Aryl-alcohol Oxidase Research Articles

Oxidation of 5-hydroxymethylfurfural with a novel aryl alcohol oxidase from Mycobacterium sp. MS1601.

SummaryBio‐based 5‐hydroxymethylfurfural (HMF) serves as an important platform for several chemicals, among which 2,5‐furan dicarboxylic acid (FDCA) has attracted considerable interest as a monomer for the production of polyethylene furanoate (PEF), a potential alternative for fossil‐based polyethylene terephthalate (PET). This study is based on the HMF oxidizing activity shown by Mycobacterium sp. MS 1601 cells and investigation of the enzyme catalysing the oxidation. The Mycobacterium whole cells oxidized the HMF to FDCA (60% yield) and hydroxymethyl furan carboxylic acid (HMFCA). A gene encoding a novel bacterial aryl alcohol oxidase, hereinafter MycspAAO, was identified in the genome and was cloned and expressed in Escherichia coli Bl21 (DE3). The purified MycspAAO displayed activity against several alcohols and aldehydes; 3,5 dimethoxy benzyl alcohol (veratryl alcohol) was the best substrate among those tested followed by HMF. 5‐Hydroxymethylfurfural was converted to 5‐formyl‐2‐furoic acid (FFCA) via diformyl furan (DFF) with optimal activity at pH 8 and 30–40°C. FDCA formation was observed during long reaction time with low HMF concentration. Mutagenesis of several amino acids shaping the active site and evaluation of the variants showed Y444F to have around 3‐fold higher kcat/Km and ~1.7‐fold lower Km with HMF.

Mycoremediation of lignocellulosic biorefinery sludge: A reinvigorating approach for organic contaminants remediation with simultaneous production of lignocellulolytic enzyme cocktail

This research work aims to valorize lignocellulosic biorefinery sludge with genetically engineered Trichoderma atroviride for simultaneous removal of organic contaminants, fermentation inhibitors, and lignocellulolytic enzyme cocktail production. Upon analysis, three phenolic compounds (42.6 ± 3.6 μg/g), two polycyclic aromatic hydrocarbons (0.42 ± 0.06 μg/g) and five fermentation inhibitors (2.5 ± 0.3 mg/g) were detected in the sludge. Bioaugmentation of sludge with 72 h-old T. atroviride (5%) results in the production of cellulase (21 U/g), xylanase (84 U/g), laccase (20 U/g), lignin peroxidase (14 U/g) and aryl alcohol oxidase (116 U/g), along with the concomitant removal of organic contaminants (phenol, 2, 4-dinitrophenol, pentchlorophenol, phenanthrene, benzo(a)pyrene) and fermentation inhibitors (furfural, 5-hydroxymethylfurfural, levulinic acid, ferulic acid, and catechol). Subsequently, the enrichment of sludge with nutrients and rhamnolipids enhanced the enzyme production by 5–6-fold and resulted in the removal of 85–95% of organic contaminants and fermentation inhibitors, which constitutes an eco-friendly process.

Agar plate assay for rapid screening of aryl-alcohol oxidase mutant libraries in Pichia pastoris

Directed evolution is a powerful tool for developing biocatalysts with tailor-made properties for biocatalytic applications. High-throughput activity screening of large mutant libraries generated by e.g. means of directed evolution is usually performed in 96-well microtiter plates requiring, however, time-consuming and laborious enzyme expression, cell harvesting and activity measurements. In addition, automated liquid handling systems are needed to cope with the high number of colonies to be screened. Herein, we developed an agar plate-based assay for simple and fast screening of H2O2-producing aryl-alcohol oxidase (AAO) mutant libraries in Pichia pastoris. Buffered minimal methanol agar plates were supplemented with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), horseradish peroxidase (HRP) and the target substrate. AAO activity is visualized by formation of green zones around AAO-secreting P. pastoris colonies due to ABTS oxidation by HRP which is fueled with H2O2 by AAO in course of substrate oxidation. Colonies were screened within 24 h for AAO activity using different AAO substrates like veratryl alcohol, p-anisyl alcohol or trans,trans-2,4-hexadien-1-ol and even low AAO activity towards 5-hydroxymethylfurfural could be detected within 48 h. The developed agar plate-based assay can be extended to other substrates and might also be applied for fast and substrate-specific screening of other H2O2-producing oxidases in P. pastoris.

