Aryl-alcohol Oxidase Research Articles

Unveiling the kinetic versatility of aryl-alcohol oxidases with different electron acceptors.

Introduction: Aryl-alcohol oxidase (AAO) shows a pronounced duality as oxidase and dehydrogenase similar to that described for other glucose-methanol-choline (GMC) oxidase/dehydrogenase superfamily proteins involved in lignocellulose decomposition. In this work, we detail the overall mechanism of AAOs from Pleurotus eryngii and Bjerkandera adusta for catalyzing the oxidation of natural aryl-alcohol substrates using either oxygen or quinones as electron acceptors and describe the crystallographic structure of AAO from B. adusta in complex with a product analogue. Methods: Kinetic studies with 4-methoxybenzyl and 3-chloro-4- methoxybenzyl alcohols, including both transient-state and steady-state analyses, along with interaction studies, provide insight into the oxidase and dehydrogenase mechanisms of these enzymes. Moreover, the resolution of the crystal structure of AAO from B. adusta allowed us to compare their overall folding and the structure of the active sites of both AAOs in relation to their activities. Results and Discussion: Although both enzymes show similar mechanistic properties, notable differences are highlighted in this study. In B. adusta, the AAO oxidase activity is limited by the reoxidation of the flavin, while in P. eryngii the slower step takes place during the reductive half-reaction, which determines the overall reaction rate. By contrast, dehydrogenase activity in both enzymes, irrespective of the alcohol participating in the reaction, is limited by the hydroquinone release from the active site. Despite these differences, both AAOs are more efficient as dehydrogenases, supporting the physiological role of this activity in lignocellulosic decay. This dual activity would allow these enzymes to adapt to different environments based on the available electron acceptors.

Exploring the potential of insect gut symbionts for polyethylene biodegradation

The global issue of synthetic plastic pollution is escalating, necessitating innovative bioremediation approaches. Specific insect species have developed symbiotic associations with intestinal microorganisms that possess the ability to degrade resistant plant materials, such as lignocellulose. This is the first study to our knowledge to isolate, characterize, and compare microbial isolates (yeast and bacteria) extracted from the guts of wood-feeding termites (WFT) and lesser waxworms (LWW). The objective was to determine their efficacy in decomposing recalcitrant pollutants, such as low-density polyethylene (LDPE). The isolates belonged to numerous taxonomic groups, as determined by molecular identification; these groups included Bacillus, Citrobacter, Cellulosimicrobium, Rhodococcus, Pseudomonas, Candida, and others. A number of isolates had the potential to degrade LDPE. After 45 days of incubation, the polymer sheets lost 19.8 % of their weight for Bacillus cereus (LDPE-DB2) and 15.4 % of their weight for Candida guilliermondii (LDPE-DY6). Additionally, LDPE degraded by LDPE-DY6 showed a tensile strength loss of 50.3 % compared to that degraded by LDPE-DB2 (58.3 %). During this time, robust biofilm formation was observed on the surfaces of the polymers. Extracellular enzymes such as cutinases, aryl alcohol oxidases, and peroxidases, secreted by specific yeast and bacterial isolates, contribute to the degradation of plastic and recalcitrant biopolymers. Notably, the isolates from LWW demonstrated a greater advantage in this regard than those from WFT. Bacillus cereus, Pseudomonas aeruginosa, Candida guilliermondii, and Sterigmatomyces halophilus were found to have the highest activities in degrading LDPE among the studied WFT and LWW gut-derived symbionts. This study paves the way for a new opportunity to enhance polyethylene degradation by insect gut bacteria and yeasts that may facilitate biotechnological applications for plastic waste remediation.

Expansion of Auxiliary Activity Family 5 sequence space via biochemical characterization of six new copper radical oxidases.

