Publications by authors named "Craig B Faulds"

58 Publications

Exploring the Diversity of Fungal DyPs in Mangrove Soils to Produce and Characterize Novel Biocatalysts.

J Fungi (Basel) 2021 Apr 21;7(5). Epub 2021 Apr 21.

INRAE, UMR1163, Biodiversité et Biotechnologie Fongiques, Aix-Marseille Université, 13288 Marseille, France.

The functional diversity of the New Caledonian mangrove sediments was examined, observing the distribution of fungal dye-decolorizing peroxidases (DyPs), together with the complete biochemical characterization of the main DyP. Using a functional metabarcoding approach, the diversity of expressed genes encoding fungal DyPs was investigated in surface and deeper sediments, collected beneath either or trees, during either the wet or the dry seasons. The highest DyP diversity was observed in surface sediments beneath the area during the wet season, and one particular operational functional unit (OFU1) was detected as the most abundant DyP isoform. This OFU was found in all sediment samples, representing 51-100% of the total DyP-encoding sequences in 70% of the samples. The complete cDNA sequence corresponding to this abundant DyP (OFU 1) was retrieved by gene capture, cloned, and heterologously expressed in . The recombinant enzyme, called DyP1, was purified and characterized, leading to the description of its physical-chemical properties, its ability to oxidize diverse phenolic substrates, and its potential to decolorize textile dyes; DyP1 was more active at low pH, though moderately stable over a wide pH range. The enzyme was very stable at temperatures up to 50 °C, retaining 60% activity after 180 min incubation. Its ability to decolorize industrial dyes was also tested on Reactive Blue 19, Acid Black, Disperse Blue 79, and Reactive Black 5. The effect of hydrogen peroxide and sea salt on DyP1 activity was studied and compared to what is reported for previously characterized enzymes from terrestrial and marine-derived fungi.
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http://dx.doi.org/10.3390/jof7050321DOI Listing
April 2021

Fungal Treatment for the Valorization of Technical Soda Lignin.

J Fungi (Basel) 2021 Jan 9;7(1). Epub 2021 Jan 9.

French National Research Institute for Agriculture, Food and Environment (INRAE), UMR1163, Biodiversité et Biotechnologie Fongiques, Aix Marseille University, 13288 Marseille, France.

Technical lignins produced as a by-product in biorefinery processes represent a potential source of renewable carbon. In consideration of the possibilities of the industrial transformation of this substrate into various valuable bio-based molecules, the biological deconstruction of a technical soda lignin by filamentous fungi was investigated. The ability of three basidiomycetes (, and ) to modify this material, the resultant structural and chemical changes, and the secreted proteins during growth on this substrate were investigated. The three fungi could grow on the technical lignin alone, and the growth rate increased when the media were supplemented with glucose or maltose. The proteomic analysis of the culture supernatants after three days of growth revealed the secretion of numerous Carbohydrate-Active Enzymes (CAZymes). The secretomic profiles varied widely between the strains and the presence of technical lignin alone triggered the early secretion of many lignin-acting oxidoreductases. The secretomes were notably rich in glycoside hydrolases and HO-producing auxiliary activity enzymes with copper radical oxidases being induced on lignin for all strains. The lignin treatment by fungi modified both the soluble and insoluble lignin fractions. A significant decrease in the amount of soluble higher molar mass compounds was observed in the case of . This strain was also responsible for the modification of the lower molar mass compounds of the lignin insoluble fraction and a 40% decrease in the thioacidolysis yield. The similarity in the activities of and in modifying the functional groups of the technical lignin were observed, the results suggest that the lignin has undergone structural changes, or at least changes in its composition, and pave the route for the utilization of filamentous fungi to functionalize technical lignins and produce the enzymes of interest for biorefinery applications.
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http://dx.doi.org/10.3390/jof7010039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827817PMC
January 2021

Enzyme Properties of a Laccase Obtained from the Transcriptome of the Marine-Derived Fungus .

Int J Mol Sci 2020 Nov 9;21(21). Epub 2020 Nov 9.

Laboratoire de Biochimie et de Génie Enzymatique des Lipases, Ecole Nationale d'Ingénieurs de Sfax, Université de Sfax, Sfax 3029, Tunisia.

Only a few studies have examined how marine-derived fungi and their enzymes adapt to salinity and plant biomass degradation. This work concerns the production and characterisation of an oxidative enzyme identified from the transcriptome of marine-derived fungus . The laccase-encoding gene Lac2 from was cloned for heterologous expression in D15#26 for protein production in the extracellular medium of around 30 mg L. The extracellular recombinant enzyme Lac2 was successfully produced and purified in three steps protocol: ultrafiltration, anion-exchange chromatography, and size exclusion chromatography, with a final recovery yield of 24%. Lac2 was characterised by physicochemical properties, kinetic parameters, and ability to oxidise diverse phenolic substrates. We also studied its activity in the presence and absence of sea salt. The molecular mass of Lac2 was about 75 kDa, consistent with that of most ascomycete fungal laccases. With syringaldazine as substrate, Lac2 showed an optimal activity at pH 6 and retained nearly 100% of its activity when incubated at 50°C for 180 min. Lac2 exhibited more than 50% of its activity with 5% wt/vol of sea salt.
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http://dx.doi.org/10.3390/ijms21218402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7664933PMC
November 2020

Characterization of the CAZy Repertoire from the Marine-Derived Fungus in Relation to Saline Conditions.

Mar Drugs 2020 Sep 9;18(9). Epub 2020 Sep 9.

Biodiversité et Biotechnologie Fongiques, Aix-Marseille Université, INRAE, UMR1163, 13288 Marseille, France.

Even if the ocean represents a large part of Earth's surface, only a few studies describe marine-derived fungi compared to their terrestrial homologues. In this ecosystem, marine-derived fungi have had to adapt to the salinity and to the plant biomass composition. This articles studies the growth of five marine isolates and the tuning of lignocellulolytic activities under different conditions, including the salinity. A de novo transcriptome sequencing and assembly were used in combination with a proteomic approach to characterize the Carbohydrate Active Enzymes (CAZy) repertoire of one of these strains. Following these approaches, was selected for its adapted growth on xylan in saline conditions, its high xylanase activity, and its improved laccase activities in seagrass-containing cultures with salt. De novo transcriptome sequencing and assembly indicated the presence of 51 putative lignocellulolytic enzymes. Its secretome composition was studied in detail when the fungus was grown on either a terrestrial or a marine substrate, under saline and non-saline conditions. Proteomic analysis of the four secretomes revealed a minimal suite of extracellular enzymes for plant biomass degradation and highlighted potential enzyme targets to be further studied for their adaptation to salts and for potential biotechnological applications.
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http://dx.doi.org/10.3390/md18090461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7551824PMC
September 2020

Screening of five marine-derived fungal strains for their potential to produce oxidases with laccase activities suitable for biotechnological applications.

