Expression and Characterization of Two α-l-Arabinofuranosidases from Talaromyces amestolkiae: Role of These Enzymes in Biomass Valorization.

α-l-arabinofuranosidases are glycosyl hydrolases that catalyze the break between α-l-arabinofuranosyl substituents or between α-l-arabinofuranosides and xylose from xylan or xylooligosaccharide backbones. While they belong to several glycosyl hydrolase (GH) families, there are only 24 characterized GH62 arabinofuranosidases, making them a small and underrepresented group, with many of their features remaining unknown. Aside from their applications in the food industry, arabinofuranosidases can also aid in the processing of complex lignocellulosic materials, where cellulose, hemicelluloses, and lignin are closely linked. These materials can be fully converted into sugar monomers to produce secondary products like second-generation bioethanol. Alternatively, they can be partially hydrolyzed to release xylooligosaccharides, which have prebiotic properties. While endoxylanases and ß-xylosidases are also necessary to fully break down the xylose backbone from xylan, these enzymes are limited when it comes to branched polysaccharides. In this article, two new GH62 α-l-arabinofuranosidases from Talaromyces amestolkiae (named ARA1 and ARA-2) have been heterologously expressed and characterized. ARA-1 is more sensitive to changes in pH and temperature, whereas ARA-2 is a robust enzyme with wide pH and temperature tolerance. Both enzymes preferentially act on arabinoxylan over arabinan, although ARA-1 has twice the catalytic efficiency of ARA-2 on this substrate. The production of xylooligosaccharides from arabinoxylan catalyzed by a T. amestolkiae endoxylanase was significantly increased upon pretreatment of the polysaccharide with ARA-1 or ARA-2, with the highest synergism values reported to date. Finally, both enzymes (ARA-1 or ARA-2 and endoxylanase) were successfully applied to enhance saccharification by combining them with a ß-xylosidase already characterized from the same fungus.

Fungal-Lactobacteria Consortia and Enzymatic Catalysis for Polylactic Acid Production.

Polylactic acid (PLA) is the main biobased plastic manufactured on an industrial scale. This polymer is synthetized by chemical methods, and there is a strong demand for the implementation of clean technologies. This work focuses on the microbial fermentation of agro-industrial waste rich in starch for the production of lactic acid (LA) in a consolidated bioprocess, followed by the enzymatic synthesis of PLA. Lactic acid bacteria (LAB) and the fungus Rhizopus oryzae were evaluated as natural LA producers in pure cultures or in fungal-lactobacteria co-cultures formed by an LAB and a fungus selected for its metabolic capacity to degrade starch and to form consortia with LAB. Microbial interaction was analyzed by scanning electron microscopy and biofilm production was quantified. The results show that the fungus Talaromyces amestolkiae and Lactiplantibacillus plantarum M9MG6-B2 establish a cooperative relationship to exploit the sugars from polysaccharides provided as carbon sources. Addition of the quorum sensing molecule dodecanol induced LA metabolism of the consortium and resulted in improved cooperation, producing 99% of the maximum theoretical yield of LA production from glucose and 65% from starch. Finally, l-PLA oligomers (up to 19-LA units) and polymers (greater than 5 kDa) were synthetized by LA polycondensation and enzymatic ring-opening polymerization catalyzed by the non-commercial lipase OPEr, naturally produced by the fungus Ophiostoma piceae.

Glycosylation of Epigallocatechin Gallate by Engineered Glycoside Hydrolases from Talaromyces amestolkiae: Potential Antiproliferative and Neuroprotective Effect of These Molecules.

