A Putative Lignin Copper Oxidase from Trichoderma reesei.

The ability of Trichoderma reesei, a fungus widely used for the commercial production of hemicellulases and cellulases, to grow and modify technical soda lignin was investigated. By quantifying fungal genomic DNA, T. reesei showed growth and sporulation in solid and liquid cultures containing lignin alone. The analysis of released soluble lignin and residual insoluble lignin was indicative of enzymatic oxidative conversion of phenolic lignin side chains and the modification of lignin structure by cleaving the ß-O-4 linkages. The results also showed that polymerization reactions were taking place. A proteomic analysis conducted to investigate secreted proteins at days 3, 7, and 14 of growth revealed the presence of five auxiliary activity (AA) enzymes in the secretome: AA6, AA9, two AA3 enzymes), and the only copper radical oxidase encoded in the genome of T. reesei. This enzyme was heterologously produced and characterized, and its activity on lignin-derived molecules was investigated. Phylogenetic characterization demonstrated that this enzyme belonged to the AA5_1 family, which includes characterized glyoxal oxidases. However, the enzyme displayed overlapping physicochemical and catalytic properties across the AA5 family. The enzyme was remarkably stable at high pH and oxidized both, alcohols and aldehydes with preference to the alcohol group. It was also active on lignin-derived phenolic molecules as well as simple carbohydrates. HPSEC and LC-MS analyses on the reactions of the produced protein on lignin dimers (SS ßß, SS ßO4 and GG ß5) uncovered the polymerizing activity of this enzyme, which was accordingly named lignin copper oxidase (TrLOx). Polymers of up 10 units were formed by hydroxy group oxidation and radical formation. The activations of lignin molecules by TrLOx along with the co-secretion of this enzyme with reductases and FAD flavoproteins oxidoreductases during growth on lignin suggest a synergistic mechanism for lignin breakdown.

Screening New Xylanase Biocatalysts from the Mangrove Soil Diversity.

Mangrove sediments from New Caledonia were screened for xylanase sequences. One enzyme was selected and characterized both biochemically and for its industrial potential. Using a specific cDNA amplification method coupled with a MiSeq sequencing approach, the diversity of expressed genes encoding GH11 xylanases was investigated beneath Avicenia marina and Rhizophora stylosa trees during the wet and dry seasons and at two different sediment depths. GH11 xylanase diversity varied more according to tree species and season, than with respect to depth. One complete cDNA was selected (OFU29) and expressed in Pichia pastoris. The corresponding enzyme (called Xyn11-29) was biochemically characterized, revealing an optimal activity at 40-50 °C and at a pH of 5.5. Xyn11-29 was stable for 48 h at 35 °C, with a half-life of 1 h at 40 °C and in the pH range of 5.5-6. Xyn11-29 exhibited a high hydrolysis capacity on destarched wheat bran, with 40% and 16% of xylose and arabinose released after 24 h hydrolysis. Its activity on wheat straw was lower, with a release of 2.8% and 6.9% of xylose and arabinose, respectively. As the protein was isolated from mangrove sediments, the effect of sea salt on its activity was studied and discussed.

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

BACKGROUND: Lavender (Lavandula angustifolia) and lavandin (a sterile hybrid of L. angustifolia × L. latifolia) 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, ß-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 Pycnoporus cinnabarinus. 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.

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

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 Aspergillus niger and the basidiomycete Neolentinus lepideus. 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 A. niger 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 N. lepideus 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 N. lepideus. The two-step process used mild conditions compatible with green chemistry.