Convergent evolution of a blood-red nectar pigment in vertebrate-pollinated flowers

Nearly 90% of flowering plants depend on animals for reproduction. One of the main rewards plants offer to pollinators for visitation is nectar. Nesocodon mauritianus (Campanulaceae) produces a blood-red nectar that has been proposed to serve as a visual attractant for pollinator visitation. Here, we show that the nectar's red color is derived from a previously undescribed alkaloid termed nesocodin. The first nectar produced is acidic and pale yellow in color, but slowly becomes alkaline before taking on its characteristic red color. Three enzymes secreted into the nectar are either necessary or sufficient for pigment production, including a carbonic anhydrase that increases nectar pH, an aryl-alcohol oxidase that produces a pigment precursor, and a ferritin-like catalase that protects the pigment from degradation by hydrogen peroxide. Our findings demonstrate how these three enzymatic activities allow for the condensation of sinapaldehyde and proline to form a pigment with a stable imine bond. We subsequently verified that synthetic nesocodin is indeed attractive to Phelsuma geckos, the most likely pollinators of Nesocodon We also identify nesocodin in the red nectar of the distantly related and hummingbird-visited Jaltomata herrerae and provide molecular evidence for convergent evolution of this trait. This work cumulatively identifies a convergently evolved trait in two vertebrate-pollinated species, suggesting that the red pigment is selectively favored and that only a limited number of compounds are likely to underlie this type of adaptation.

Overview of Mycelial Fungi - Lignin Destructors

In this work, the following genuses of mycelial fungi, capable of producing ligninolytic enzymes of various actions, were considered:Penicillium, Aspergillus, Fusariumand Altermaria. Fungi of the genus Aspergilluswere capable of producing laccase, manganese peroxidase and lignin peroxidase in the medium. Penicillium mostly produced laccase. Fusariumproduced laccase, aryl alcohol oxidase, manganesedependent peroxidase, manganese-independent peroxidase and lignin peroxidase. Alternariaproduced laccase, lignin peroxidase and manganese peroxidase. The results demonstrated the possibility of using specific substrates in the study of enzyme activity, as well as the influence of some factors introduced into the medium on the synthesis of enzymes. The auxiliary influence of these fungi on the synthesis of ligninolytic enzymes in symbiosis with otherswas considered.
 Keywords: mycelial fungi, ligninolytic enzymes, Penicillium, Aspergillus, Fusarium, Altermaria

Characterization of a thermotolerant aryl-alcohol oxidase from Moesziomyces antarcticus oxidizing 5-hydroxymethyl-2-furancarboxylic acid

The development of enzymatic processes for the environmentally friendly production of 2,5-furandicarboxylic acid (FDCA), a renewable precursor for bioplastics, from 5-hydroxymethylfurfural (HMF) has gained increasing attention over the last years. Aryl-alcohol oxidases (AAOs) catalyze the oxidation of HMF to 5-formyl-2-furancarboxylic acid (FFCA) through 2,5-diformylfuran (DFF) and have thus been applied in enzymatic reaction cascades for the production of FDCA. AAOs are flavoproteins that oxidize a broad range of benzylic and aliphatic allylic primary alcohols to the corresponding aldehydes, and in some cases further to acids, while reducing molecular oxygen to hydrogen peroxide. These promising biocatalysts can also be used for the synthesis of flavors, fragrances, and chemical building blocks, but their industrial applicability suffers from low production yield in natural and heterologous hosts. Here we report on heterologous expression of a new aryl-alcohol oxidase, MaAAO, from Moesziomyces antarcticus at high yields in the methylotrophic yeast Pichia pastoris (recently reclassified as Komagataella phaffii). Fed-batch fermentation of recombinant P. pastoris yielded around 750 mg of active enzyme per liter of culture. Purified MaAAO was highly stable at pH 2–9 and exhibited high thermal stability with almost 95% residual activity after 48 h at 57.5 °C. MaAAO accepts a broad range of benzylic primary alcohols, aliphatic allylic alcohols, and furan derivatives like HMF as substrates and some oxidation products thereof like piperonal or perillaldehyde serve as building blocks for pharmaceuticals or show health-promoting effects. Besides this, MaAAO oxidized 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) to FFCA, which has not been shown for any other AAO so far. Combining MaAAO with an unspecific peroxygenase oxidizing HMFCA to FFCA in one pot resulted in complete conversion of HMF to FDCA within 144 h. MaAAO is thus a promising biocatalyst for the production of precursors for bioplastics and bioactive compounds.Key points• MaAAO from M. antarcticus was expressed in P. pastoris at 750 mg/l.• MaAAO oxidized 5-hydroxymethyl-2-furancarboxylic acid (HMFCA).• Complete conversion of HMF to 2,5-furandicarboxylic acid by combining MaAAO and UPO.