BMC Biotechnol 2020 05 12;20(1):27. Epub 2020 May 12.

Ecole Nationale d'Ingénieurs de Sfax, Laboratoire de Biochimie et de Génie enzymatique des lipases, Université de Sfax, Sfax, Tunisie.

Background: Environmental pollution is one of the major problems that the world is facing today. Several approaches have been taken, from physical and chemical methods to biotechnological strategies (e.g. the use of oxidoreductases). Oxidative enzymes from microorganisms offer eco-friendly, cost-effective processes amenable to biotechnological applications, such as in industrial dye decolorization. The aim of this study was to screen marine-derived fungal strains isolated from three coastal areas in Tunisia to identify laccase-like activities, and to produce and characterize active cell-free supernatants of interest for dye decolorization.

Results: Following the screening of 20 fungal strains isolated from the harbors of Sfax and Monastir (Tunisia), five strains were identified that displayed laccase-like activities. Molecular-based taxonomic approaches identified these strains as belonging to the species Trichoderma asperellum, Stemphylium lucomagnoense and Aspergillus nidulans. Among these five isolates, one T. asperellum strain (T. asperellum 1) gave the highest level of secreted oxidative activities, and so was chosen for further studies. Optimization of the growth medium for liquid cultures was first undertaken to improve the level of laccase-like activity in culture supernatants. Finally, the culture supernatant of T. asperellum 1 decolorized different synthetic dyes belonging to diverse dye families, in the presence or absence of 1-hydroxybenzotriazole (HBT) as a mediator.

Conclusions: The optimal growth conditions to produce laccase-like active cell-free supernatants from T. asperellum 1 were 1.8 mM CuSO as an inducer, 1% NaCl to mimic a seawater environment and 3% sucrose as a carbon source. The culture supernatant of T. asperellum 1 effectively decolorized different synthetic dyes belonging to diverse chemical classes, and the presence of HBT as a mediator improved the decolorization process.
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http://dx.doi.org/10.1186/s12896-020-00617-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7218534PMC
May 2020

Characterization and Dye Decolorization Potential of Two Laccases from the Marine-Derived Fungus sp

Int J Mol Sci 2019 Apr 15;20(8). Epub 2019 Apr 15.

Aix-Marseille Université, INRA UMR1163, Biodiversité et Biotechnologie Fongiques, 13288 Marseille, France.

Two laccase-encoding genes from the marine-derived fungus sp. have been cloned in for heterologous production, and the recombinant enzymes have been characterized to study their physicochemical properties, their ability to decolorize textile dyes for potential biotechnological applications, and their activity in the presence of sea salt. The optimal pH and temperature of Lac1 and Lac2 differed in relation to the substrates tested, and both enzymes were shown to be extremely stable at temperatures up to 50 °C, retaining 100% activity after 3 h at 50 °C. Both enzymes were stable between pH 4-6. Different substrate specificities were exhibited, and the lowest and highest catalytic efficiency values were obtained against syringaldazine and 2,6-dimethoxyphenol (DMP) for Lac1 and Lac2, respectively. The industrially important dyes-Acid Yellow, Bromo Cresol Purple, Nitrosulfonazo III, and Reactive Black 5-were more efficiently decolorized by Lac1 in the presence of the redox mediator 1-hydroxybenzotriazole (HBT). Activities were compared in saline conditions, and Lac2 seemed more adapted to the presence of sea salt than Lac1. The overall surface charges of the predicted Lac three-dimensional models showed large negatively charged surfaces for Lac2, as found in proteins for marine organisms, and more balanced solvent exposed charges for Lac1, as seen in proteins from terrestrial organisms.
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http://dx.doi.org/10.3390/ijms20081864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6515530PMC
April 2019

glyoxal oxidases display differential catalytic efficiencies on 5-hydroxymethylfurfural and its oxidized derivatives.

Fungal Biol Biotechnol 2019 1;6. Epub 2019 Apr 1.

1INRA, UMR1163 Biodiversité et Biotechnologie Fongiques (BBF), Aix Marseille Université, 13009 Marseille, France.

Background: 5-Hydroxymethylfurfural (HMF), a major residual component of a lignocellulosic bio-refinery process, can be transformed into fundamental building blocks for green chemistry via oxidation. While chemical methods are well established, interest is also being directed into the enzymatic oxidation of HMF into the bio-plastic precursor 2,5-furandicarboxylic acid (FDCA).

Results: We demonstrate that three glyoxal oxidases (GLOX) isoenzymes from the Basidiomycete fungus were able to oxidize HMF, with GLOX2 and GLOX3 being the most efficient. The major reaction product obtained with the three isoenzymes was 5-hydroxymethyl-2-furancarboxylic (HMFCA), a precursor in polyesters and pharmaceuticals production, and very little subsequent conversion of this compound was observed. However, small concentrations of FDCA, a substitute for terephthalic acid in the production of polyesters, were also obtained. The oxidation of HMF was significantly boosted in the presence of catalase for GLOX2, leading to 70% HMFCA yield. The highest conversion percentages were observed on 2,5-furandicarboxaldehyde (DFF), a minor product from the reaction of GLOX on HMF. To bypass HMFCA accumulation and exploit the efficiency of GLOX in oxidizing DFF and 5-formyl-2-furan carboxylic acid (FFCA) towards FDCA production, HMF was oxidized in a cascade reaction with an aryl alcohol oxidase (AAO). After 2 h of reaction, AAO completely oxidized HMF to DFF and further to FFCA, with FDCA only being detected when GLOX3 was added to the reaction. The maximum yield of 16% FDCA was obtained 24 h after the addition of GLOX3 in the presence of catalase.

Conclusions: At least two conversion pathways for HMF oxidation can be considered for GLOX; however, the highest selectivity was seen towards the production of the valuable polyester precursor HMFCA. The three isoenzymes showed differences in their catalytic efficiencies and substrate specificities when reacted with HMF derivatives.
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http://dx.doi.org/10.1186/s40694-019-0067-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442418PMC
April 2019

Feruloyl esterases: Biocatalysts to overcome biomass recalcitrance and for the production of bioactive compounds.

Bioresour Technol 2019 Apr 17;278:408-423. Epub 2019 Jan 17.

