The unusual structure of Ruminococcin C1 antimicrobial peptide confers clinical properties.

The emergence of superbugs developing resistance to antibiotics and the resurgence of microbial infections have led scientists to start an antimicrobial arms race. In this context, we have previously identified an active RiPP, the Ruminococcin C1, naturally produced by Ruminococcus gnavus E1, a symbiont of the healthy human intestinal microbiota. This RiPP, subclassified as a sactipeptide, requires the host digestive system to become active against pathogenic Clostridia and multidrug-resistant strains. Here we report its unique compact structure on the basis of four intramolecular thioether bridges with reversed stereochemistry introduced posttranslationally by a specific radical-SAM sactisynthase. This structure confers to the Ruminococcin C1 important clinical properties including stability to digestive conditions and physicochemical treatments, a higher affinity for bacteria than simulated intestinal epithelium, a valuable activity at therapeutic doses on a range of clinical pathogens, mediated by energy resources disruption, and finally safety for human gut tissues.

2D and 3D maximum-quantum NMR and diffusion spectroscopy for the characterization of enzymatic reaction mixtures.

1D 1H NMR spectroscopy has been widely used to monitor enzymatic activity by recording the evolution of the spectra of substrates and/or products, thanks to the linear response of NMR. For complex systems involving the coexistence of multiple compounds (substrate, final product and various intermediates), the identification and quantification can be a more arduous task. Here, we present a simple analytical method for the rapid characterization of reaction mixtures involving enzymatic complexes using Maximum Quantum (MaxQ) NMR, accelerated with the Non-Uniform Sampling (NUS) acquisition procedure. Specifically, this approach enables, in the first analytical step, the counting of the molecules present in the samples. We also show, using two different enzymatic systems, that the implementation of these pulse sequences implies precautions related to the short relaxation times due to the presence of metallo-enzymes or paramagnetic catalysts. Finally, the combination of MaxQ and diffusion experiments, which leads to a 3D chart, greatly improves the resolution and offers an extreme simplification of the spectra while giving valuable indications on the affinity of the enzymes to the different compounds present in the reaction mixture.

A survey of substrate specificity among Auxiliary Activity Family 5 copper radical oxidases.

There is significant contemporary interest in the application of enzymes to replace or augment chemical reagents toward the development of more environmentally sound and sustainable processes. In particular, copper radical oxidases (CRO) from Auxiliary Activity Family 5 Subfamily 2 (AA5_2) are attractive, organic cofactor-free catalysts for the chemoselective oxidation of alcohols to the corresponding aldehydes. These enzymes were first defined by the archetypal galactose-6-oxidase (GalOx, EC 1.1.3.13) from the fungus Fusarium graminearum. The recent discovery of specific alcohol oxidases (EC 1.1.3.7) and aryl alcohol oxidases (EC 1.1.3.47) within AA5_2 has indicated a potentially broad substrate scope among fungal CROs. However, only relatively few AA5_2 members have been characterized to date. Guided by sequence similarity network and phylogenetic analysis, twelve AA5_2 homologs have been recombinantly produced and biochemically characterized in the present study. As defined by their predominant activities, these comprise four galactose 6-oxidases, two raffinose oxidases, four broad-specificity primary alcohol oxidases, and two non-carbohydrate alcohol oxidases. Of particular relevance to applications in biomass valorization, detailed product analysis revealed that two CROs produce the bioplastics monomer furan-2,5-dicarboxylic acid (FDCA) directly from 5-hydroxymethylfurfural (HMF). Furthermore, several CROs could desymmetrize glycerol (a by-product of the biodiesel industry) to D- or L-glyceraldehyde. This study furthers our understanding of CROs by doubling the number of characterized AA5_2 members, which may find future applications as biocatalysts in diverse processes.

Identification of Copper-Containing Oxidoreductases in the Secretomes of Three Colletotrichum Species with a Focus on Copper Radical Oxidases for the Biocatalytic Production of Fatty Aldehydes.