Two adjacent C-terminal mutations enable expression of aryl-alcohol oxidase from Pleurotus eryngii in Pichia pastoris

Fungal aryl-alcohol oxidases (AAOs) are attractive biocatalysts because they selectively oxidize a broad range of aromatic and aliphatic allylic primary alcohols while yielding hydrogen peroxide as the only by-product. However, their use is hampered by challenging and often unsuccessful heterologous expression. Production of PeAAO1 from Pleurotus eryngii ATCC 90787 in Pichia pastoris failed, while PeAAO2 from P. eryngii P34 with an amino acid identity of 99% was expressed at high yields. By successively introducing mutations in PeAAO1 to mimic the sequence of PeAAO2, the double mutant PeAAO1 ER with mutations K583E and Q584R was constructed, that was successfully expressed in P. pastoris. Functional expression was enhanced up to 155 U/l via further replacements D361N (variant NER) or V367A (variant AER). Fed-batch cultivation of recombinant P. pastoris yielded up to 116 mg/l of active variants. Glycosylated PeAAO1 variants demonstrated high stability and catalytic efficiencies similar to PeAAO2. Interestingly, P. pastoris expressing PeAAO1 variant ER contained roughly 13 gene copies but showed similar volumetric activity as NER and AER with one to two gene copies and four times lower mRNA levels. Additional H-bonds and salt bridges introduced by mutations K583E and Q584R might facilitate heterologous expression by enhanced protein folding.Key points• PeAAO1 not expressed in P. pastoris and PeAAO2 well-expressed in Pichia differ at 7 positions.• Expression of PeAAO1 in P. pastoris achieved through mutagenesis based on PeAAO2 sequence.• Combination of K583E and Q584R is essential for expression of PeAAO1 in P. pastoris.

Retracted and Republished from: "Substrate-Specific Differential Gene Expression and RNA Editing in the Brown Rot Fungus Fomitopsis pinicola"

Wood-decaying fungi tend to have characteristic substrate ranges that partly define their ecological niche. Fomitopsis pinicola is a brown rot species of Polyporales that is reported on 82 species of softwoods and 42 species of hardwoods. We analyzed gene expression levels of F. pinicola from submerged cultures with ground wood powder (sampled at 5 days) or solid wood wafers (sampled at 10 and 30 days), using aspen, pine, and spruce substrates (aspen was used only in submerged cultures). Fomitopsis pinicola expressed similar sets of wood-degrading enzymes typical of brown rot fungi across all culture conditions and time points. Nevertheless, differential gene expression was observed across all pairwise comparisons of substrates and time points. Genes exhibiting differential expression encode diverse enzymes with known or potential function in brown rot decay, including laccase, benzoquinone reductase, aryl alcohol oxidase, cytochrome P450s, and various glycoside hydrolases. Comparing transcriptomes from submerged cultures and wood wafers, we found that culture conditions had a greater impact on global expression profiles than substrate wood species. These findings highlight the need for standardization of culture conditions in studies of gene expression in wood-decaying fungi. IMPORTANCE All species of wood-decaying fungi occur on a characteristic range of substrates (host plants), which may be broad or narrow. Understanding the mechanisms that allow fungi to grow on particular substrates is important for both fungal ecology and applied uses of different feedstocks in industrial processes. We grew the wood-decaying polypore Fomitopsis pinicola on three different wood species—aspen, pine, and spruce—under various culture conditions. We found that F. pinicola is able to modify gene expression (transcription levels) across different substrate species and culture conditions. Many of the genes involved encode enzymes with known or predicted functions in wood decay. This study provides clues to how wood-decaying fungi may adjust their arsenal of decay enzymes to accommodate different host substrates.