Department of Biochemistry, State University of Maringá, Maringá, Paraná, Brazil. Electronic address:

Ferulic acid and its hydroxycinnamate derivatives represent one of the most abundant forms of low molecular weight phenolic compounds in plant biomass. Feruloyl esterases are part of a microorganism's plant cell wall-degrading enzymatic arsenal responsible for cleaving insoluble wall-bound hydroxycinnamates and soluble cytosolic conjugates. Stimulated by industrial requirements, accelerating scientific discoveries and knowledge transfer, continuous improvement efforts have been made to identify, create and repurposed biocatalysts dedicated to plant biomass conversion and biosynthesis of high-added value molecules. Here we review the basic knowledge and recent advances in biotechnological characteristics and the gene content encoding for feruloyl esterases. Information about several enzymes is systematically organized according to their function, biochemical properties, substrate specificity, and biotechnological applications. This review contributes to further structural, functional, and biotechnological R&D both for obtaining hydroxycinnamates from agricultural by-products as well as for lignocellulose biomass treatments aiming for production of bioethanol and other derivatives of industrial interest.
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http://dx.doi.org/10.1016/j.biortech.2019.01.064DOI Listing
April 2019

Arthrospira maxima OF15 biomass cultivation at laboratory and pilot scale from sugarcane vinasse for potential biological new peptides production.

Bioresour Technol 2019 Feb 31;273:103-113. Epub 2018 Oct 31.

Bioprocess Engineering and Biotechnology Department, Federal University of Parana (UFPR), 81531-990 Curitiba, PR, Brazil. Electronic address:

An environmental friendly process was developed to produce Arthrospira maxima's biomass from sugarcane vinasse, which was generated in a bioethanol production chain, at laboratory and pilot scale. Peptides fractions were than obtained from enzymatically hydrolyzed biomass. High microalgae biomass productivities were reached (0.150 g L day) coupled with a significant reduction of BOD and COD (89.2 and 81%, respectively). Three peptide fractions were obtained from microalgae biomass through single or sequential enzymatic hydrolysis. Antioxidant, antimicrobial, anti-inflammatory, and/or anti-collagenase activities of biopetides' fractions were observed. The PHS showed multi-biological activities. The three peptides fractions could be potential candidates for different applications in pharmaceutical, cosmetic and food industry.
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http://dx.doi.org/10.1016/j.biortech.2018.10.081DOI Listing
February 2019

Lavender- and lavandin-distilled straws: an untapped feedstock with great potential for the production of high-added value compounds and fungal enzymes.

Biotechnol Biofuels 2018 2;11:217. Epub 2018 Aug 2.

1UMR1163 BBF Biodiversité et Biotechnologie Fongiques, INRA, Aix Marseille Univ, 13288 Marseille Cedex 09, France.

Background: Lavender () and lavandin (a sterile hybrid of  × ) essential oils are among those most commonly used in the world for various industrial purposes, including perfumes, pharmaceuticals and cosmetics. The solid residues from aromatic plant distillation such as lavender- and lavandin-distilled straws are generally considered as wastes, and consequently either left in the fields or burnt. However, lavender- and lavandin-distilled straws are a potentially renewable plant biomass as they are cheap, non-food materials that can be used as raw feedstocks for green chemistry industry. The objective of this work was to assess different pathways of valorization of these straws as bio-based platform chemicals and fungal enzymes of interest in biorefinery.

Results: Sugar and lignin composition analyses and saccharification potential of the straw fractions revealed that these industrial by-products could be suitable for second-generation bioethanol prospective. The solvent extraction processes, developed specifically for these straws, released terpene derivatives (e.g. τ-cadinol, β-caryophyllene), lactones (e.g. coumarin, herniarin) and phenolic compounds of industrial interest, including rosmarinic acid which contributed to the high antioxidant activity of the straw extracts. Lavender and lavandin straws were also suitable inducers for the secretion of a wide panel of lignocellulose-acting enzymes (cellulases, hemicellulases and oxido-reductases) from the white-rot model fungus Interestingly, high amounts of laccase and several lytic polysaccharide monooxygenases were identified in the lavender and lavandin straw secretomes using proteomics.

Conclusions: The present study demonstrated that the distilled straws of lavender and lavandin are lignocellulosic-rich materials that can be used as raw feedstocks for producing high-added value compounds (antioxidants, aroma) and fungal oxidative enzymes, which represent opportunities to improve the decomposition of recalcitrant lignocellulose into biofuel. Hence, the structure and the physico-chemical properties of these straws clearly open new perspectives for use in biotechnological processes involving especially filamentous fungi. These approaches represent sustainable strategies to foster the development of a local circular bioeconomy.
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http://dx.doi.org/10.1186/s13068-018-1218-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071384PMC
August 2018

Enzyme Activities of Two Recombinant Heme-Containing Peroxidases, DyP1 and VP2, Identified from the Secretome of Trametes versicolor.

Appl Environ Microbiol 2018 04 2;84(8). Epub 2018 Apr 2.

INRA, Aix-Marseille Université, UMR1163, Biodiversité et Biotechnologie Fongiques, Marseille, France

is a wood-inhabiting agaricomycete known for its ability to cause strong white-rot decay on hardwood and for its high tolerance of phenolic compounds. The goal of the present work was to gain insights into the molecular biology and biochemistry of the heme-including class II and dye-decolorizing peroxidases secreted by this fungus. Proteomic analysis of the secretome of BRFM 1218 grown on oak wood revealed a set of 200 secreted proteins, among which were the dye-decolorizing peroxidase DyP1 and the versatile peroxidase VP2. Both peroxidases were heterologously produced in , biochemically characterized, and tested for the ability to oxidize complex substrates. Both peroxidases were found to be active against several substrates under acidic conditions, and DyP1 was very stable over a relatively large pH range of 2.0 to 6.0, while VP2 was more stable at pH 5.0 to 6.0 only. The thermostability of both enzymes was also tested, and DyP1 was globally found to be more stable than VP2. After 180 min of incubation at temperatures ranging from 30 to 50°C, the activity of VP2 drastically decreased, with 10 to 30% of the initial activity retained. Under the same conditions, DyP1 retained 20 to 80% of its enzyme activity. The two proteins were catalytically characterized, and VP2 was shown to accept a wider range of reducing substrates than DyP1. Furthermore, both enzymes were found to be active against two flavonoids, quercetin and catechin, found in oak wood, with VP2 displaying more rapid oxidation of the two compounds. They were tested for the ability to decolorize five industrial dyes, and VP2 presented a greater ability to oxidize and decolorize the dye substrates than DyP1. is a wood-inhabiting agaricomycete known for its ability to cause strong white-rot decay on hardwood and for its high tolerance of phenolic compounds. Among white-rot fungi, the basidiomycete has been extensively studied for its ability to degrade wood, specifically lignin, thanks to an extracellular oxidative enzymatic system. The corresponding oxidative system was previously studied in several works for classical lignin and manganese peroxidases, and in this study, two new components of the oxidative system of , one dye-decolorizing peroxidase and one versatile peroxidase, were biochemically characterized in depth and compared to other fungal peroxidases.
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http://dx.doi.org/10.1128/AEM.02826-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5881066PMC
April 2018

A Two-Step Bioconversion Process for Canolol Production from Rapeseed Meal Combining an Aspergillus niger Feruloyl Esterase and the Fungus Neolentinus lepideus.