Copper radical alcohol oxidases (CRO-AlcOx), which have been recently discovered among fungal phytopathogens, are attractive for the production of fragrant fatty aldehydes. With the initial objective to investigate the secretion of CRO-AlcOx by natural fungal strains, we undertook time course analyses of the secretomes of three Colletotrichum species (C. graminicola, C. tabacum, and C. destructivum) using proteomics. The addition of a copper-manganese-ethanol mixture in the absence of any plant-biomass mimicking compounds to Colletotrichum cultures unexpectedly induced the secretion of up to 400 proteins, 29 to 52% of which were carbohydrate-active enzymes (CAZymes), including a wide diversity of copper-containing oxidoreductases from the auxiliary activities (AA) class (AA1, AA3, AA5, AA7, AA9, AA11, AA12, AA13, and AA16). Under these specific conditions, while a CRO-glyoxal oxidase from the AA5_1 subfamily was among the most abundantly secreted proteins, the targeted AA5_2 CRO-AlcOx were secreted at lower levels, suggesting heterologous expression as a more promising strategy for CRO-AlcOx production and utilization. C. tabacum and C. destructivum CRO-AlcOx were thus expressed in Pichia pastoris, and their preference toward both aromatic and aliphatic primary alcohols was assessed. The CRO-AlcOx from C. destructivum was further investigated in applied settings, revealing a full conversion of C6 and C8 alcohols into their corresponding fragrant aldehydes. IMPORTANCE In the context of the industrial shift toward greener processes, the biocatalytic production of aldehydes is of utmost interest owing to their importance for their use as flavor and fragrance ingredients. Copper radical alcohol oxidases (CRO-AlcOx) have the potential to become platform enzymes for the oxidation of alcohols to aldehydes. However, the secretion of CRO-AlcOx by natural fungal strains has never been explored, while the use of crude fungal secretomes is an appealing approach for industrial applications to alleviate various costs pertaining to biocatalyst production. While investigating this primary objective, the secretomics studies revealed unexpected results showing that under the oxidative stress conditions we probed, Colletotrichum species can secrete a broad diversity of copper-containing enzymes (laccases, sugar oxidoreductases, and lytic polysaccharide monooxygenases [LPMOs]) usually assigned to "plant cell wall degradation," despite the absence of any plant-biomass mimicking compound. However, in these conditions, only small amounts of CRO-AlcOx were secreted, pointing out recombinant expression as the most promising path for their biocatalytic application.

The Multifunctional Sactipeptide Ruminococcin C1 Displays Potent Antibacterial Activity In Vivo as Well as Other Beneficial Properties for Human Health.

The world is on the verge of a major antibiotic crisis as the emergence of resistant bacteria is increasing, and very few novel molecules have been discovered since the 1960s. In this context, scientists have been exploring alternatives to conventional antibiotics, such as ribosomally synthesized and post-translationally modified peptides (RiPPs). Interestingly, the highly potent in vitro antibacterial activity and safety of ruminococcin C1, a recently discovered RiPP belonging to the sactipeptide subclass, has been demonstrated. The present results show that ruminococcin C1 is efficient at curing infection and at protecting challenged mice from Clostridium perfringens with a lower dose than the conventional antibiotic vancomycin. Moreover, antimicrobial peptide (AMP) is also effective against this pathogen in the complex microbial community of the gut environment, with a selective impact on a few bacterial genera, while maintaining a global homeostasis of the microbiome. In addition, ruminococcin C1 exhibits other biological activities that could be beneficial for human health, as well as other fields of applications. Overall, this study, by using an in vivo infection approach, confirms the antimicrobial clinical potential and highlights the multiple functional properties of ruminococcin C1, thus extending its therapeutic interest.

The Quaternary Structure of a Glycoside Hydrolase Dictates Specificity toward ß-Glucans.