Crystal structure and functional characterization of an oligosaccharide dehydrogenase from Pycnoporus cinnabarinus provides insights into fungal breakdown of lignocellulose

BackgroundFungal glucose dehydrogenases (GDHs) are FAD-dependent enzymes belonging to the glucose-methanol-choline oxidoreductase superfamily. These enzymes are classified in the “Auxiliary Activity” family 3 (AA3) of the Carbohydrate-Active enZymes database, and more specifically in subfamily AA3_2, that also includes the closely related flavoenzymes aryl-alcohol oxidase and glucose 1-oxidase. Based on sequence similarity to known fungal GDHs, an AA3_2 enzyme active on glucose was identified in the genome of Pycnoporus cinnabarinus, a model Basidiomycete able to completely degrade lignin.ResultsIn our work, substrate screening and functional characterization showed an unexpected preferential activity of this enzyme toward oligosaccharides containing a β(1→3) glycosidic bond, with the highest efficiency observed for the disaccharide laminaribiose. Despite its sequence similarity to GDHs, we defined a novel enzymatic activity, namely oligosaccharide dehydrogenase (ODH), for this enzyme. The crystallographic structures of ODH in the sugar-free form and in complex with glucose and laminaribiose unveiled a peculiar saccharide recognition mechanism which is not shared with previously characterized AA3 oxidoreductases and accounts for ODH preferential activity toward oligosaccharides. The sugar molecules in the active site of ODH are mainly stabilized through CH-π interactions with aromatic residues rather than through hydrogen bonds with highly conserved residues, as observed instead for the fungal glucose dehydrogenases and oxidases characterized to date. Finally, three sugar-binding sites were identified on ODH external surface, which were not previously observed and might be of importance in the physiological scenario.ConclusionsStructure–function analysis of ODH is consistent with its role as an auxiliary enzyme in lignocellulose degradation and unveils yet another enzymatic function within the AA3 family of the Carbohydrate-Active enZymes database. Our findings allow deciphering the molecular determinants of substrate binding and provide insight into the physiological role of ODH, opening new perspectives to exploit biodiversity for lignocellulose transformation into fuels and chemicals.

Exploring lignin depolymerization by a bi-enzyme system containing aryl alcohol oxidase and lignin peroxidase in aqueous biocompatible ionic liquids

Enzymatic depolymerization of lignin to produce low molecular weight products requires mild reaction conditions and exhibits higher selectivity compared to thermochemical lignin depolymerization. However, it remains challenging to depolymerize lignin enzymatically, partially due to the low solubility of lignin in aqueous phase. This study aimed to develop a novel approach to combine aqueous lignin extraction with enzymatic lignin depolymerization in biocompatible ionic liquids. A bi-enzyme system containing aryl alcohol oxidase (AAO) and lignin peroxidase (LiP) was developed to depolymerize lignin. Temperature and pH profiles for LiP and AAO were determined. Biocompatibilities of LiP and AAO in different deep eutectic solvents and ionic liquids were investigated. Aqueous cholinium glycinate was found to be an efficient and suitable solvent to solubilize lignin and serve as a biocompatible medium for enzymes to work. LiP and AAO together reduced lignin molecular weight in both solid and liquid phase after enzymatic lignin depolymerization.

Two Fusarium copper radical oxidases with high activity on aryl alcohols

BackgroundBiomass valorization has been suggested as a sustainable alternative to petroleum-based energy and commodities. In this context, the copper radical oxidases (CROs) from Auxiliary Activity Family 5/Subfamily 2 (AA5_2) are attractive biocatalysts for the selective oxidation of primary alcohols to aldehydes. Originally defined by the archetypal galactose 6-oxidase from Fusarium graminearum, fungal AA5_2 members have recently been shown to comprise a wide range of specificities for aromatic, aliphatic and furan-based alcohols. This suggests a broader substrate scope of native CROs for applications. However, only 10% of the annotated AA5_2 members have been characterized to date.ResultsHere, we define two homologues from the filamentous fungi Fusarium graminearum and F. oxysporum as predominant aryl alcohol oxidases (AAOs) through recombinant production in Pichia pastoris, detailed kinetic characterization, and enzyme product analysis. Despite possessing generally similar active-site architectures to the archetypal FgrGalOx, FgrAAO and FoxAAO have weak activity on carbohydrates, but instead efficiently oxidize specific aryl alcohols. Notably, both FgrAAO and FoxAAO oxidize hydroxymethyl furfural (HMF) directly to 5-formyl-2-furoic acid (FFCA), and desymmetrize the bioproduct glycerol to the uncommon L-isomer of glyceraldehyde.ConclusionsThis work expands understanding of the catalytic diversity of CRO from AA5_2 to include unique representatives from Fusarium species that depart from the well-known galactose 6-oxidase activity of this family. Detailed enzymological analysis highlights the potential biotechnological applications of these orthologs in the production of renewable plastic polymer precursors and other chemicals.