Bacterial and fungal copper radical oxidases (CROs) from Auxiliary Activity Family 5 (AA5) are implicated in morphogenesis and pathogenesis. The unique catalytic properties of CROs also make these enzymes attractive biocatalysts for the transformation of small molecules and biopolymers. Despite a recent increase in the number of characterized AA5 members, especially from subfamily 2 (AA5_2), the catalytic diversity of the family as a whole remains underexplored. In the present study, phylogenetic analysis guided the selection of six AA5_2 members from diverse fungi for recombinant expression in Komagataella pfaffii (syn. Pichia pastoris) and biochemical characterization in vitro. Five of the targets displayed predominant galactose 6-oxidase activity (EC 1.1.3.9), and one was a broad-specificity aryl alcohol oxidase (EC 1.1.3.7) with maximum activity on the platform chemical 5-hydroxymethyl furfural (EC 1.1.3.47). Sequence alignment comparing previously characterized AA5_2 members to those from this study indicated various amino acid substitutions at active site positions implicated in the modulation of specificity.IMPORTANCEEnzyme discovery and characterization underpin advances in microbial biology and the application of biocatalysts in industrial processes. On one hand, oxidative processes are central to fungal saprotrophy and pathogenesis. On the other hand, controlled oxidation of small molecules and (bio)polymers valorizes these compounds and introduces versatile functional groups for further modification. The biochemical characterization of six new copper radical oxidases further illuminates the catalytic diversity of these enzymes, which will inform future biological studies and biotechnological applications.

Combined use of versatile peroxidase and aryl alcohol oxidase of Pleurotus eryngii to decolorize melanin on the skin

Melanin plays an important role in protecting skin cells from harmful UV radiation, but its uneven pigmentation in the skin sometimes demands cosmetic resolution. As a safe and effective way of evening the skin tone, enzymatic decolorization of melanin at the stratum corneum has been proposed. In this regard, the use of lignin peroxidase, in the presence of hydrogen peroxide and veratryl alcohol, an electron mediator, has been explored. Here, we first show that versatile peroxidase (VP) and aryl alcohol oxidase (AAO) purified from the Pleurotus eryngii liquid culture can be used to decolorize melanin without exogenous hydrogen peroxide. This resembles the oxidative degradation pathway of lignin in nature; AAO generates hydrogen peroxide from veratryl alcohol, and VP utilizes generated hydrogen peroxide while using veratryl alcohol as a mediator. We further explored the use of POX_Pe, the crude peroxidase preparation of the Pleurotus eryngii liquid culture, for melanin decolorization because of its better stability. Using POX_Pe and veratryl alcohol, over 60 % of melanin decolorization was obtained in 1 h in the absence of exogenous hydrogen peroxide addition. Furthermore, POX_Pe could decolorize melanin in a 3D human pigmented epidermis model, demonstrating its possible applications in cosmetics.

Substrate specificity mapping of fungal CAZy AA3_2 oxidoreductases

BackgroundOxidative enzymes targeting lignocellulosic substrates are presently classified into various auxiliary activity (AA) families within the carbohydrate-active enzyme (CAZy) database. Among these, the fungal AA3 glucose–methanol–choline (GMC) oxidoreductases with varying auxiliary activities are attractive sustainable biocatalysts and important for biological function. CAZy AA3 enzymes are further subdivided into four subfamilies, with the large AA3_2 subfamily displaying diverse substrate specificities. However, limited numbers of enzymes in the AA3_2 subfamily are currently biochemically characterized, which limits the homology-based mining of new AA3_2 oxidoreductases. Importantly, novel enzyme activities may be discovered from the uncharacterized parts of this large subfamily.ResultsIn this study, phylogenetic analyses employing a sequence similarity network (SSN) and maximum likelihood trees were used to cluster AA3_2 sequences. A total of 27 AA3_2 proteins representing different clusters were selected for recombinant production. Among them, seven new AA3_2 oxidoreductases were successfully produced, purified, and characterized. These enzymes included two glucose dehydrogenases (TaGdhA and McGdhA), one glucose oxidase (ApGoxA), one aryl alcohol oxidase (PsAaoA), two aryl alcohol dehydrogenases (AsAadhA and AsAadhB), and one novel oligosaccharide (gentiobiose) dehydrogenase (KiOdhA). Notably, two dehydrogenases (TaGdhA and KiOdhA) were found with the ability to utilize phenoxy radicals as an electron acceptor. Interestingly, phenoxy radicals were found to compete with molecular oxygen in aerobic environments when serving as an electron acceptor for two oxidases (ApGoxA and PsAaoA), which sheds light on their versatility. Furthermore, the molecular determinants governing their diverse enzymatic functions were discussed based on the homology model generated by AlphaFold.ConclusionsThe phylogenetic analyses and biochemical characterization of AA3_2s provide valuable guidance for future investigation of AA3_2 sequences and proteins. A clear correlation between enzymatic function and SSN clustering was observed. The discovery and biochemical characterization of these new AA3_2 oxidoreductases brings exciting prospects for biotechnological applications and broadens our understanding of their biological functions.