Glycoside hydrolases (GHs) are enzymes that hydrolyze glycosidic bonds, but some of them can also catalyze the synthesis of glycosides by transglycosylation. However, the yields of this reaction are generally low since the glycosides formed end up being hydrolyzed by these same enzymes. For this reason, mutagenic variants with null or drastically reduced hydrolytic activity have been developed, thus enhancing their synthetic ability. Two mutagenic variants, a glycosynthase engineered from a ß-glucosidase (BGL-1-E521G) and a thioglycoligase from a ß-xylosidase (BxTW1-E495A), both from the ascomycete Talaromyces amestolkiae, were used to synthesize three novel epigallocatechin gallate (EGCG) glycosides. EGCG is a phenolic compound from green tea known for its antioxidant effects and therapeutic benefits, whose glycosylation could increase its bioavailability and improve its bioactive properties. The glycosynthase BGL-1-E521G produced a ß-glucoside and a ß-sophoroside of EGCG, while the thioglycoligase BxTW1-E495A formed the ß-xyloside of EGCG. Glycosylation occurred in the 5″ and 4″ positions of EGCG, respectively. In this work, the reaction conditions for glycosides' production were optimized, achieving around 90% conversion of EGCG with BGL-1-E521G and 60% with BxTW1-E495A. The glycosylation of EGCG caused a slight loss of its antioxidant capacity but notably increased its solubility (between 23 and 44 times) and, in the case of glucoside, also improved its thermal stability. All three glycosides showed better antiproliferative properties on breast adenocarcinoma cell line MDA-MB-231 than EGCG, and the glucosylated and sophorylated derivatives induced higher neuroprotection, increasing the viability of SH-S5Y5 neurons exposed to okadaic acid.

A Fungal Versatile GH10 Endoxylanase and Its Glycosynthase Variant: Synthesis of Xylooligosaccharides and Glycosides of Bioactive Phenolic Compounds.

The study of endoxylanases as catalysts to valorize hemicellulosic residues and to obtain glycosides with improved properties is a topic of great industrial interest. In this work, a GH10 ß-1,4-endoxylanase (XynSOS), from the ascomycetous fungus Talaromyces amestolkiae, has been heterologously produced in Pichia pastoris, purified, and characterized. rXynSOS is a highly glycosylated monomeric enzyme of 53 kDa that contains a functional CBM1 domain and shows its optimal activity on azurine cross-linked (AZCL)-beechwood xylan at 70 °C and pH 5. Substrate specificity and kinetic studies confirmed its versatility and high affinity for beechwood xylan and wheat arabinoxylan. Moreover, rXynSOS was capable of transglycosylating phenolic compounds, although with low efficiencies. For expanding its synthetic capacity, a glycosynthase variant of rXynSOS was developed by directed mutagenesis, replacing its nucleophile catalytic residue E236 by a glycine (rXynSOS-E236G). This novel glycosynthase was able to synthesize ß-1,4-xylooligosaccharides (XOS) of different lengths (four, six, eight, and ten xylose units), which are known to be emerging prebiotics. rXynSOS-E236G was also much more active than the native enzyme in the glycosylation of a broad range of phenolic compounds with antioxidant properties. The interesting capabilities of rXynSOS and its glycosynthase variant make them promising tools for biotechnological applications.

Synthesis and Characterization of a Novel Resveratrol Xylobioside Obtained Using a Mutagenic Variant of a GH10 Endoxylanase.

Resveratrol is a natural polyphenol with antioxidant activity and numerous health benefits. However, in vivo application of this compound is still a challenge due to its poor aqueous solubility and rapid metabolism, which leads to an extremely low bioavailability in the target tissues. In this work, rXynSOS-E236G glycosynthase, designed from a GH10 endoxylanase of the fungus Talaromyces amestolkiae, was used to glycosylate resveratrol by using xylobiosyl-fluoride as a sugar donor. The major product from this reaction was identified by NMR as 3-O-ꞵ-d-xylobiosyl resveratrol, together with other glycosides produced in a lower amount as 4'-O-ꞵ-d-xylobiosyl resveratrol and 3-O-ꞵ-d-xylotetraosyl resveratrol. The application of response surface methodology made it possible to optimize the reaction, producing 35% of 3-O-ꞵ-d-xylobiosyl resveratrol. Since other minor glycosides are obtained in addition to this compound, the transformation of the phenolic substrate amounted to 70%. Xylobiosylation decreased the antioxidant capacity of resveratrol by 2.21-fold, but, in return, produced a staggering 4,866-fold improvement in solubility, facilitating the delivery of large amounts of the molecule and its transit to the colon. A preliminary study has also shown that the colonic microbiota is capable of releasing resveratrol from 3-O-ꞵ-d-xylobiosyl resveratrol. These results support the potential of mutagenic variants of glycosyl hydrolases to synthesize highly soluble resveratrol glycosides, which could, in turn, improve the bioavailability and bioactive properties of this polyphenol.