The Termite Fungal Cultivar Termitomyces Combines Diverse Enzymes and Oxidative Reactions for Plant Biomass Conversion.

ABSTRACTMacrotermitine termites have domesticated fungi in the genus Termitomyces as their primary food source using predigested plant biomass. To access the full nutritional value of lignin-enriched plant biomass, the termite-fungus symbiosis requires the depolymerization of this complex phenolic polymer. While most previous work suggests that lignocellulose degradation is accomplished predominantly by the fungal cultivar, our current understanding of the underlying biomolecular mechanisms remains rudimentary. Here, we provide conclusive omics and activity-based evidence that Termitomyces employs not only a broad array of carbohydrate-active enzymes (CAZymes) but also a restricted set of oxidizing enzymes (manganese peroxidase, dye decolorization peroxidase, an unspecific peroxygenase, laccases, and aryl-alcohol oxidases) and Fenton chemistry for biomass degradation. We propose for the first time that Termitomyces induces hydroquinone-mediated Fenton chemistry (Fe2+ + H2O2 + H+ → Fe3+ + •OH + H2O) using a herein newly described 2-methoxy-1,4-dihydroxybenzene (2-MH2Q, compound 19)-based electron shuttle system to complement the enzymatic degradation pathways. This study provides a comprehensive depiction of how efficient biomass degradation by means of this ancient insect’s agricultural symbiosis is accomplished.

A Multiomic Approach to Understand How Pleurotus eryngii Transforms Non-Woody Lignocellulosic Material.

Pleurotus eryngii is a grassland-inhabiting fungus of biotechnological interest due to its ability to colonize non-woody lignocellulosic material. Genomic, transcriptomic, exoproteomic, and metabolomic analyses were combined to explain the enzymatic aspects underlaying wheat–straw transformation. Up-regulated and constitutive glycoside–hydrolases, polysaccharide–lyases, and carbohydrate–esterases active on polysaccharides, laccases active on lignin, and a surprisingly high amount of constitutive/inducible aryl–alcohol oxidases (AAOs) constituted the suite of extracellular enzymes at early fungal growth. Higher enzyme diversity and abundance characterized the longer-term growth, with an array of oxidoreductases involved in depolymerization of both cellulose and lignin, which were often up-regulated since initial growth. These oxidative enzymes included lytic polysaccharide monooxygenases (LPMOs) acting on crystalline polysaccharides, cellobiose dehydrogenase involved in LPMO activation, and ligninolytic peroxidases (mainly manganese-oxidizing peroxidases), together with highly abundant H2O2-producing AAOs. Interestingly, some of the most relevant enzymes acting on polysaccharides were appended to a cellulose-binding module. This is potentially related to the non-woody habitat of P. eryngii (in contrast to the wood habitat of many basidiomycetes). Additionally, insights into the intracellular catabolism of aromatic compounds, which is a neglected area of study in lignin degradation by basidiomycetes, were also provided. The multiomic approach reveals that although non-woody decay does not result in dramatic modifications, as revealed by detailed 2D-NMR and other analyses, it implies activation of the complete set of hydrolytic and oxidative enzymes characterizing lignocellulose-decaying basidiomycetes.

Phenolic acid-degrading Paraburkholderia prime decomposition in forest soil

Plant-derived phenolic acids are catabolized by soil microorganisms whose activity may enhance the decomposition of soil organic carbon (SOC). We characterized whether phenolic acid-degrading bacteria enhance SOC mineralization in forest soils when primed with 13C-labeled p-hydroxybenzoic acid (pHB). We further tested whether pHB-induced priming could explain differences in SOC content among mono-specific tree plantations in a 70-year-old common garden experiment. pHB addition primed significant losses of SOC (3–13 µmols C g−1 dry wt soil over 7 days) compared to glucose, which reduced mineralization (-3 to -8 µmols C g−1 dry wt soil over 7 days). The principal degraders of pHB were Paraburkholderia and Caballeronia in all plantations regardless of tree species or soil type, with one predominant phylotype (RP11ASV) enriched 23-fold following peak pHB respiration. We isolated and confirmed the phenolic degrading activity of a strain matching this phylotype (RP11T), which encoded numerous oxidative enzymes, including secretion signal-bearing laccase, Dyp-type peroxidase and aryl-alcohol oxidase. Increased relative abundance of RP11ASV corresponded with higher pHB respiration and expression of pHB monooxygenase (pobA), which was inversely proportional to SOC content among plantations. pobA expression proved a responsive measure of priming activity. We found that stimulating phenolic-acid degrading bacteria can prime decomposition and that this activity, corresponding with differences in tree species, is a potential mechanism in SOC cycling in forests. Overall, this study highlights the ecology and function of Paraburkholderia whose associations with plant roots and capacity to degrade phenolics suggest a role for specialized bacteria in the priming effect.