Microorganisms 2017 Oct 14;5(4). Epub 2017 Oct 14.

INRA Institut National de la Recherche Agronomique, Aix Marseille Univ., UMR1163 BBF Biodiversité et Biotechnologie Fongiques, 163 Avenue de Luminy, 13288 Marseille CEDEX 09, France.

Rapeseed meal is a cheap and abundant raw material, particularly rich in phenolic compounds of biotechnological interest. In this study, we developed a two-step bioconversion process of naturally occurring sinapic acid (4-hydroxy-3,5-dimethoxycinnamic acid) from rapeseed meal into canolol by combining the complementary potentialities of two filamentous fungi, the micromycete and the basidiomycete . Canolol could display numerous industrial applications because of its high antioxidant, antimutagenic and anticarcinogenic properties. In the first step of the process, the use of the enzyme feruloyl esterase type-A (named AnFaeA) produced with the recombinant strain BRFM451 made it possible to release free sinapic acid from the raw meal by hydrolysing the conjugated forms of sinapic acid in the meal (mainly sinapine and glucopyranosyl sinapate). An amount of 39 nkat AnFaeA per gram of raw meal, at 55 °C and pH 5, led to the recovery of 6.6 to 7.4 mg of free sinapic acid per gram raw meal, which corresponded to a global hydrolysis yield of 68 to 76% and a 100% hydrolysis of sinapine. Then, the XAD2 adsorbent (a styrene and divinylbenzene copolymer resin), used at pH 4, enabled the efficient recovery of the released sinapic acid, and its concentration after elution with ethanol. In the second step, 3-day-old submerged cultures of the strain BRFM15 were supplied with the recovered sinapic acid as the substrate of bioconversion into canolol by a non-oxidative decarboxylation pathway. Canolol production reached 1.3 g/L with a molar yield of bioconversion of 80% and a productivity of 100 mg/L day. The same XAD2 resin, when used at pH 7, allowed the recovery and purification of canolol from the culture broth of . The two-step process used mild conditions compatible with green chemistry.
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http://dx.doi.org/10.3390/microorganisms5040067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5748576PMC
October 2017

Rheology and microstructure of gels based on wheat arabinoxylans enzymatically modified in arabinose to xylose ratio.

J Sci Food Agric 2018 Feb 28;98(3):914-922. Epub 2017 Aug 28.

INRA, UMR1163 Biodiversité et Biotechnologie Fongiques, Marseille, France.

Background: Arabinoxylans (AX) are polysaccharides consisting of a backbone of xyloses with arabinose substituents ester-linked to ferulic acid (FA). The arabinose to xylose ratio (A/X) in AX may vary from 0.3 to 1.1. AX form covalent gels by cross-linking of FA but physical interactions between AX chains also contribute to the network formation. The present study aimed to investigate the rheological and microstructural characteristics of gels based on AX enzymatically modified in A/X.

Results: Tailored AX presented A/X ranging from 0.68 to 0.51 and formed covalent gels. Dimers of FA content and elasticity (G') increased from 0.31 to 0.39 g kg AX and from 106 to 164 Pa when the A/X in the polysaccharide decreased from 0.68 to 0.51. Atomic force microscopy images of AX gels showed a sponge-like microstructure at A/X = 0.68, whereas, at lower values, gels presented a more compact microstructure. Scanning electron microscopy analysis of AX gels show an arrangement of different morphology, passing from an imperfect honeycomb (A/X = 0.68) to a flake-like microstructure (A/X = 0.51).

Conclusion: Lower A/X values favor the aggregation of AX chains resulting in an increase in di-FA content, which improves the rheological and microstructural characteristics of the gel formed. © 2017 Society of Chemical Industry.
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http://dx.doi.org/10.1002/jsfa.8537DOI Listing
February 2018

Oxidoreductases on their way to industrial biotransformations.

Biotechnol Adv 2017 Nov 15;35(6):815-831. Epub 2017 Jun 15.

CLEA Technologies BV, Delft, The Netherlands.

Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and the new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate proteins. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological interest due to their ability to catalyze a variety of regio- and stereo-selective monooxygenation reactions with HO as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating HO that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other HO generating enzymes - copper-radical oxidases - together with classical laccases that are the oxidoreductases with the largest number of reported applications to date. However, the recently described lytic polysaccharide monooxygenases have attracted the highest attention among copper oxidoreductases, since they are capable of oxidatively breaking down crystalline cellulose, the disintegration of which is still a major bottleneck in lignocellulose biorefineries, along with lignin degradation. Interestingly, some flavin-containing dehydrogenases also play a key role in cellulose breakdown by directly/indirectly "fueling" electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, combining rational and computational design with directed evolution, to attain the selectivity, catalytic efficiency and stability properties required for their industrial utilization. Indeed, using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site in biophysical simulations, and electron transfer efficiency in biochemical simulations, reducing in orders of magnitude the time of experimental work in oxidoreductase screening and engineering. What has been set out above is illustrated by a series of remarkable oxyfunctionalization and oxidation reactions developed in the frame of an intersectorial and multidisciplinary European RTD project. The optimized reactions include enzymatic synthesis of 1-naphthol, 25-hydroxyvitamin D, drug metabolites, furandicarboxylic acid, indigo and other dyes, and conductive polyaniline, terminal oxygenation of alkanes, biomass delignification and lignin oxidation, among others. These successful case stories demonstrate the unexploited potential of oxidoreductases in medium and large-scale biotransformations.
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http://dx.doi.org/10.1016/j.biotechadv.2017.06.003DOI Listing
November 2017

Glyoxal oxidases: their nature and properties.

World J Microbiol Biotechnol 2017 May 7;33(5):87. Epub 2017 Apr 7.

Aix Marseille University, INRA, UMR1163 Biodiversité et Biotechnologie Fongiques (BBF), Marseille, France.