Lytic Polysaccharide Monooxygenase from Talaromyces amestolkiae with an Enigmatic Linker-like Region: The Role of This Enzyme on Cellulose Saccharification.

The first lytic polysaccharide monooxygenase (LPMO) detected in the genome of the widespread ascomycete Talaromyces amestolkiae (TamAA9A) has been successfully expressed in Pichia pastoris and characterized. Molecular modeling of TamAA9A showed a structure similar to those from other AA9 LPMOs. Although fungal LPMOs belonging to the genera Penicillium or Talaromyces have not been analyzed in terms of regioselectivity, phylogenetic analyses suggested C1/C4 oxidation which was confirmed by HPAEC. To ascertain the function of a C-terminal linker-like region present in the wild-type sequence of the LPMO, two variants of the wild-type enzyme, one without this sequence and one with an additional C-terminal carbohydrate binding domain (CBM), were designed. The three enzymes (native, without linker and chimeric variant with a CBM) were purified in two chromatographic steps and were thermostable and active in the presence of H2O2. The transition midpoint temperature of the wild-type LPMO (Tm = 67.7 °C) and its variant with only the catalytic domain (Tm = 67.6 °C) showed the highest thermostability, whereas the presence of a CBM reduced it (Tm = 57.8 °C) and indicates an adverse effect on the enzyme structure. Besides, the potential of the different T. amestolkiae LPMO variants for their application in the saccharification of cellulosic and lignocellulosic materials was corroborated.

Fungal glycosyl hydrolases for sustainable plant biomass valorization: Talaromyces amestolkiae as a model fungus.

As the main decomposers and recyclers in nature, fungi secrete complex mixtures of extracellular enzymes for degradation of plant biomass, which is essential for mobilization of the organic carbon fixed by the photosynthesis in vegetal cells. Biotechnology can emulate the closed natural biological cycles, using lignocellulosic biomass as a renewable resource and lignocellulolytic fungal enzymes as catalysts to sustainably produce consumer goods. Cellulose and hemicellulose are the major polysaccharides on Earth, and the main enzymes involved in their hydrolytic depolymerization are cellulases (endoglucanases, cellobiohydrolases, and ß-glucosidases) and hemicellulases (mainly endoxylanases and ß-xylosidases). This work will focus on the enzymes secreted by the filamentous ascomycete Talaromyces amestolkiae and on some of their biotechnological applications. Their excellent hydrolytic activity was demonstrated by the partial degradation of xylans to prebiotic oligosaccharides by the endoxylanase XynN, or by the saccharification of lignocellulosic wastes to monosaccharides (fermentable to ethanol) either by the whole secretomes or by isolated enzymes used as supplements of commercial cocktails. However, apart from their expected hydrolytic activity, some of the ß-glycosidases produced by this strain catalyze the transfer of a sugar molecule to specific aglycons by transglycosylation. As the synthesis of customized glycoconjugates is a major goal for biocatalysis, mutant variants of the ß-xyloxidase BxTW1 and the ß-glucosidases BGL-1 and BGL-2 were obtained by directed mutagenesis, substantially improving the regioselective production yields of bioactive glycosides since they showed reduced or null hydrolytic activity.

Production of a ß-Glucosidase-Rich Cocktail from Talaromyces amestolkiae Using Raw Glycerol: Its Role for Lignocellulose Waste Valorization.