Identification of redox activators for continuous reactivation of glyoxal oxidase from Trametes versicolor in a two-enzyme reaction cascade

Glyoxal oxidases, belonging to the group of copper radical oxidases (CROs), oxidize aldehydes to carboxylic acids, while reducing O2 to H2O2. Their activity on furan derivatives like 5-hydroxymethylfurfural (HMF) makes these enzymes promising biocatalysts for the environmentally friendly synthesis of the bioplastics precursor 2,5-furandicarboxylic acid (FDCA). However, glyoxal oxidases suffer from inactivation, which requires the identification of suitable redox activators for efficient substrate conversion. Furthermore, only a few glyoxal oxidases have been expressed and characterized so far. Here, we report on a new glyoxal oxidase from Trametes versicolor (TvGLOX) that was expressed at high levels in Pichia pastoris (reclassified as Komagataella phaffii). TvGLOX was found to catalyze the oxidation of aldehyde groups in glyoxylic acid, methyl glyoxal, HMF, 2,5-diformylfuran (DFF) and 5-formyl-2-furancarboxylic acid (FFCA), but barely accepted alcohol groups as in 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), preventing formation of FDCA from HMF. Various redox activators were tested for TvGLOX reactivation during catalyzed reactions. Among them, a combination of horseradish peroxidase and its substrate 2,2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) most efficiently reactivated TvGLOX. Through continuous reactivation of TvGLOX in a two-enzyme system employing a recombinant Moesziomyces antarcticus aryl-alcohol oxidase (MaAAO) almost complete conversion of 8 mM HMF to FDCA was achieved within 24 h.

Label-free comparative proteomic analysis of Pleurotus eryngii grown on sawdust, bagasse, and peanut shell substrates

The white rot fungi Pleurotus eryngii are environmental microorganisms that can effectively break down lignocellulosic biomass. However, understanding of the mechanisms by which P. eryngii is effective in degrading lignocellulose is still limited. This work aimed to examine the extracellular secretory proteins implicated in the breakdown of lignocellulose in P. eryngii and identify degradation tactics across various cultivation substrates. Thus, a comparative analysis of the secretory proteins based on Nanoliquid chromatography combined with tandem mass spectrometry was conducted among P. eryngii cultivated on sawdusts, bagasse, peanut shells, and glucose. In total, 647, 616, 604, and 511 proteins were identified from the four samples, respectively. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis of protein expression differences identified pathways (hydrolytic enzymes, catalytic activity, metabolic processes, cellular processes, and response to stimuli) significantly enriched in proteins associated with lignocellulose degradation in P. eryngii. An integrated analysis of proteome data revealed specifically or differentially expressed genes secreted by P. eryngii in different cultivation substrates. The most prevalent carbohydrate-active enzymes involved in lignocellulose degradation in the secretome of the four samples were laccase (Lac), manganese peroxidase (MnP), aryl alcohol oxidase (AaO), and copper radical oxidase (CRO). Among them, Lac 2 mainly involved in the lignin degradation of sawdust peanut shells, and bagasse by P. eryngii, and Mnp 3 was mainly involved in the degradation of peanut shells. AaO and Lac 4 were mainly involved in glucose substrate defense and oxidative stress. It was found that exogenous addition of sawdust and peanut shells significantly increased lignolytic enzyme abundance. These findings provide insight and guidance for improving agricultural waste resource recovery.In this study, the secretomes of P. eryngii grown on four different carbon sources were compared. The findings revealed the extracellular enzymes implicated in the degradation of lignocellulose, offering avenues for further investigation into the biotransformation mechanisms of P. eryngii biomass and the potential utilization of agricultural wastes. SignificanceThe cost of the substrate for mushroom cultivation has increased as the production of edible fungus has risen year after year. Therefore, the use of these locally available lignocellulosic wastes as substrates offers a cost-cutting option.Further, the overuse of wood for the cultivation of edible mushrooms is also detrimental to the conservation of forest resources or the ecological environment.Consequently, the use of other agricultural wastes as an alternative to sawdust or other woody substrates is a viable approach for cultivating P. eryngii.The distribution of extracellular lignocellulosic degrading enzymes, inferred in the present study could help improve the cultivation efficiency of P. eryngii vis-à-vis managing agricultural waste.