HO has been found to be required for the activity of the main microbial enzymes responsible for lignin oxidative cleavage, peroxidases. Along with other small radicals, it is implicated in the early attack of plant biomass by fungi. Among the few extracellular HO-generating enzymes known are the glyoxal oxidases (GLOX). GLOX is a copper-containing enzyme, sharing high similarity at the level of active site structure and chemistry with galactose oxidase. Genes encoding GLOX enzymes are widely distributed among wood-degrading fungi especially white-rot degraders, plant pathogenic and symbiotic fungi. GLOX has also been identified in plants. Although widely distributed, only few examples of characterized GLOX exist. The first characterized fungal GLOX was isolated from Phanerochaete chrysosporium. The GLOX from Utilago maydis has a role in filamentous growth and pathogenicity. More recently, two other glyoxal oxidases from the fungus Pycnoporus cinnabarinus were also characterized. In plants, GLOX from Vitis pseudoreticulata was found to be implicated in grapevine defence mechanisms. Fungal GLOX were found to be activated by peroxidases in vitro suggesting a synergistic and regulatory relationship between these enzymes. The substrates oxidized by GLOX are mainly aldehydes generated during lignin and carbohydrates degradation. The reactions catalysed by this enzyme such as the oxidation of toxic molecules and the production of valuable compounds (organic acids) makes GLOX a promising target for biotechnological applications. This aspect on GLOX remains new and needs to be investigated.
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http://dx.doi.org/10.1007/s11274-017-2254-1DOI Listing
May 2017

Hydrolysis of Nonpolar n-Alkyl Ferulates by Feruloyl Esterases.

J Agric Food Chem 2016 Nov 7;64(45):8549-8554. Epub 2016 Nov 7.

Institute of Food, Nutrition and Health, ETH Zurich , Schmelzbergstrasse 9, CH-8092 Zurich, Switzerland.

Ferulic acid is one of the major phenolic acids in plants and can be found esterified to plant cell wall components, but also as long-chain n-alkyl and steryl esters. Microbial feruloyl esterases may play a role in the bioavailability of phenolic acids during human and animal digestion. It is therefore of interest if feruloyl esterases are capable of hydrolyzing nonpolar ferulic acid esters. A series of n-alkyl ferulates with increasing lipophilicity were enzymatically synthesized, and the kinetic constants of their hydrolysis by four feruloyl esterases and a lipase as control were determined. A decrease in K and k could be observed with decreased substrate polarity for all of the feruloyl esterases. Only one feruloyl esterase and the control lipase showed hydrolytic activity toward octadecyl ferulate. These results led to the conclusion that lipophilic ferulates are poor substrates for known feruloyl esterases and more specific esterases/lipases need to be identified.
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http://dx.doi.org/10.1021/acs.jafc.6b02694DOI Listing
November 2016

Heterologous Production and Characterization of Two Glyoxal Oxidases from Pycnoporus cinnabarinus.

Appl Environ Microbiol 2016 08 29;82(16):4867-75. Epub 2016 Jul 29.

Aix Marseille Université, INRA, BBF (Biodiversité et Biotechnologie Fongiques), Marseille, France

Unlabelled: The genome of the white rot fungus Pycnoporus cinnabarinus includes a large number of genes encoding enzymes implicated in lignin degradation. Among these, three genes are predicted to encode glyoxal oxidase, an enzyme previously isolated from Phanerochaete chrysosporium The glyoxal oxidase of P. chrysosporium is physiologically coupled to lignin-oxidizing peroxidases via generation of extracellular H2O2 and utilizes an array of aldehydes and α-hydroxycarbonyls as the substrates. Two of the predicted glyoxal oxidases of P. cinnabarinus, GLOX1 (PciGLOX1) and GLOX2 (PciGLOX2), were heterologously produced in Aspergillus niger strain D15#26 (pyrG negative) and purified using immobilized metal ion affinity chromatography, yielding 59 and 5 mg of protein for PciGLOX1 and PciGLOX2, respectively. Both proteins were approximately 60 kDa in size and N-glycosylated. The optimum temperature for the activity of these enzymes was 50°C, and the optimum pH was 6. The enzymes retained most of their activity after incubation at 50°C for 4 h. The highest relative activity and the highest catalytic efficiency of both enzymes occurred with glyoxylic acid as the substrate. The two P. cinnabarinus enzymes generally exhibited similar substrate preferences, but PciGLOX2 showed a broader substrate specificity and was significantly more active on 3-phenylpropionaldehyde.

Importance: This study addresses the poorly understood role of how fungal peroxidases obtain an in situ supply of hydrogen peroxide to enable them to oxidize a variety of organic and inorganic compounds. This cooperative activity is intrinsic in the living organism to control the amount of toxic H2O2 in its environment, thus providing a feed-on-demand scenario, and can be used biotechnologically to supply a cheap source of peroxide for the peroxidase reaction. The secretion of multiple glyoxal oxidases by filamentous fungi as part of a lignocellulolytic mechanism suggests a controlled system, especially as these enzymes utilize fungal metabolites as the substrates. Two glyoxal oxidases have been isolated and characterized to date, and the differentiation of the substrate specificity of the two enzymes produced by Pycnoporus cinnabarinus illustrates the alternative mechanisms existing in a single fungus, together with the utilization of these enzymes to prepare platform chemicals for industry.
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http://dx.doi.org/10.1128/AEM.00304-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4968546PMC
August 2016

Salt-responsive lytic polysaccharide monooxygenases from the mangrove fungus Pestalotiopsis sp. NCi6.

Biotechnol Biofuels 2016 20;9:108. Epub 2016 May 20.

INRA, UMR1163 Biodiversité et Biotechnologie Fongiques, Aix-Marseille Université, Polytech Marseille, 163 Avenue de Luminy, CP 925, 13288 Marseille Cedex 09, France ; UMR1163 Biodiversité et Biotechnologie Fongiques, Faculté des Sciences de Luminy-Polytech Marseille, Aix-Marseille Université, 163 Avenue de Luminy, CP 925, 13288 Marseille Cedex 09, France.

Background: Lytic polysaccharide monooxygenases (LPMOs) belong to the "auxiliary activities (AA)" enzyme class of the CAZy database. They are known to strongly improve the saccharification process and boost soluble sugar yields from lignocellulosic biomass, which is a key step in the efficient production of sustainable economic biofuels. To date, most LPMOs have been characterized from terrestrial fungi, but novel fungal LPMOs isolated from more extreme environments such as an estuary mangrove ecosystem could offer enzymes with unique properties in terms of salt tolerance and higher stability under harsh condition.

Results: Two LPMOs secreted by the mangrove-associated fungus Pestalotiopsis sp. NCi6 (PsLPMOA and PsLPMOB) were expressed in the yeast Pichia pastoris and produced in a bioreactor with >85 mg L(-1) for PsLPMOA and >260 mg L(-1) for PsLPMOB. Structure-guided homology modeling of the PsLPMOs showed a high abundance of negative surface charges, enabling enhanced protein stability and activity in the presence of sea salt. Both PsLPMOs were activated by a cellobiose dehydrogenase (CDH) from Neurospora crassa, with an apparent optimum of interaction at pH 5.5. Investigation into their regioselective mode of action revealed that PsLPMOA released C1- and C4-oxidized cello-oligosaccharide products, while PsLPMOB released only C4-oxidized products. PsLPMOA was found to cleave polymeric cellulose in the presence of up to 6 % sea salt, which emphasizes the use of sea water in the industrial saccharification process with improved ecological footprints.