As ß-glucosidases represent the major bottleneck for the industrial degradation of plant biomass, great efforts are being devoted to discover both novel and robust versions of these enzymes, as well as to develop efficient and inexpensive ways to produce them. In this work, raw glycerol from chemical production of biodiesel was tested as carbon source for the fungus Talaromyces amestolkiae with the aim of producing enzyme ß-glucosidase-enriched cocktails. Approximately 11 U/mL ß-glucosidase was detected in these cultures, constituting the major cellulolytic activity. Proteomic analysis showed BGL-3 as the most abundant protein and the main ß-glucosidase. This crude enzyme was successfully used to supplement a basal commercial cellulolytic cocktail (Celluclast 1.5 L) for saccharification of pretreated wheat straw, corroborating that even hardly exploitable industrial wastes, such as glycerol, can be used as secondary raw materials to produce valuable enzymatic preparations in a framework of the circular economy.

Optimization of ß-1,4-Endoxylanase Production by an Aspergillus niger Strain Growing on Wheat Straw and Application in Xylooligosaccharides Production.

Plant biomass constitutes the main source of renewable carbon on the planet. Its valorization has traditionally been focused on the use of cellulose, although hemicellulose is the second most abundant group of polysaccharides on Earth. The main enzymes involved in plant biomass degradation are glycosyl hydrolases, and filamentous fungi are good producers of these enzymes. In this study, a new strain of Aspergillus niger was used for hemicellulase production under solid-state fermentation using wheat straw as single-carbon source. Physicochemical parameters for the production of an endoxylanase were optimized by using a One-Factor-at-a-Time (OFAT) approach and response surface methodology (RSM). Maximum xylanase yield after RSM optimization was increased 3-fold, and 1.41- fold purification was achieved after ultrafiltration and ion-exchange chromatography, with about 6.2% yield. The highest activity of the purified xylanase was observed at 50 °C and pH 6. The enzyme displayed high thermal and pH stability, with more than 90% residual activity between pH 3.0-9.0 and between 30-40 °C, after 24 h of incubation, with half-lives of 30 min at 50 and 60 °C. The enzyme was mostly active against wheat arabinoxylan, and its kinetic parameters were analyzed (Km = 26.06 mg·mL-1 and Vmax = 5.647 U·mg-1). Wheat straw xylan hydrolysis with the purified ß-1,4 endoxylanase showed that it was able to release xylooligosaccharides, making it suitable for different applications in food technology.

Characterization of a Dye-Decolorizing Peroxidase from Irpex lacteus Expressed in Escherichia coli: An Enzyme with Wide Substrate Specificity Able to Transform Lignosulfonates.

A dye-decolorizing peroxidase (DyP) from Irpex lacteus was cloned and heterologously expressed as inclusion bodies in Escherichia coli. The protein was purified in one chromatographic step after its in vitro activation. It was active on ABTS, 2,6-dimethoxyphenol (DMP), and anthraquinoid and azo dyes as reported for other fungal DyPs, but it was also able to oxidize Mn2+ (as manganese peroxidases and versatile peroxidases) and veratryl alcohol (VA) (as lignin peroxidases and versatile peroxidases). This corroborated that I. lacteus DyPs are the only enzymes able to oxidize high redox potential dyes, VA and Mn+2. Phylogenetic analysis grouped this enzyme with other type D-DyPs from basidiomycetes. In addition to its interest for dye decolorization, the results of the transformation of softwood and hardwood lignosulfonates suggest a putative biological role of this enzyme in the degradation of phenolic lignin.

Gene family expansions and transcriptome signatures uncover fungal adaptations to wood decay.

