninolytic enzymes production during polycyclic aromatic hydrocarbons degradation: effect of soil pH, soil amendments and fungal co-cultivation.

Soil microorganisms play an important role in the degradation of PAHs and use various metabolic pathways for this process. The effect of soil pH, different soil amendments and the co-cultivation of fungi on the degradation of PAHs in soil and on the activity of ligninolytic enzymes was evaluated. For that purpose, three fungi were studied: Trichoderma viride, Penicillium chrysogenum and Agrocybe aegerita. Biodegradation assays with a mixture of 200 ppm PAHs (fluorene, pyrene, chrysene, and benzo[a]pyrene-50 ppm each) were set up at room temperature for 8 weeks. The maximum laccase activity by solid state fermentation-SSF (7.43 U/g) was obtained by A. aegerita on kiwi peels with 2 weeks and the highest manganese peroxidase activity (7.21 U/g) was reached in 4 weeks, both at pH 7. Fluorene, pyrene, and benzo[a]pyrene achieved higher degradation rates in soil at pH 5, while chrysene was more degradable at pH 7. About 85-90% of the PAHs were degraded by fungal remediation. The highest degradation of fluorene was achieved by co-cultivation of A. aegerita and P. chrysogenum, remaining 14% undegradable. Around 13% of pyrene stay undegradable by A. aegerita and T. viride and by A. aegerita and P. chrysogenum, both systems supported in kiwi peels, while 11% of chrysene remained in soil by the co-cultivation of these fungi, supported by peanut shells. Regarding benzo[a]pyrene, 13% remained in soil after treatment with A. aegerita. Despite the increase in degradation of some PAHs with co-cultivation, higher enzyme production during degradation was observed when fungi were cultivated alone.

Chromoselective Photocatalysis Enables Stereocomplementary Biocatalytic Pathways*.

Controlling the selectivity of a chemical reaction with external stimuli is common in thermal processes, but rare in visible-light photocatalysis. Here we show that the redox potential of a carbon nitride photocatalyst (CN-OA-m) can be tuned by changing the irradiation wavelength to generate electron holes with different oxidation potentials. This tuning was the key to realizing photo-chemo-enzymatic cascades that give either the (S)- or the (R)-enantiomer of phenylethanol. In combination with an unspecific peroxygenase from Agrocybe aegerita, green light irradiation of CN-OA-m led to the enantioselective hydroxylation of ethylbenzene to (R)-1-phenylethanol (99 % ee). In contrast, blue light irradiation triggered the photocatalytic oxidation of ethylbenzene to acetophenone, which in turn was enantioselectively reduced with an alcohol dehydrogenase from Rhodococcus ruber to form (S)-1-phenylethanol (93 % ee).

Comprehensive evaluation of alkali-extracted polysaccharides from Agrocybe cylindracea: Comparison on structural characterization.

Agrocybe cylindracea is a common source of active polysaccharides, but their fine structures are not clearly elucidated. In the present study, four fractions were purified from the alkaline extract of A. cylindracea (JACP), and their chemical components and structures were compared by HPAEC-PAD, methylation combined with GC-MS, and 1D/2D NMR analysis. Results showed the purified fractions' physicochemical properties, including monosaccharide compositions, molecular weights, viscosities and surface morphology considerably varied. JACP-30 was identified as a fucoglucogalactan with a α-(1 → 6)-galactopyranosyl as main chain. JACP-50p and JACP-80r were characterized as ß-(1 → 6)-glucans with side chains composed of terminal and 3-substituted ß-glucopyranosyl residues attached at O-3 for every three residues. Similarly, the backbone of JACP-80 was ß-(1 → 6)-linked glucopyranosyl and ß-(1 → 3,6)-linked glucopyranosyl residues at a ratio of 4:1. This work provides more information to the understanding of polysaccharides from A. cylindracea, further guiding its biological researches and developing the application in food and biomedicine industries.

Modeling and simulation-based design of electroenzymatic batch processes catalyzed by unspecific peroxygenase from A. aegerita.

Unspecific peroxygenases have attracted interest due to their ability to catalyze the oxygenation of various types of C-H bonds using only hydrogen peroxide as a cosubstrate. Due to the instability of these enzymes at even low hydrogen peroxide concentrations, careful fed-batch addition of the cosubstrate or ideally in situ production is required. While various approaches for hydrogen peroxide addition have been qualitatively assessed, only limited kinetic data concerning enzyme inactivation and peroxide accumulation has been reported so far. To obtain quantitative insights into the kinetics of such a process, a detailed data set for a peroxygenase-catalyzed benzylic hydroxylation coupled with electrochemical hydrogen peroxide production is presented. Based on this data set, we set out to model such an electroenzymatic process. For this, initial velocity data for the benzylic hydroxylation is collected and an extended Ping-Pong-Bi-Bi type rate equation is established, which sufficiently describes the enzyme kinetic. Moreover, we propose an empirical inactivation term based on the collected data set. Finally, we show that the full model does not only describe the process with sufficient accuracy, but can also be used predictively to control hydrogen peroxide feeding rates To limit the concentration of this critical cosubstrate in the system.

A novel serine protease with anticoagulant and fibrinolytic activities from the fruiting bodies of mushroom Agrocybe aegerita.