In the Carbohydrate-Active Enzyme (CAZy) database, glycoside hydrolase family 5 (GH5) is a large family with more than 6,000 sequences. Among the 51 described GH5 subfamilies, subfamily GH5_26 contains members that display either endo-ß(1,4)-glucanase or ß(1,3;1,4)-glucanase activities. In this study, we focused on the GH5_26 enzyme fromSaccharophagus degradans(SdGluc5_26A), a marine bacterium known for its capacity to degrade a wide diversity of complex polysaccharides.SdGluc5_26A displays lichenase activity toward ß(1,3;1,4)-glucans with a side cellobiohydrolase activity toward ß(1,4)-glucans. The three-dimensional structure ofSdGluc5_26A adopts a stable trimeric quaternary structure also observable in solution. The N-terminal region ofSdGluc5_26A protrudes into the active site of an adjacent monomer. To understand whether this occupation of the active site could influence its activity, we conducted a comprehensive enzymatic characterization ofSdGluc5_26A and of a mutant truncated at the N terminus. Ligand complex structures and kinetic analyses reveal that the N terminus governs the substrate specificity ofSdGluc5_26A. Its deletion opens the enzyme cleft at the -3 subsite and turns the enzyme into an endo-ß(1,4)-glucanase. This study demonstrates that experimental approaches can reveal structure-function relationships out of reach of current bioinformatic predictions.

Four GH11 xylanases from the xylanolytic fungus Talaromyces versatilis act differently on (arabino)xylans.

The filamentous fungus Talaromyces versatilis produces a wide range of cellulolytic and hemicellulolytic enzymes such as xylanases. The recent accessibility to the T. versatilis genome allows identifying two new genes, xynE and xynF, encoding glycoside-hydrolases from family GH11. Both genes were cloned and expressed in the methylotrophic yeast Pichia pastoris in order to compare these new xylanases with two other GH11 xylanases from T. versatilis (XynB and XynC) that were previously reported. High-level expression of recombinant enzymes was obtained for the four enzymes that were purified to homogeneity. The XynB, XynC, XynE and XynF enzymes have molecular masses of 34, 22, 45 and 23 kDa, an optimal pH between 3.5 and 4.5 and an optimal temperature between 50 °C and 60 °C. Interestingly, XynF has shown the best thermal stability at 50 °C for at least 180 min with a weak loss of activity. The four xylanases catalysed hydrolysis of low viscosity arabinoxylan (LVAX) with K m(app) between 11.5 and 23.0 mg.mL(-1) and k cat/K m(app) 170 and 3,963 s(-1) mg(-1).mL. Further investigations on the rate and pattern of hydrolysis of the four enzymes on LVAX showed the predominant production of xylose, xylobiose and some (arabino)xylo-oligosaccharides as end products. The initial rate data from the hydrolysis of short xylo-oligosaccharides indicated that the catalytic efficiency increased with increasing degree of polymerisation of oligomer up to 6, suggesting that the specificity region of XynE and XynF spans at least six xylose residues. Because of their attractive properties, T. versatilis xylanases might be considered for biotechnological applications.

Carbohydrate-active enzymes in animal feed.

Considering an ever-growing global population, which hit 8 billion people in the fall of 2022, it is essential to find solutions to avoid the competition between human food and animal feed for croplands. Agricultural co-products have become important components of the circular economy with their use in animal feed. Their implementation was made possible by the addition of exogenous enzymes in the diet, especially carbohydrate-active enzymes (CAZymes). In this review, we describe the diversity and versatility of microbial CAZymes targeting non-starch polysaccharides to improve the nutritional potential of diets containing cereals and protein meals. We focused our attention on cellulases, hemicellulases, pectinases which were often found to be crucial in vivo. We also highlight the performance and health benefits brought by the exogenous addition of enzymatic cocktails containing CAZymes in the diets of monogastric animals. Taking the example of the well-studied commercial cocktail Rovabio™, we discuss the evolution, constraints and future challenges faced by feed enzymes suppliers. We hope that this review will promote the use and development of enzyme solutions for industries to sustainably feed humans in the future.