Evolved Peroxygenase–Aryl Alcohol Oxidase Fusions for Self-Sufficient Oxyfunctionalization Reactions

Fungal peroxygenases are deemed emergent biocatalysts for selective C-H bond oxyfunctionalization reactions. In this study, we have engineered a functional and stable self-sufficient chimeric peroxygenase-oxidase fusion. The bifunctional biocatalyst carried a laboratory-evolved version of the fungal peroxygenase fused to an evolved fungal aryl-alcohol oxidase that supplies H2O2 in situ. Enzyme fusion libraries with peptide linkers of different sizes and amino acid compositions were designed, while attached leader sequences favored secretion in yeast. The most promising functional enzyme fusions were characterized biochemically and further tested for the synthesis of dextrorphan, a metabolite of the antitussive drug dextromethorphan. This reaction system was optimized to control the aromatic alcohol transformation rate, and therefore the H2O2 supply, to achieve total turnover numbers of 62,000, the highest value reported for the biocatalytic synthesis of dextrorphan to date. Accordingly, our study opens an avenue for the use of peroxygenase-aryl alcohol oxidase fusions in the pharmaceutical and chemical sectors.

High-level expression of aryl-alcohol oxidase 2 from Pleurotus eryngii in Pichia pastoris for production of fragrances and bioactive precursors

The fungal secretome comprises various oxidative enzymes participating in the degradation of lignocellulosic biomass as a central step in carbon recycling. Among the secreted enzymes, aryl-alcohol oxidases (AAOs) are of interest for biotechnological applications including production of bio-based precursors for plastics, bioactive compounds, and flavors and fragrances. Aryl-alcohol oxidase 2 (PeAAO2) from the fungus Pleurotus eryngii was heterologously expressed and secreted at one of the highest yields reported so far of 315 mg/l using the methylotrophic yeast Pichia pastoris (recently reclassified as Komagataella phaffii). The glycosylated PeAAO2 exhibited a high stability in a broad pH range between pH 3.0 and 9.0 and high thermal stability up to 55 °C. Substrate screening with 41 compounds revealed that PeAAO2 oxidized typical AAO substrates like p-anisyl alcohol, veratryl alcohol, and trans,trans-2,4-hexadienol with up to 8-fold higher activity than benzyl alcohol. Several compounds not yet reported as substrates for AAOs were oxidized by PeAAO2 as well. Among them, cumic alcohol and piperonyl alcohol were oxidized to cuminaldehyde and piperonal with high catalytic efficiencies of 84.1 and 600.2 mM−1 s−1, respectively. While the fragrance and flavor compound piperonal also serves as starting material for agrochemical and pharmaceutical building blocks, various positive health effects have been attributed to cuminaldehyde including anticancer, antidiabetic, and neuroprotective effects. PeAAO2 is thus a promising biocatalyst for biotechnological applications.Key points• Aryl-alcohol oxidase PeAAO2 from P. eryngii was produced in P. pastoris at 315 mg/l.• Purified enzyme exhibited stability over a broad pH and temperature range.• Oxidation products cuminaldehyde and piperonal are of biotechnological interest.Graphical abstract

Aspergillus nidulans: A Potential Resource of the Production of the Native and Heterologous Enzymes for Industrial Applications.

Aspergillus nidulans is a filamentous fungus that is a potential resource for industrial enzymes. It is a versatile fungal cell factory that can synthesize various industrial enzymes such as cellulases, β-glucosidases, hemicellulases, laccases, lipases, proteases, β-galactosidases, tannases, keratinase, cutinases, and aryl alcohol oxidase. A. nidulans has shown the potential to utilize low-cost substrates such as wheat bran, rice straw, sugarcane bagasse, rice bran, coir pith, black gram residue, and chicken feathers to produce enzymes cost-effectively. A. nidulans has also been known as a model organism for the production of heterologous enzymes. Several studies reported genetically engineered strains of A. nidulans for the production of different enzymes. Native as well as heterologous enzymes of A. nidulans have been employed for various industrial processes.