Conclusions: Two new LPMOs from the mangrove fungus Pestalotiopsis sp. NCi6 were found to be fully reactive against cellulose. The combined hydrolytic activities of these salt-responsive LPMOs could therefore facilitate the saccharification process using sea water as a reaction medium for large-scale biorefineries.
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http://dx.doi.org/10.1186/s13068-016-0520-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4875668PMC
May 2016

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

Appl Environ Microbiol 2016 Apr 4;82(8):2411-2423. Epub 2016 Apr 4.

INRA, UMR 1163 Biotechnologie des Champignons Filamenteux, Polytech Marseille, Marseille, France.

Auxiliary activities family 3 subfamily 2 (AA3_2) from the CAZy database comprises various functions related to ligninolytic enzymes, such as fungal aryl alcohol oxidases (AAO) and glucose oxidases, both of which are flavoenzymes. The recent study of the Pycnoporus cinnabarinus CIRM BRFM 137 genome combined with its secretome revealed that four AA3_2 enzymes are secreted during biomass degradation. One of these AA3_2 enzymes, scf184803.g17, has recently been produced heterologously in Aspergillus niger Based on the enzyme's activity and specificity, it was assigned to the glucose dehydrogenases (PcinnabarinusGDH [PcGDH]). Here, we analyze the distribution of the other three AA3_2 enzymes (scf185002.g8, scf184611.g7, and scf184746.g13) to assess their putative functions. These proteins showed the highest homology with aryl alcohol oxidase from Pleurotus eryngii Biochemical characterization demonstrated that they were also flavoenzymes harboring flavin adenine dinucleotide (FAD) as a cofactor and able to oxidize a wide variety of phenolic and nonphenolic aryl alcohols and one aliphatic polyunsaturated primary alcohol. Though presenting homology with fungal AAOs, these enzymes exhibited greater efficiency in reducing electron acceptors (quinones and one artificial acceptor) than molecular oxygen and so were defined as aryl-alcohol:quinone oxidoreductases (AAQOs) with two enzymes possessing residual oxidase activity (PcAAQO2 and PcAAQO3). Structural comparison of PcAAQO homology models with P. eryngii AAO demonstrated a wider substrate access channel connecting the active-site cavity to the solvent, explaining the absence of activity with molecular oxygen. Finally, the ability of PcAAQOs to reduce radical intermediates generated by laccase from P. cinnabarinus was demonstrated, shedding light on the ligninolytic system of this fungus.
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http://dx.doi.org/10.1128/AEM.03761-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4959486PMC
April 2016

Essential oils and distilled straws of lavender and lavandin: a review of current use and potential application in white biotechnology.

Appl Microbiol Biotechnol 2015 Apr 13;99(8):3375-85. Epub 2015 Mar 13.

INRA, UMR 1163 Biodiversité et Biotechnologie Fongiques, 13009, Marseille, France.

The Lavandula genus, which includes lavender (Lavandula angustifolia) and lavandin (L. angustifolia × Lavandula latifolia), is cultivated worldwide for its essential oils, which find applications in perfumes, cosmetics, food processing and, more recently, in aromatherapy products. The chemical composition of lavender and lavandin essential oils, usually produced by steam distillation from the flowering stems, is characterized by the presence of terpenes (e.g. linalool and linalyl acetate) and terpenoids (e.g. 1,8-cineole), which are mainly responsible for their characteristic flavour and their biological and therapeutic properties. Lavender and lavandin distilled straws, the by-products of oil extraction, were traditionally used for soil replenishment or converted to a fuel source. They are mineral- and carbon-rich plant residues and, therefore, a cheap, readily available source of valuable substances of industrial interest, especially aroma and antioxidants (e.g. terpenoids, lactones and phenolic compounds including coumarin, herniarin, α-bisabolol, rosmarinic and chlorogenic acids). Accordingly, recent studies have emphasized the possible uses of lavender and lavandin straws in fermentative or enzymatic processes involving various microorganisms, especially filamentous fungi, for the production of antimicrobials, antioxidants and other bioproducts with pharmaceutical and cosmetic activities, opening up new challenging perspectives in white biotechnology applications.
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http://dx.doi.org/10.1007/s00253-015-6511-7DOI Listing
April 2015

Effects of enzymatic removal of plant cell wall acylation (acetylation, p-coumaroylation, and feruloylation) on accessibility of cellulose and xylan in natural (non-pretreated) sugar cane fractions.

Biotechnol Biofuels 2014 15;7(1):153. Epub 2014 Oct 15.

Departamento de Biotecnologia, Escola de Engenharia de Lorena, Universidade de São Paulo, 12602-810 Lorena, SP Brasil.

Background: Sugar cane internodes can be divided diagonally into four fractions, of which the two innermost ones are the least recalcitrant pith and the moderately accessible pith-rind interface. These fractions differ in enzymatic hydrolyzability due to structural differences. In general, cellulose hydrolysis in plants is hindered by its physical interaction with hemicellulose and lignin. Lignin is believed to be linked covalently to hemicellulose through hydroxycinnamic acids, forming a compact matrix around the polysaccharides. Acetyl xylan esterase and three feruloyl esterases were evaluated for their potential to fragment the lignocellulosic network in sugar cane and to indirectly increase the accessibility of cellulose.

Results: The hydrolyzability of the pith and pith-rind interface fractions of a low-lignin-containing sugar cane clone (H58) was compared to that of a reference cultivar (RC). Acetyl xylan esterase enhanced the rate and overall yield of cellulose and xylan hydrolysis in all four substrates. Of the three feruloyl esterases tested, only TsFaeC was capable of releasing p-coumaric acid, while AnFaeA and NcFaeD released ferulic acid from both the pith and interface fractions. Ferulic acid release was higher from the less recalcitrant clone (H58)/fraction (pith), whereas more p-coumaric acid was released from the clone (RC)/fraction (interface) with a higher lignin content. In addition, a compositional analysis of the four fractions revealed that p-coumaroyl content correlated with lignin, while feruloyl content correlated with arabinose content, suggesting different esterification patterns of these two hydroxycinnamic acids. Despite the extensive release of phenolic acids, feruloyl esterases only moderately promoted enzyme access to cellulose or xylan.