Rational engineering of AA5_2 copper radical oxidases to probe the molecular determinants governing their substrate selectivity.

Fungal copper radical oxidases (CROs) from the Auxiliary Activity family 5 (AA5) constitute a group of metalloenzymes that oxidize a wide panel of natural compounds, such as galactose-containing saccharides or primary alcohols, into product derivatives exhibiting promising biotechnological interests. Despite a well-conserved first copper-coordination sphere and overall fold, some members of the AA5_2 subfamily are incapable of oxidizing galactose and galactosides but conversely efficiently catalyse the oxidation of diverse aliphatic alcohols. The objective of this study was to understand which residues dictate the substrate preferences between alcohol oxidases and galactose oxidases within the AA5_2 subfamily. Based on structural differences and molecular modelling predictions between the alcohol oxidase from Colletotrichum graminicola (CgrAlcOx) and the archetypal galactose oxidase from Fusarium graminearum (FgrGalOx), a rational mutagenesis approach was developed to target regions or residues potentially driving the substrate specificity of these enzymes. A set of 21 single and multiple CgrAlcOx variants was produced and characterized leading to the identification of six residues (W39, F138, M173, F174, T246, L302), in the vicinity of the active site, crucial for substrate recognition. Two multiple CgrAlcOx variants, i.e. M4F (W39F, F138W, M173R and T246Q) and M6 (W39F, F138W, M173R, F174Y, T246Q and L302P), exhibited a similar affinity for carbohydrate substrates when compared to FgrGalOx. In conclusion, using a rational site-directed mutagenesis approach, we identified key residues involved in the substrate selectivity of AA5_2 enzymes towards galactose-containing saccharides.

Evaluation of the Efficiency of Random and Diblock Methacrylate-Based Amphiphilic Cationic Polymers against Major Bacterial Pathogens Associated with Cystic Fibrosis.

Cystic fibrosis (CF) is associated with repeated lung bacterial infection, mainly by Pseudomonas aeruginosa, Staphylococcus aureus, and Mycobacterium abscessus, all known to be or becoming resistant to several antibiotics, often leading to therapeutic failure and death. In this context, antimicrobial peptides and antimicrobial polymers active against resistant strains and less prompt to cause resistance, appear as a good alternative to conventional antibiotics. In the present study, methacrylate-based copolymers obtained by radical chemistry were evaluated against CF-associated bacterial strains. Results showed that the type (Random versus Diblock) and the size of the copolymers affected their antibacterial activity and toxicity. Among the different copolymers tested, four (i.e., Random10200, Random15000, Random23900, and Diblock9500) were identified as the most active and the safest molecules and were further investigated. Data showed that they inserted into bacterial lipids, leading to a rapid membranolytic effect and killing of the bacterial. In relation with their fast bactericidal action and conversely to conventional antibiotics, those copolymers did not induce a resistance and remained active against antibiotic-resistant strains. Finally, the selected copolymers possessed a preventive effect on biofilm formation, although not exhibiting disruptive activity. Overall, the present study demonstrates that methacrylate-based copolymers are an interesting alternative to conventional antibiotics in the treatment of CF-associated bacterial infection.

The diversification of the antimicrobial peptides from marine worms is driven by environmental conditions.