Because they comprise some of the most efficient wood-decayers, Polyporales fungi impact carbon cycling in forest environment. Despite continuous discoveries on the enzymatic machinery involved in wood decomposition, the vision on their evolutionary adaptation to wood decay and genome diversity remains incomplete. We combined the genome sequence information from 50 Polyporales species, including 26 newly sequenced genomes and sought for genomic and functional adaptations to wood decay through the analysis of genome composition and transcriptome responses to different carbon sources. The genomes of Polyporales from different phylogenetic clades showed poor conservation in macrosynteny, indicative of genome rearrangements. We observed different gene family expansion/contraction histories for plant cell wall degrading enzymes in core polyporoids and phlebioids and captured expansions for genes involved in signalling and regulation in the lineages of white rotters. Furthermore, we identified conserved cupredoxins, thaumatin-like proteins and lytic polysaccharide monooxygenases with a yet uncharacterized appended module as new candidate players in wood decomposition. Given the current need for enzymatic toolkits dedicated to the transformation of renewable carbon sources, the observed genomic diversity among Polyporales strengthens the relevance of mining Polyporales biodiversity to understand the molecular mechanisms of wood decay.

Hemicellulases from Penicillium and Talaromyces for lignocellulosic biomass valorization: A review.

The term hemicellulose groups different polysaccharides with heterogeneous structures, mannans, xyloglucans, mixed-linkage ß-glucans and xylans, which differ in their backbone and branches, and in the type and distribution of glycosidic linkages. The enzymatic degradation of these complex polymers requires the concerted action of multiple hemicellulases and auxiliary enzymes. Most commercial enzymes are produced by Trichoderma and Aspergillus species, but recent studies have disclosed Penicillium and Talaromyces as promising sources of hemicellulases. In this review, we summarize the current knowledge on the hemicellulolytic system of these genera, and the role of hemicellulases in the disruption and synthesis of glycosidic bonds. In both cases, the enzymes from Penicillium and Talaromyces represent an interesting alternative for valorization of lignocellulosic biomass in the current framework of circular economy.

Enzymatic glycosylation of bioactive acceptors catalyzed by an immobilized fungal ß-xylosidase and its multi-glycoligase variant.

A recombinant ß-xylosidase (rBxTW1) from the ascomycete Talaromyces amestolkiae and a mutant derived from it, with mostly synthetic activity, have been immobilized as magnetic cross-linked enzyme aggregates (mCLEAs). The mCLEAs of rBxTW1 kept the excellent hydrolytic and O-transxylosylating activities of the free enzyme and had improved thermal and pH stability. The mCLEAs of the mutant also maintained or improved the catalytic properties of the soluble enzyme, synthetizing the O-xylosides of vanillin and (-)-epigallocatechin gallate, and the N- and S-xyloside of 3,5-dibromo-1,2,4-triazole and thiophenol, respectively. The mCLEAs were recyclable across 4 cycles of synthesis of the O-xylosides through a green and highly selective process. The magnetic properties of the scaffold used for immobilization may allow the easy recovery and reuse of the biocatalyst even from reactions containing insoluble lignocellulosic biomass.

Thioglycoligase derived from fungal GH3 ß-xylosidase is a multi-glycoligase with broad acceptor tolerance.

The synthesis of customized glycoconjugates constitutes a major goal for biocatalysis. To this end, engineered glycosidases have received great attention and, among them, thioglycoligases have proved useful to connect carbohydrates to non-sugar acceptors. However, hitherto the scope of these biocatalysts was considered limited to strong nucleophilic acceptors. Based on the particularities of the GH3 glycosidase family active site, we hypothesized that converting a suitable member into a thioglycoligase could boost the acceptor range. Herein we show the engineering of an acidophilic fungal ß-xylosidase into a thioglycoligase with broad acceptor promiscuity. The mutant enzyme displays the ability to form O-, N-, S- and Se- glycosides together with sugar esters and phosphoesters with conversion yields from moderate to high. Analyses also indicate that the pKa of the target compound was the main factor to determine its suitability as glycosylation acceptor. These results expand on the glycoconjugate portfolio attainable through biocatalysis.

A glucotolerant ß-glucosidase from the fungus Talaromyces amestolkiae and its conversion into a glycosynthase for glycosylation of phenolic compounds.