Conclusions: Acetyl xylan esterase TrAXE was more efficient in enhancing the overall saccharification of sugar cane, compared to the feruloyl esterases AnFaeA, TsFaeC, and NcFaeD. The hydroxycinnamic acid composition of sugar cane fractions and the hydrolysis data together suggest that feruloyl groups are more likely to decorate xylan, while p-coumaroyl groups are rather linked to lignin. The three different feruloyl esterases had distinct product profiles on non-pretreated sugar cane substrate, indicating that sugar cane pith could function as a possible natural substrate for feruloyl esterase activity measurements. Hydrolysis data suggest that TsFaeC was able to release p-coumaroyl groups esterifying lignin.
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http://dx.doi.org/10.1186/s13068-014-0153-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4201721PMC
October 2014

Enzymatic degradation of Elephant grass (Pennisetum purpureum) stems: influence of the pith and bark in the total hydrolysis.

Bioresour Technol 2014 Sep 24;167:469-75. Epub 2014 Jun 24.

Centro de Investigaciones Biológicas, Campus Universidad, Ramiro de Maeztu 9, 28040 Madrid, Spain; INRA, UMR 1163 Biotechnologie des Champignons Filamenteux, 163 avenue de Luminy, 13288 Marseille cedex 09, France; Aix-Marseille Université, POLYTECH Marseille, UMR 1163 Biotechnologie des Champignons Filamenteux, 163 avenue de Luminy, 13288 Marseille cedex 09, France. Electronic address:

The internal pith of a high energy plant, Elephant grass (EG), was more extensively degraded (>50% dry matter) compared to the outer cortex (31%) or the whole stem (35%) by an enzyme preparation from Humicola insolens, Ultraflo. Reducing sugars and acetic acid release from the pith was also higher compared to the cortex. Supplementation of Ultraflo with a type-C feruloyl esterase increased the level of deacetylation but also led to reduced solubilisation. The addition of 20% dimethyl sulfoxide (DMSO) as a co-solvent also reduced the solubility of EG by Ultraflo, although acetic acid release was increased, complimenting previous results found on model substrates. The presence of DMSO was also shown to have a protective effect on xylanase activity but not acetyl esterase activity in Ultraflo. Xylan in the biomass was preferentially solubilised by DMSO, while Ultraflo removed more glucose than xylose.
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http://dx.doi.org/10.1016/j.biortech.2014.06.018DOI Listing
September 2014

A novel glucose dehydrogenase from the white-rot fungus Pycnoporus cinnabarinus: production in Aspergillus niger and physicochemical characterization of the recombinant enzyme.

Appl Microbiol Biotechnol 2014 Dec 26;98(24):10105-18. Epub 2014 Jun 26.

INRA, UMR 1163 Biotechnologie des Champignons Filamenteux, Polytech Marseille, 163 Avenue de Luminy, 13228, Marseille cedex 09, France,

Data on glucose dehydrogenases (GDHs) are scarce and availability of these enzymes for application purposes is limited. This paper describes a new GDH from the fungus Pycnoporus cinnabarinus CIRM BRFM 137 that is the first reported GDH from a white-rot fungus belonging to the Basidiomycota. The enzyme was recombinantly produced in Aspergillus niger, a well-known fungal host producing an array of homologous or heterologous enzymes for industrial applications. The full-length gene that encodes GDH from P. cinnabarinus (PcGDH) consists of 2,425 bp and codes for a deduced protein of 620 amino acids with a calculated molecular mass of 62.5 kDa. The corresponding complementary DNA was cloned and placed under the control of the strong and constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. The signal peptide of the glucoamylase prepro sequence of A. niger was used to target PcGDH secretion into the culture medium, achieving a yield of 640 mg L(-1), which is tenfold higher than any other reported value. The recombinant PcGDH was purified twofold to homogeneity in a one-step procedure with a 41 % recovery using a Ni Sepharose column. The identity of the recombinant protein was further confirmed by immunodetection using western blot analysis and N-terminal sequencing. The molecular mass of the native PcGDH was 130 kDa, suggesting a homodimeric form. Optimal pH and temperature were found to be similar (5.5 and 60 °C, respectively) to those determined for the previously characterized GDH, i.e., from Glomerella cingulata. However PcGDH exhibits a lower catalytic efficiency of 67 M(-1) s(-1) toward glucose. This substrate is by far the preferred substrate, which constitutes an advantage over other sugar oxidases in the case of blood glucose monitoring. The substrate-binding domain of PcGDH turns out to be conserved as compared to other glucose-methanol-choline (GMCs) oxidoreductases. In addition, the ability of PcGDH to reduce oxidized quinones or radical intermediates was clearly demonstrated, which raises prospects for applying this enzyme to detoxify toxic compounds formed during the degradation of lignin.
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http://dx.doi.org/10.1007/s00253-014-5891-4DOI Listing
December 2014

Biorefining of waste paper biomass: increasing the concentration of glucose by optimising enzymatic hydrolysis.

Appl Biochem Biotechnol 2014 Apr 22;172(7):3621-34. Epub 2014 Feb 22.

The Biorefinery Centre, Institute of Food Research, Norwich, NR4 7UA, UK.

Waste copier paper is a potential substrate for the production of glucose relevant for manufacture of platform chemicals and intermediates, being composed of 51 % glucan. The yield and concentration of glucose arising from the enzymatic saccharification of solid ink-free copier paper as cellulosic substrate was studied using a range of commercial cellulase preparations. The results show that in all cellulase preparations examined, maximum hydrolysis was only achieved with the addition of beta-glucosidase, despite its presence in the enzyme mixtures. With the use of Accellerase® (cellulase), high substrate loading decreased conversion yield. However, this was overcome if the enzyme was added between 12.5 and 20 FPU g substrate(-1). Furthermore, this reaction condition facilitated continual stirring and enabled sequential additions (up to 50 % w/v) of paper to be made to the hydrolysis reaction, degrading nearly all (99 %) of the cellulose fibres and increasing the final concentration of glucose whilst simultaneously making high substrate concentrations achievable. Under optimal conditions (50 °C, pH 5.0, 72 h), digestions facilitate the production of glucose to much improved concentrations of up to 1.33 mol l(-1).
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http://dx.doi.org/10.1007/s12010-014-0767-8DOI Listing
April 2014

Comprehensive study of valuable lipophilic phytochemicals in wheat bran.

J Agric Food Chem 2014 Feb 4;62(7):1664-73. Epub 2014 Feb 4.

Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC , P.O. Box 1052, E- 41080 Seville, Spain.

Wheat bran, the major side-stream generated in the milling of wheat grains in the production of white flour, contains significant quantities of carbohydrate and proteins. While not interfering with flour utilization, the bran could be considered as an important feedstock within a biorefinery concept. Wheat bran also contains some amounts of lipids that can be used as a source of valuable phytochemicals. Gas chromatography and mass spectrometry analysis of the lipid composition of destarched wheat bran demonstrated that the predominant lipids found in wheat bran were free fatty acids (ca. 40% of total lipids), followed by acylglycerols (40%). Additionally, important amounts of alkylresorcinols (13% of total lipids) and steroid compounds (hydrocarbons, ketones, free sterols, sterol glycosides, sterol esters, and sterol ferulates) (7% of total lipids) were also present among the lipids of wheat bran. The use of wheat bran as a valuable source of phytochemicals of interest in the context of a wheat bran biorefinery is discussed.
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http://dx.doi.org/10.1021/jf404772bDOI Listing
February 2014

Compositional analysis of Chinese water chestnut (Eleocharis dulcis) cell-wall material from parenchyma, epidermis, and subepidermal tissues.