Antimicrobial peptides (AMPs) play a key role in the external immunity of animals, offering an interesting model for studying the influence of the environment on the diversification and evolution of immune effectors. Alvinellacin (ALV), arenicin (ARE) and polaricin (POL, a novel AMP identified here), characterized from three marine worms inhabiting contrasted habitats ('hot' vents, temperate and polar respectively), possess a well conserved BRICHOS domain in their precursor molecule despite a profound amino acid and structural diversification of the C-terminal part containing the core peptide. Data not only showed that ARE, ALV and POL display an optimal bactericidal activity against the bacteria typical of the habitat where each worm species lives but also that this killing efficacy is optimal under the thermochemical conditions encountered by their producers in their environment. Moreover, the correlation between species habitat and the cysteine contents of POL, ARE and ALV led us to investigate the importance of disulfide bridges in their biological efficacy as a function of abiotic pressures (pH and temperature). The construction of variants using non-proteinogenic residues instead of cysteines (α-aminobutyric acid variants) leading to AMPs devoid of disulfide bridges, provided evidence that the disulfide pattern of the three AMPs allows for a better bactericidal activity and suggests an adaptive way to sustain the fluctuations of the worm's environment. This work shows that the external immune effectors exemplified here by BRICHOS AMPs are evolving under strong diversifying environmental pressures to be structurally shaped and more efficient/specific under the ecological niche of their producer.

Mechanistic and functional aspects of the Ruminococcin C sactipeptide isoforms.

In a scenario where the discovery of new molecules to fight antibiotic resistance is a public health concern, ribosomally synthesized and post-translationally modified peptides constitute a promising alternative. In this context, the Gram-positive human gut symbiont Ruminococcus gnavus E1 produces five sactipeptides, Ruminococcins C1 to C5 (RumC1-C5), co-expressed with two radical SAM maturases. RumC1 has been shown to be effective against various multidrug resistant Gram-positives clinical isolates. Here, after adapting the biosynthesis protocol to obtain the four mature RumC2-5 we then evaluate their antibacterial activities. Establishing first that both maturases exhibit substrate tolerance, we then observed a variation in the antibacterial efficacy between the five isoforms. We established that all RumCs are safe for humans with interesting multifunctionalities. While no synergies where observed for the five RumCs, we found a synergistic action with conventional antibiotics targeting the cell wall. Finally, we identified crucial residues for antibacterial activity of RumC isoforms.

Characterization of a broad-specificity ß-glucanase acting on ß-(1,3)-, ß-(1,4)-, and ß-(1,6)-glucans that defines a new glycoside hydrolase family.

Here we report the cloning of the Pa_3_10940 gene from the coprophilic fungus Podospora anserina, which encodes a C-terminal family 1 carbohydrate binding module (CBM1) linked to a domain of unknown function. The function of the gene was investigated by expression of the full-length protein and a truncated derivative without the CBM1 domain in the yeast Pichia pastoris. Using a library of polysaccharides of different origins, we demonstrated that the full-length enzyme displays activity toward a broad range of ß-glucan polysaccharides, including laminarin, curdlan, pachyman, lichenan, pustulan, and cellulosic derivatives. Analysis of the products released from polysaccharides revealed that this ß-glucanase is an exo-acting enzyme on ß-(1,3)- and ß-(1,6)-linked glucan substrates and an endo-acting enzyme on ß-(1,4)-linked glucan substrates. Hydrolysis of short ß-(1,3), ß-(1,4), and ß-(1,3)/ß-(1,4) gluco-oligosaccharides confirmed this striking feature and revealed that the enzyme performs in an exo-type mode on the nonreducing end of gluco-oligosaccharides. Excision of the CBM1 domain resulted in an inactive enzyme on all substrates tested. To our knowledge, this is the first report of an enzyme that displays bifunctional exo-ß-(1,3)/(1,6) and endo-ß-(1,4) activities toward beta-glucans and therefore cannot readily be assigned to existing Enzyme Commission groups. The amino acid sequence has high sequence identity to hypothetical proteins within the fungal taxa and thus defines a new family of glycoside hydrolases, the GH131 family.

Biocatalytic oxidation of fatty alcohols into aldehydes for the flavors and fragrances industry.