BACKGROUND: The interest for finding novel ß-glucosidases that can improve the yields to produce second-generation (2G) biofuels is still very high. One of the most desired features for these enzymes is glucose tolerance, which enables their optimal activity under high-glucose concentrations. Besides, there is an additional focus of attention on finding novel enzymatic alternatives for glycoside synthesis, for which a mutated version of glycosidases, named glycosynthases, has gained much interest in recent years. RESULTS: In this work, a glucotolerant ß-glucosidase (BGL-1) from the ascomycete fungus Talaromyces amestolkiae has been heterologously expressed in Pichia pastoris, purified, and characterized. The enzyme showed good efficiency on p-nitrophenyl glucopyranoside (pNPG) (Km= 3.36 ± 0.7 mM, kcat= 898.31 s-1), but its activity on cellooligosaccharides, the natural substrates of these enzymes, was much lower, which could limit its exploitation in lignocellulose degradation applications. Interestingly, when examining the substrate specificity of BGL-1, it showed to be more active on sophorose, the ß-1,2 disaccharide of glucose, than on cellobiose. Besides, the transglycosylation profile of BGL-1 was examined, and, for expanding its synthetic capacities, it was converted into a glycosynthase. The mutant enzyme, named BGL-1-E521G, was able to use α-D-glucosyl-fluoride as donor in glycosylation reactions, and synthesized glucosylated derivatives of different pNP-sugars in a regioselective manner, as well as of some phenolic compounds of industrial interest, such as epigallocatechin gallate (EGCG). CONCLUSIONS: In this work, we report the characterization of a novel glucotolerant 1,2-ß-glucosidase, which also has a considerable activity on 1,4-ß-glucosyl bonds, that has been cloned in P. pastoris, produced, purified and characterized. In addition, the enzyme was converted into an efficient glycosynthase, able to transfer glucose molecules to a diversity of acceptors for obtaining compounds of interest. The remarkable capacities of BGL-1 and its glycosynthase mutant, both in hydrolysis and synthesis, suggest that it could be an interesting tool for biotechnological applications.

Exploiting xylan as sugar donor for the synthesis of an antiproliferative xyloside using an enzyme cascade.

BACKGROUND: Currently, industrial societies are seeking for green alternatives to conventional chemical synthesis. This demand has merged with the efforts to convert lignocellulosic biomass into value-added products. In this context, xylan, as one of main components of lignocellulose, has emerged as a raw material with high potential for advancing towards a sustainable economy. RESULTS: In this study, the recombinant endoxylanase rXynM from the ascomycete Talaromyces amestolkiae has been heterologously expressed in Pichia pastoris and used as one of the catalysts of an enzyme cascade developed to synthesize the antiproliferative 2-(6-hydroxynaphthyl) ß-D-xylopyranoside, by transglycosylation of 2,6-dihydroxynaphthalene. The approach combines the use of two fungal xylanolytic enzymes, rXynM and the ß-xylosidase rBxTW1 from the same fungus, with the cost-effective substrate xylan. The reaction conditions for the cascade were optimized by a Central Composite Design. Maximal productions of 0.59 and 0.38 g/L were reached using beechwood xylan and birchwood xylan, respectively. For comparison, xylans from other sources were tested in the same reaction, suggesting that a specific optimization is required for each xylan variety. The results obtained using this enzyme cascade and xylan were similar or better to those previously reported for a single catalyst and xylobiose, an expensive sugar donor. CONCLUSIONS: Beechwood and birchwood xylan, two polysaccharides easily available from biomass, were used in a novel enzyme cascade to synthetize an antiproliferative agent. The approach represents a green alternative to the conventional chemical synthesis of 2-(6-hydroxynaphthyl) ß-D-xylopyranoside using a cost-effective substrate. The work highlights the role of xylan as a raw material for producing value-added products and the potential of fungal xylanolytic enzymes in the biomass conversion.

Transglycosylation products generated by Talaromyces amestolkiae GH3 ß-glucosidases: effect of hydroxytyrosol, vanillin and its glucosides on breast cancer cells.