Production of high-value oxidative enzymes by Cyathus bulleri on agricultural and agri-food wastes for application in the textile sector.

Ligninolytic and other oxidative enzymes have emerged as promising biocatalysts in several industries. Since their production at a low cost is necessary for any large-scale application, we demonstrate the use of rice bran (RB), an agricultural waste and agri-food wastes such as potato peelings (PP), banana peelings (BP), and green pea peelings (GPP) for their production. High activity of laccase (12 U/ml), manganese peroxidase (16.11 ± 1.43 U/ml), and aryl alcohol oxidase (1.25 U/ml) was obtained on the PP on the 12th day of growth and ~ 6 U/ml of lytic polysaccharide monooxygenase was obtained on the 14th day of growth demonstrating PP to be a good substrate for their production. RB served as the next best substrate for the production of these enzymes. While the GPP was effective for the production of laccase (9.2 U/ml), this and the BP were not good substrates for the production of other enzymes. Efficient (48-82%) decolorization of several azo-, triarylmethane- dyes, and real textile effluent, without the addition of any mediator, demonstrated the high oxidative ability of the crude culture filtrate produced on the PP (CF-PP), which was a significant improvement compared to the treatment given by the previously reported culture filtrate obtained on wheat bran (CF-WB). An extensive breakdown of Reactive Orange (RO) 16 was demonstrated using CF-PP resulting in the formation of a new product at m/z of 294.05 (6-acetamido-3,4-dioxo-3,4-dihydronapthalene-2-sulfonate), previously reported to be produced on ozonation/advanced oxidation of RO16. The predominant laccase and manganese peroxidase isoforms produced on the PP were also identified.

Cellulose degradation by lytic polysaccharide monooxygenase fueled by an aryl-alcohol oxidase

Cellulose degradation by lytic polysaccharide monooxygenase fueled by an aryl-alcohol oxidase

Fungal lyophilisates as catalysts for organic synthesis: Preparative oxidations with the white-rot fungus Bjerkandera adusta

A clean and selective “on-water-on-air” oxidation of primary alcohols to the corresponding aldehydes with mycelium lyophilisates of the non-toxic white-rot fungus Bjerkandera adusta was developed. The biocatalytic protocol is simple and the catalyst is readily accessible as a powder, which could be stored at least for 7 months without changes of activity. The catalytic activity of the fungus is associated with the presence of aryl-alcohol oxidase, that was isolated from the lyophilisate and characterised. Remarkably, a dichotomy in the fungal reactivity was observed, as the corresponding growing cultures catalyse the reverse process, selectively reducing aldehydes to the corresponding alcohols. Enzymatic oxidations by B. adusta are an excellent non-toxic alternative to metal-mediated oxidations in the lab with potential in food, beverage, cosmetic, and pharmaceutical industries, as it tolerates many functional groups.

Structure elucidation and characterization of patulin synthase, insights into the formation of a fungal mycotoxin.