A novel fibrinolytic enzyme, ACase was isolated from fruiting bodies of a mushroom, Agrocybe aegerita. ACase was purified by using ammonium sulfate precipitation, gel filtration, ion exchange and hydrophobic chromatographies to 237.12 fold with a specific activity of 1716.77 U/mg. ACase was found to be a heterodimer with molecular mass of 31.4 and 21.2 kDa by SDS-PAGE and appeared as a single band on Native-PAGE and fibrin-zymogram. The N-terminal sequence of the two subunits of ACase was AIVTQTNAPWGL (subunit 1) and SNADGNGHGTHV (subunit 2). ACase had maximal activity at 47 °C and pH 7.6. It's activity was improved by Cu2+, Na+, Fe3+, Zn2+, Ba2+, K+ and Mn2+, but inhibited by Fe2+, Mg2+ and Ca2+. PMSF, SBTI, aprotinine and Lys inhibited the enzyme activity, which suggested that ACase was a serine protease. ACase could degrade all three chains (α, ß and γ) of fibrinogen. Moreover, the enzyme acted as both, a plasmin-like fibrinolytic enzyme and a plasminogen activator. It could hydrolyze human thrombin slightly, which indicated that the ACase could inhibit the activity of thrombin and acted as an anticoagulant to prevent thrombosis. Based on these results, ACase might act as a therapeutic agent for treating thrombosis, or as a functional food. Further investigation of the enzyme is underway.

Gene Organization, Expression, and Localization of Ribotoxin-Like Protein Ageritin in Fruiting Body and Mycelium of Agrocybe aegerita.

The edible mushroom Agrocybe aegerita produces a ribotoxin-like protein known as Ageritin. In this work, the gene encoding Ageritin was characterized by sequence analysis. It contains several typical features of fungal genes such as three short introns (60, 55 and 69 bp) located at the 5' region of the coding sequence and typical splice junctions. This sequence codes for a precursor of 156 amino acids (~17-kDa) containing an additional N-terminal peptide of 21 amino acid residues, absent in the purified toxin (135 amino acid residues; ~15-kDa). The presence of 17-kDa and 15-kDa forms was investigated by Western blot in specific parts of fruiting body and in mycelia of A. aegerita. Data show that the 15-kDa Ageritin is the only form retrieved in the fruiting body and the principal form in mycelium. The immunolocalization by confocal laser scanning microscopy and transmission electron microscopy proves that Ageritin has vacuolar localization in hyphae. Coupling these data with a bioinformatics approach, we suggest that the N-terminal peptide of Ageritin (not found in the purified toxin) is a new signal peptide in fungi involved in intracellular routing from endoplasmic reticulum to vacuole, necessary for self-defense of A. aegerita ribosomes from Ageritin toxicity.

Determination of Tissue Protective and Antioxidant Activities of Two Medicinal Mushrooms from Turkey against CCl4-lnduced Experimental Oxidative Stress in Rats.

This study aimed at determining hepatoprotective and antioxidants effects of Lactarius deliciosus and Agrocybe cylindracea against CCl4-induced oxidative stress (OS) in rats. Herein, 36 rats were divided into 6 groups: (I) Control, (II) CCl4, (III) CCl4 + L. deliciosus, (IV) L. deliciosus, (V) CCl4 + A. cylindracea, and (VI) A. cylindracea. According to the 7-week study results, aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutathione S-transferase (GST) (brain), superoxide dismutase (SOD) (kidney), malondialdehyde (MDA) (kidney), MDA (spleen), MDA (erythrocyte), GST (erythrocyte), and GST (liver) in group III (at 10%, 26%, 34%, 57%, 7%, 25%, 42%, 27%, and 35%, respectively) were significantly lower than in group II. Moreover, the reduced glutathione (GSH) (spleen) and catalase (spleen) in group III (at 20% and 38%, respectively) were significantly higher than in group II. AST, ALT, triglyceride, glutathione reductase (GR) (brain), GR (kidney), SOD (spleen), and SOD (liver) in group V (at 194%, 147%, 38%, 36%, 66%, 8%, and 7%, respectively) were significantly higher than in group II. However, MDA (kidney), MDA (erythrocyte), (GSH) (erythrocyte), MDA (liver), GSH (liver), and GST (liver) in group V (at 48%, 35%, 34%, 15%, 30%, and 41%, respectively) were significantly lower than in group II. Hence, while L. deliciosus may exhibit tissue-protective against CCl4-induced OS in rats, A. cylindracea does not.

Exploring the Role of Phenylalanine Residues in Modulating the Flexibility and Topography of the Active Site in the Peroxygenase Variant PaDa-I.

Unspecific peroxygenases (UPOs) are fungal heme-thiolate enzymes able to catalyze a wide range of oxidation reactions, such as peroxidase-like, catalase-like, haloperoxidase-like, and, most interestingly, cytochrome P450-like. One of the most outstanding properties of these enzymes is the ability to catalyze the oxidation a wide range of organic substrates (both aromatic and aliphatic) through cytochrome P450-like reactions (the so-called peroxygenase activity), which involves the insertion of an oxygen atom from hydrogen peroxide. To catalyze this reaction, the substrate must access a channel connecting the bulk solution to the heme group. The composition, shape, and flexibility of this channel surely modulate the catalytic ability of the enzymes in this family. In order to gain an understanding of the role of the residues comprising the channel, mutants derived from PaDa-I, a laboratory-evolved UPO variant from Agrocybe aegerita, were obtained. The two phenylalanine residues at the surface of the channel, which regulate the traffic towards the heme active site, were mutated by less bulky residues (alanine and leucine). The mutants were experimentally characterized, and computational studies (i.e., molecular dynamics (MD)) were performed. The results suggest that these residues are necessary to reduce the flexibility of the region and maintain the topography of the channel.