J Agric Food Chem 2013 Oct 25;61(40):9680-8. Epub 2013 Sep 25.

Biorefinery Centre, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom.

Chinese water chestnut (Eleocharis dulcis (Burman f.) Trin ex Henschel) is a corm consumed globally in Oriental-style cuisine. The corm consists of three main tissues, the epidermis, subepidermis, and parenchyma; the cell walls of which were analyzed for sugar, phenolic, and lignin content. Sugar content, measured by gas chromatography, was higher in the parenchyma cell walls (931 μg/mg) than in the subepidermis (775 μg/mg) or epidermis (685 μg/mg). The alkali-extractable phenolic content, measured by high-performance liquid chromatography, was greater in the epidermal (32.4 μg/mg) and subepidermal cell walls (21.7 μg/mg) than in the cell walls of the parenchyma (12.3 μg/mg). The proportion of diferulic acids was higher in the parenchyma. The Klason lignin content of epidermal and subepidermal cell walls was ~15%. Methylation analysis of Chinese water chestnut cell-wall polysaccharides identified xyloglucan as the predominant hemicellulose in the parenchyma for the first time, and also a significant pectin component, similar to other nongraminaceous monocots.
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http://dx.doi.org/10.1021/jf401863nDOI Listing
October 2013

Interactions of a lignin-rich fraction from brewer's spent grain with gut microbiota in vitro.

J Agric Food Chem 2013 Jul 26;61(27):6754-62. Epub 2013 Jun 26.

Bio and Process Technology, VTT Technical Research Centre of Finland , P.O. Box 1000, FI-02044 VTT Espoo, Finland.

Lignin is a constituent of plant cell walls and thus is classified as part of dietary fiber. However, little is known about the role of lignin in gastrointestinal fermentation. In this work, a lignin-rich fraction was prepared from brewer's spent grain and subjected to an in vitro colon model to study its potential bioconversions and interactions with fecal microbiota. No suppression of microbial conversion by the fraction was observed in the colon model, as measured as short-chain fatty acid production. Furthermore, no inhibition on the growth was observed when the fraction was incubated with strains of lactobacilli and bifidobacteria. In fact, the lignin-rich fraction enabled bifidobacteria to survive longer than with glucose. Several transiently appearing phenolic compounds, very likely originating from lignin, were observed during the fermentation. This would indicate that the gut microbiota was able to partially degrade lignin and metabolize the released compounds.
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http://dx.doi.org/10.1021/jf401738xDOI Listing
July 2013

Versatile peroxidase as a valuable tool for generating new biomolecules by homogeneous and heterogeneous cross-linking.

Enzyme Microb Technol 2013 May 19;52(6-7):303-11. Epub 2013 Mar 19.

Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.

The modification and generation of new biomolecules intended to give higher molecular-mass species for biotechnological purposes, can be achieved by enzymatic cross-linking. The versatile peroxidase (VP) from Pleurotus eryngii is a high redox-potential enzyme with oxidative activity on a wide variety of substrates. In this study, VP was successfully used to catalyze the polymerization of low molecular mass compounds, such as lignans and peptides, as well as larger macromolecules, such as protein and complex polysaccharides. Different analytical, spectroscopic, and rheological techniques were used to determine structural changes and/or variations of the physicochemical properties of the reaction products. The lignans secoisolariciresinol and hydroxymatairesinol were condensed by VP forming up to 8 unit polymers in the presence of organic co-solvents and Mn(2+). Moreover, 11 unit of the peptides YIGSR and VYV were homogeneously cross-linked. The heterogeneous cross-linking of one unit of the peptide YIGSR and several lignan units was also achieved. VP could also induce gelation of feruloylated arabinoxylan and the polymerization of β-casein. These results demonstrate the efficacy of VP to catalyze homo- and hetero-condensation reactions, and reveal its potential exploitation for polymerizing different types of compounds.
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http://dx.doi.org/10.1016/j.enzmictec.2013.03.010DOI Listing
May 2013

Pre-hydrolysis with carbohydrases facilitates the release of protein from brewer's spent grain.

Bioresour Technol 2013 May 18;136:529-34. Epub 2013 Mar 18.

VTT Technical Research Centre of Finland, P.O. Box 1000, FI-02044 VTT, Finland.

Brewer's spent grain (BSG) is the most abundant side-stream from brewing. It is food-grade being rich in dietary fibre and protein and thus having potential as their source for both food and non-food applications. Initial treatment of milled BSG with a carbohydrase cocktail from Humicola insolens significantly enhanced the subsequent solubilisation of protein from the residual biomass. When treated with an alkaline protease, 76% of BSG protein was solubilized, whereas the yields were significantly lower with neutral or acidic proteases. In alkaline conditions significant amount of protein (53%) as predominantly low molecular weight protein was solubilized even without any protease addition. The degree of protein solubilisation was influenced by the time of exposure of modified BSG to the alkaline environment. The non-enzymatic protein solubilisation was, however, only observed when BSG had been initially treated with the carbohydrase, suggesting the protein is surrounded by cell wall polysaccharides restricting its initial release.
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http://dx.doi.org/10.1016/j.biortech.2013.03.076DOI Listing
May 2013

Characterization of lipids and lignans in brewer's spent grain and its enzymatically extracted fraction.

J Agric Food Chem 2012 Oct 20;60(39):9910-7. Epub 2012 Sep 20.

Bio and Process Technology, VTT Technical Research Centre of Finland, P.O. Box 1000, FI-02044 VTT, Espoo, Finland.

Brewer's spent grain (BSG), the major side stream of brewing, consists of the husks and the residual parts of malts after the mashing process. BSG was enzymatically fractionated by a two-step treatment with carbohydrate- and protein-degrading enzymes, which solubilized 66% of BSG. BSG contained 11% lipids, which were mostly triglycerides, but also a notable amount of free fatty acids was present. Lipids were mostly solubilized due to the alkaline pH applied in the protease treatment. The main fatty acids were linoleic, palmitic, and oleic acids. Several lignans were identified in BSG, syringaresinol and secoisolariciresinol being the most abundant, many associated with the cell wall matrix and released by the alkaline-protease treatment.
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http://dx.doi.org/10.1021/jf302684xDOI Listing
October 2012