Patulin synthase (PatE) from Penicillium expansum is a flavin-dependent enzyme that catalyses the last step in the biosynthesis of the mycotoxin patulin. This secondary metabolite is often present in fruit and fruit-derived products, causing postharvest losses. The patE gene was expressed in Aspergillus niger allowing purification and characterization of PatE. Thisconfirmed that PatE is active not only on the proposed patulin precursorascladiol but also on several aromatic alcohols including 5-hydroxymethylfurfural. By elucidating its crystal structure, details on its catalytic mechanism were revealed. Several aspects of the active site architecture are reminiscent of that of fungal aryl-alcohol oxidases. Yet, PatE is most efficient with ascladiol as substrate confirming its dedicated role in biosynthesis of patulin.

The secretome of Agaricus bisporus: Temporal dynamics of plant polysaccharides and lignin degradation

Despite substantial lignocellulose conversion during mycelial growth, previous transcriptome and proteome studies have not yet revealed how secretomes from the edible mushroom Agaricus bisporus develop and whether they modify lignin models invitro. To clarify these aspects, A.bisporus secretomes collected throughout a 15-day industrial substrate production and from axenic lab-cultures were subjected to proteomics, and tested on polysaccharides and lignin models. Secretomes (day 6-15) comprised A.bisporus endo-acting and substituent-removing glycoside hydrolases, whereas β-xylosidase and glucosidase activities gradually decreased. Laccases appeared from day 6 onwards. From day 10 onwards, many oxidoreductases were found, with numerous multicopper oxidases (MCO), aryl alcohol oxidases (AAO), glyoxal oxidases (GLOX), a manganese peroxidase (MnP), and unspecific peroxygenases (UPO). Secretomes modified dimeric lignin models, thereby catalyzing syringylglycerol-β-guaiacyl ether (SBG) cleavage, guaiacylglycerol-β-guaiacyl ether (GBG) polymerization, and non-phenolic veratrylglycerol-β-guaiacyl ether (VBG) oxidation. We explored A.bisporus secretomes and insights obtained can help to better understand biomass valorization.

Eco-friendly detoxification of hazardous Congo red dye using novel fungal strain Trametes flavida WTFP2: Deduced enzymatic biomineralization process through combinatorial in-silico and in-vitro studies

Growing textile industry is a major global concern, owing to the presence of recalcitrant hazardous pollutants, like synthetic dyes in discharged effluents. To explore new bioresources for mycoremediation, a high laccase-producing novel white-rot fungus (WRF), Trametes flavida WTFP2, was employed. T. flavida is an underexplored member of Polyporales. Using bioinformatic tools, 8 different cis-acting RNA elements were identified in the 5.8 S ITS gene sequence, where CRISPR (CRISPR-DR15), sRNA (RUF1), and snoRNA (ceN111) are uniquely present. Molecular docking was adopted to predict the catalytic interaction of chosen toxic diazo colorant, Congo red (CR), with four dye-degrading enzymes (laccase, lignin peroxidase, azoreductase, and aryl alcohol oxidase). With 376.41 × 103 U/L laccase production, novel WRF exhibited dye-decolorization potential. WTFP2 effectively removed 99.48 ± 0.04% CR (100 mg/L) and demonstrated remarkable recyclability and persistence in consecutive remediation trials. Mycelial dye adsorption was not only substantial driver of colorant elimination; decolorization using active T. flavida was regulated by enzymatic catalysis, as outlined by in-vitro growth, induction of extracellular enzymes, and FESEM. Fifteen metabolites were identified using HRLCMS-QTOF, and novel CR degradation pathway was proposed. Furthermore, microbial and phyto-toxicity tests of metabolites suggested complete detoxification of toxic dye, making the process clean, green, and economically sustainable.

Structure-Based Redesign of a Methanol Oxidase into an "Aryl Alcohol Oxidase" for Enzymatic Synthesis of Aromatic Flavor Compounds.