From Egyptian mummies to the Chanel n°5 perfume, fatty aldehydes have long been used and keep impacting our senses in a wide range of foods, beverages and perfumes. Natural sources of fatty aldehydes are threatened by qualitative and quantitative variability while traditional chemical routes are insufficient to answer the society shift toward more sustainable and natural products. The production of fatty aldehydes using biotechnologies is therefore the most promising alternative for the flavors and fragrances industry. In this review, after drawing the portrait of the origin and characteristics of fragrant fatty aldehydes, we present the three main classes of enzymes that catalyze the reaction of fatty alcohols oxidation into aldehydes, namely alcohol dehydrogenases, flavin-dependent alcohol oxidases and copper radical alcohol oxidases. The constraints, challenges and opportunities to implement these oxidative enzymes in the flavors and fragrances industry are then discussed. By setting the scene on the biocatalytic production of fatty aldehydes, and providing a critical assessment of its potential, we expect this review to contribute to the development of biotechnology-based solutions in the flavors and fragrances industry.

Tunable Production of (R)- or (S)-Citronellal from Geraniol via a Bienzymatic Cascade Using a Copper Radical Alcohol Oxidase and Old Yellow Enzyme.

Biocatalytic pathways for the synthesis of (-)-menthol, the most sold flavor worldwide, are highly sought-after. To access the key intermediate (R)-citronellal used in current major industrial production routes, we established a one-pot bienzymatic cascade from inexpensive geraniol, overcoming the problematic biocatalytic reduction of the mixture of (E/Z)-isomers in citral by harnessing a copper radical oxidase (CgrAlcOx) and an old yellow enzyme (OYE). The cascade using OYE2 delivered 95.1% conversion to (R)-citronellal with 95.9% ee, a 62 mg scale-up affording high yield and similar optical purity. An alternative OYE, GluER, gave (S)-citronellal from geraniol with 95.3% conversion and 99.2% ee.

A simple and direct ionic chromatography method to monitor galactose oxidase activity.

Galactose oxidase (GalOx, EC.1.1.3.9) is one of the most extensively studied copper radical oxidases (CROs). The reaction catalyzed by GalOx leads to the oxidation of the C-6 hydroxyl group of galactose and galactosides (including galactosylated polysaccharides and glycoproteins) to the corresponding aldehydes, coupled to the reduction of dioxygen to hydrogen peroxide. Despite more than 60 years of research including mechanistic studies, enzyme engineering and application development, GalOx activity remains primarily monitored by indirect measurement of the co-product hydrogen peroxide. Here, we describe a simple direct method to measure GalOx activity through the identification of galactosylated oxidized products using high-performance anion-exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD). Using galactose and lactose as representative substrates, we were able to separate and detect the C-6 oxidized products, which were confirmed by LC-MS and NMR analyses to exist in their hydrated (geminal-diol) forms. We show that the HPAEC-PAD method is superior to other methods in terms of sensitivity as we could detect down to 0.08 µM of LacOX (eq. 30 µg L-1). We believe the method will prove useful for qualitative detection of galactose oxidase activity in biological samples or for quantitative purposes to analyze enzyme kinetics or to compare enzyme variants in directed evolution programs.

Tandem metalloenzymes gate plant cell entry by pathogenic fungi.

Global food security is endangered by fungal phytopathogens causing devastating crop production losses. Many of these pathogens use specialized appressoria cells to puncture plant cuticles. Here, we unveil a pair of alcohol oxidase-peroxidase enzymes to be essential for pathogenicity. Using Colletotrichum orbiculare, we show that the enzyme pair is cosecreted by the fungus early during plant penetration and that single and double mutants have impaired penetration ability. Molecular modeling, biochemical, and biophysical approaches revealed a fine-tuned interplay between these metalloenzymes, which oxidize plant cuticular long-chain alcohols into aldehydes. We show that the enzyme pair is involved in transcriptional regulation of genes necessary for host penetration. The identification of these infection-specific metalloenzymes opens new avenues on the role of wax-derived compounds and the design of oxidase-specific inhibitors for crop protection.