Enzymatic versatility and thermostability of a new aryl-alcohol oxidase from Thermothelomyces thermophilus M77

BackgroundFungal aryl-alcohol oxidases (AAOx) are extracellular flavoenzymes that belong to glucose-methanol-choline oxidoreductase family and are responsible for the selective conversion of primary aromatic alcohols into aldehydes and aromatic aldehydes to their corresponding acids, with concomitant production of hydrogen peroxide (H2O2) as by-product. The H2O2 can be provided to lignin degradation pathway, a biotechnological property explored in biofuel production. In the thermophilic fungus Thermothelomyces thermophilus (formerly Myceliophthora thermophila), just one AAOx was identified in the exo-proteome.MethodsThe glycosylated and non-refolded crystal structure of an AAOx from T. thermophilus at 2.6 Å resolution was elucidated by X-ray crystallography combined with small-angle X-ray scattering (SAXS) studies. Moreover, biochemical analyses were carried out to shed light on enzyme substrate specificity and thermostability.ResultsThis flavoenzyme harbors a flavin adenine dinucleotide as a cofactor and is able to oxidize aromatic substrates and 5-HMF. Our results also show that the enzyme has similar oxidation rates for bulky or simple aromatic substrates such as cinnamyl and veratryl alcohols. Moreover, the crystal structure of MtAAOx reveals an open active site, which might explain observed specificity of the enzyme.ConclusionsMtAAOx shows previously undescribed structural differences such as a fully accessible catalytic tunnel, heavy glycosylation and Ca2+ binding site providing evidences for thermostability and activity of the enzymes from AA3_2 subfamily.General significanceStructural and biochemical analyses of MtAAOx could be important for comprehension of aryl-alcohol oxidases structure-function relationships and provide additional molecular tools to be used in future biotechnological applications.

Process optimization and scale-up production of fungal aryl alcohol oxidase from genetically modified Aspergillus nidulans in stirred-tank bioreactor

Microbial production of aryl alcohol oxidase (AAO) has attracted increasing attention due to the central role of AAO in enzymatic lignin depolymerization. However, large-scale production of AAO has not been reached because of the low yield and inefficient fermentation process. This study aims to optimize the process parameters and scale-up production of AAO using Aspergillus nidulans in a stirred-tank bioreactor. Effects of pH and dissolved oxygen on AAO production at bioreactor scale were particularly investigated. Results revealed that pH control significantly affected protein production and increasing dissolved oxygen level stimulated AAO production. The greatest AAO activity (1906 U/L) and protein concentration (1.19 g/L) were achieved in 48 h at 60% dissolved oxygen with pH controlled at 6.0. The yield and productivity (in 48 h) were 31.2 U/g maltose and 39.7 U/L/h, respectively. In addition, crude AAO was concentrated and partially purified by ultrafiltration and verified by protein identification.

Novel Routes in Transformation of Lignocellulosic Biomass to Furan Platform Chemicals: From Pretreatment to Enzyme Catalysis

The constant depletion of fossil fuels along with the increasing need for novel materials, necessitate the development of alternative routes for polymer synthesis. Lignocellulosic biomass, the most abundant carbon source on the planet, can serve as a renewable starting material for the design of environmentally-friendly processes for the synthesis of polyesters, polyamides and other polymers with significant value. The present review provides an overview of the main processes that have been reported throughout the literature for the production of bio-based monomers from lignocellulose, focusing on physicochemical procedures and biocatalysis. An extensive description of all different stages for the production of furans is presented, starting from physicochemical pretreatment of biomass and biocatalytic decomposition to monomeric sugars, coupled with isomerization by enzymes prior to chemical dehydration by acid Lewis catalysts. A summary of all biotransformations of furans carried out by enzymes is also described, focusing on galactose, glyoxal and aryl-alcohol oxidases, monooxygenases and transaminases for the production of oxidized derivatives and amines. The increased interest in these products in polymer chemistry can lead to a redirection of biomass valorization from second generation biofuels to chemical synthesis, by creating novel pathways to produce bio-based polymers.