Alcohol oxidases (AOxs) catalyze the aerobic oxidation of alcohols to the corresponding carbonyl products (aldehydes or ketones), producing only H2O2 as the byproduct. The majority of known AOxs, however, have a strong preference for small, primary alcohols, limiting their broad applicability, e.g., in the food industry. To broaden the product scope of AOxs, we performed structure-guided enzyme engineering of a methanol oxidase from Phanerochaete chrysosporium (PcAOx). The substrate preference was extended from methanol to a broad range of benzylic alcohols by modifying the substrate binding pocket. A mutant (PcAOx-EFMH) with four substitutions exhibited improved catalytic activity toward benzyl alcohols with increased conversion and kcat toward the benzyl alcohol from 11.3 to 88.9% and from 0.5 to 2.6 s-1, respectively. The molecular basis for the change of substrate selectivity was analyzed by molecular simulation.

Bienzymatic Cascade Combining a Peroxygenase with an Oxidase for the Synthesis of Aromatic Aldehydes from Benzyl Alcohols

Aromatic aldehydes are important aromatic compounds for the flavour and fragrance industry. In this study, a parallel cascade combining aryl alcohol oxidase from Pleurotus eryngii (PeAAOx) and unspecific peroxygenase from the basidiomycete Agrocybe aegerita (AaeUPO) to convert aromatic primary alcohols into high-value aromatic aldehydes is proposed. Key influencing factors in the process of enzyme cascade catalysis, such as enzyme dosage, pH and temperature, were investigated. The universality of PeAAOx coupled with AaeUPO cascade catalysis for the synthesis of aromatic aldehyde flavour compounds from aromatic primary alcohols was evaluated. In a partially optimised system (comprising 30 μM PeAAOx, 2 μM AaeUPO at pH 7 and 40 °C) up to 84% conversion of 50 mM veratryl alcohol into veratryl aldehyde was achieved in a self-sufficient aerobic reaction. Promising turnover numbers of 2800 and 21,000 for PeAAOx and AaeUPO, respectively, point towards practical applicability.

Complete oxidation of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid by a novel enzyme–nanozyme hybrid catalyst

• A robust and efficient nanocatalyst for the cascade oxidation of HMF to FDCA. • Latent activity of AAO on FFCA was activated by the designed nanozyme. • Novel oxidizability of HP-7@Fe 3 (PO 4 ) 2 nanozyme on FFCA was discovered. • A complete conversion of HMF with excellent (100%) selectivity toward FDCA. 2,5-Furandicarboxylic acid (FDCA) is a well-known biobased platform chemical to produce sustainable polyethylene furanoate (PEF) and other valuable furanic chemicals. The eco-friendly enzyme cascade reactions have been adopted to synthesize FDCA using the multistep oxidation of biomass-derived 5-hydroxymethylfurfural (HMF) based on different oxidoreductases. However, the coordination of natural oxidoreductases in cascade reactions presents challenges because of their different adaptabilities and catalytic environments. A robust and efficient catalyst herein was developed by the hybridization of a designed hemin–peptide (HP-7) complex and natural aryl-alcohol oxidase (AAO) in iron phosphate solution to achieve the cascade oxidation of HMF to FDCA. HMF is first transformed in 5-formylfurancarboxylic acid (FFCA) based on the oxidizability of AAO toward benzylic alcohol, and then the latent activity of AAO on FFCA was activated by the catalase activity of the HP-7@Fe 3 (PO 4 ) 2 nanozyme with the in situ consumption of H 2 O 2 initially produced by AAO. Moreover, closer inspection demonstrated the oxidizability of the HP-7@Fe 3 (PO 4 ) 2 nanozyme on FFCA in both the presence and absence of H 2 O 2 for the first time. Consequently, the hybrid catalyst AAO/HP-7@Fe 3 (PO 4 ) 2 enabled the self-activated and efficient oxidation of HMF by integrating multiple reaction processes. A complete conversion of HMF with excellent selectivity (100%) toward FDCA was finally observed.