GH10 xylanase D from Penicillium funiculosum: biochemical studies and xylooligosaccharide production.

BACKGROUND: The filamentous fungus Penicillium funiculosum produces a range of glycoside hydrolases (GH). The XynD gene, encoding the sole P. funiculosum GH10 xylanase described so far, was cloned into the pPICZαA vector and expressed in methylotrophe yeast Pichia pastoris, in order to compare the results obtained with the P. funiculosum GH11 xylanases data. RESULTS: High level expression of recombinant XynD was obtained with a secretion of around 60 mg.L-1. The protein was purified to homogeneity using one purification step. The apparent size on SDS-PAGE was around 64 kDa and was 46 kDa by mass spectrometry thus higher than the expected molecular mass of 41 kDa. The recombinant protein was N- and O-glycosylated, as demonstrated using glycoprotein staining and deglycosylation reactions, which explained the discrepancy in molecular mass. Enzyme-catalysed hydrolysis of low viscosity arabinoxylan (LVAX) was maximal at pH 5.0 with Km(app) and kcat/Km(app) of 3.7 ± 0.2 (mg.mL-1) and 132 (s-1mg-1.mL), respectively. The activity of XynD was optimal at 80°C and the recombinant enzyme has shown an interesting high thermal stability at 70°C for at least 180 min without loss of activity. The enzyme had an endo-mode of action on xylan forming mainly xylobiose and short-chain xylooligosaccharides (XOS). The initial rate data from the hydrolysis of short XOS indicated that the catalytic efficiency increased slightly with increasing their chain length with a small difference of the XynD catalytic efficiency against the different XOS. CONCLUSION: Because of its attractive properties XynD might be considered for biotechnological applications. Moreover, XOS hydrolysis suggested that XynD possess four catalytic subsites with a high energy of interaction with the substrate and a fifth subsite with a small energy of interaction, according to the GH10 xylanase literature data.

An integrative in vitro approach to analyse digestion of wheat polysaccharides and the effect of enzyme supplementation.

The digestion of polysaccharides from the wheat cultivars Caphorn and Isengrain was investigated, and the efficiency of an enzyme preparation was tested using the TNO gastrointestinal model (TIM-1). The apparent digestibility (AD) of carbohydrates was determined based on the measurement of organic matter (OM), total monosaccharides, reducing ends (RE) and end products (EP: glucose, maltose and xylobiose). The AD of the OM from Caphorn and Isengrain measured using caecectomised cockerels did not differ from that measured using TIM-1: 72.0 (SD 2.6) v. 70.6 (SD 0.6) % for Caphorn (P = 0.580) and 73.0 (SD 2.3) v. 71.1 (SD 1.9) % for Isengrain (P = 0.252). After the 6 h TIM-1 digestion, 41.4-58.9 % of the OM, RE and EP were recovered from the jejunal compartment and 18.3-27.1 % from the ileal compartment, while ileal deliveries and digestive residues constituted the remainder. A commercial enzyme cocktail tested at 0.2 µl/g of wheat improved TIM-1 digestibility of Caphorn and Isengrain polysaccharides: 3.9 % (P = 0.0203) and 3.4 % (P = 0.0058) based on the OM; 9.7 % (P < 0.0001) and 3.1 % (P = 0.031) based on the total glucose; 47.2 % (P < 0.0001) and 14.2 % (P = 0.0004) based on the RE, respectively. The enzyme cocktail improved the release of the EP for Caphorn (3.8 %, P = 0.008) but not for Isengrain ( − 0.8 %, P = 0.561). The higher efficiency of the enzyme supplementation on the digestion of Caphorn polysaccharides compared with Isengrain seems to be linked to the higher soluble carbohydrate contents and/or less ramified arabinoxylan of Caphorn.

Two Fusarium copper radical oxidases with high activity on aryl alcohols.

BACKGROUND: Biomass 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. RESULTS: Here, 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. CONCLUSIONS: This 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.