Metagenomic and proteomic analysis of bacterial retting community and proteome profile in the degumming process of kenaf bast

BackgroundData on the microbial community and functional proteins associated with degumming in kenaf remains scant. Here, we analyzed the microbial communities associated with kenaf (Hibiscus cannabinus) bast fibers during retting to identify potential candidate degumming bacteria. Retting liquids were collected and analyzed at 0 days, 10 days, and 34 days and then evaluated the yield and quality of kenaf fiber at the different retting times. Besides, the microbial communities were characterized using metagenomic and proteomic analysis by LC–MS/MS technology.ResultsThe data showed that increase in the retting time significantly improves the softness, dispersion, and fiber whiteness of the kenaf fiber. The relative abundance of Acinetobacter increased from 2.88% at the baseline to 6.64% at the 34th retting. On the other hand, some members of Clostridium were reduced from 3% at the baseline to 2% at the 34th retting. Analysis of carbohydrate active enzymes showed constant changes in the utilization of carbohydrates. Besides, benzoquinone reductase, cellobiose dehydrogenase, glucose 1-oxidase, aryl alcohol oxidase and alcohol oxidase were the top five most abundant enzymes in the retting liquids. This present results demonstrated that the expressions of B7GYR8, Q6RYW5 and Q6FFK2 proteins were suppressed in Acinetobacter with the retting time. On the contrary, P05149 was upregulated with the retting time. In Clostridium, P37698, P52040 and P54937 proteins were upregulated with the retting time.ConclusionIn addition, bacteria Acinetobacter and Clostridium might be playing important roles in the kenaf degumming process. Similarly, up-regulation of P37698, P52040 and P54937 proteins is an important manifestation and mediates important roles in the degumming process.

Active-Site Engineering Switches Carbohydrate Regiospecificity in a Fungal Copper Radical Oxidase.

Copper radical oxidases (CROs) from Auxiliary Activity Family 5, Subfamily 2 (AA5_2), are organic cofactor-free biocatalysts for the selective oxidation of alcohols to the corresponding aldehydes. AA5_2 CROs comprise canonical galactose-6-oxidases as well as the more recently discovered general alcohol oxidases and aryl alcohol oxidases. Guided by primary and tertiary protein structural analyses, we targeted a distinct extended loop in the active site of a Colletotrichum graminicola aryl alcohol oxidase (CgrAAO) to explore its effect on catalysis in the broader context of AA5_2. Deletion of this loop, which is bracketed by a conserved disulfide bridge, significantly reduced the inherent activity of the enzyme toward extended galacto-oligosaccharides, as anticipated from molecular modeling. Unexpectedly, kinetic and product analysis on a range of monosaccharides and disaccharides revealed that an altered carbohydrate specificity in CgrAAO-Δloop was accompanied by a complete change in regiospecificity from C-6 to C-1 oxidation, thereby generating aldonic acids. C-1 regiospecificity is unprecedented in AA5 enzymes and is classically associated with flavin-dependent carbohydrate oxidases of Auxiliary Activity Family 3. Thus, this work further highlights the catalytic adaptability of the unique mononuclear copper radical active site and provides a basis for the design of improved biocatalysts for diverse potential applications.

Front Cover Picture: Aryl‐Alcohol‐Oxidase‐Mediated Synthesis of Piperonal and Other Valuable Aldehydes (Adv. Synth. Catal. 14/2022)

The front cover image illustrates the oxidation of primary aromatic and aliphatic allylic alcohols to valuable aldehydes catalyzed by a recombinant aryl-alcohol oxidase of Pleurotus eryngii P34. This versatile enzyme demonstrates high stability against the by-product of the reaction, H2O2, and in the presence of organic solvents. After reaction optimization, a space-time-yield of 9.5 g piperonal/l/h was achieved on a preparative scale. The produced aldehydes are of great interest as fragrances or building blocks in the synthesis of pharmaceuticals. Further details can be found in the Research Article by Urlacher and co-workers (N. Jankowski, K. Koschorreck, V. B. Urlacher, Adv. Synth. Catal. 2022, 364, XXXX–XXXX; DOI: 10.1002/adsc.202200381)

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.