BACKGROUND: Transglycosylation represents one of the most promising approaches for obtaining novel glycosides, and plant phenols and polyphenols are emerging as one of the best targets for creating new molecules with enhanced capacities. These compounds can be found in diet and exhibit a wide range of bioactivities, such as antioxidant, antihypertensive, antitumor, neuroprotective and anti-inflammatory, and the eco-friendly synthesis of glycosides from these molecules can be a suitable alternative for increasing their health benefits. RESULTS: Transglycosylation experiments were carried out using different GH3 ß-glucosidases from the fungus Talaromyces amestolkiae. After a first screening with a wide variety of potential transglycosylation acceptors, mono-glucosylated derivatives of hydroxytyrosol, vanillin alcohol, 4-hydroxybenzyl alcohol, and hydroquinone were detected. The reaction products were analyzed by thin-layer chromatography, high-pressure liquid chromatography, and mass spectrometry. Hydroxytyrosol and vanillyl alcohol were selected as the best options for transglycosylation optimization, with a final conversion yield of 13.8 and 19% of hydroxytyrosol and vanillin glucosides, respectively. NMR analysis confirmed the structures of these compounds. The evaluation of the biological effect of these glucosides using models of breast cancer cells, showed an enhancement in the anti-proliferative capacity of the vanillin derivative, and an improved safety profile of both glucosides. CONCLUSIONS: GH3 ß-glucosidases from T. amestolkiae expressed in P. pastoris were able to transglycosylate a wide variety of acceptors. Between them, phenolic molecules like hydroxytyrosol, vanillin alcohol, 4-hydroxybenzyl alcohol, and hydroquinone were the most suitable for its interesting biological properties. The glycosides of hydroxytyrosol and vanillin were tested, and they improved the biological activities of the original aglycons on breast cancer cells.

Purification and characterization of a fungal laccase from the ascomycete Thielavia sp. and its role in the decolorization of a recalcitrant dye.

A laccase-producing ascomycete was isolated from arid soil in Tunisia. This fungus was identified as Thielavia sp. using the phylogenetic analysis of rDNA internal transcribed spacers. The extracellular laccase produced by the fungus was purified to electrophoretic homogeneity, showing a molecular mass around 70 kDa. The enzyme had an optimum pH of 5.0 and 6.0 for ABTS and 2,6­DMP, respectively and it showed remarkable high thermal stability, showing its optimal temperature at 70 °C (against 2,6­DMP). It presented slight inhibiting effect by EDTA, SDS and l­cyst although this effect was more marked by sodium azide (0.1 mM). On the other hand, it showed tolerance to up to 300 mM NaCl, retaining around 50% of its activity at 900 mM. Among the metal ions tested on TaLac1, Mn2+ showed an activating effect. Their kinetic parameters Km and kcat were 23.7 µM and 4.14 s-1 for ABTS, and 24.3 µM and 3.46 s-1 towards 2,6­DMP. The purified enzyme displayed greater efficiency in Remazol Brilliant Blue R decolorization (90%) in absence of redox mediator, an important property for biotechnological applications.

The ß-glucosidase secreted by Talaromyces amestolkiae under carbon starvation: a versatile catalyst for biofuel production from plant and algal biomass.

BACKGROUND: In the last years, the most outstanding trend for obtaining high added-value components and second-generation (2G) biofuels consisted on exploitation of plant biomass. But recently, 3G biofuels, based in algae biomass, have emerged as a great alternative for production of energy. RESULTS: In this work, a versatile ß-glucosidase from the ascomycete fungus Talaromyces amestolkiae has been purified, characterized, and heterologously expressed. The synthesis of this ß-glucosidase (BGL-3) was not induced by cellulose, and the presence of a specific carbon source is not required for its production, which is uncommon for ß-glucosidases. BGL-3, which was obtained from a basal medium with glucose as carbon source, was profusely secreted under carbon starvation conditions, which was corroborated by qRT-PCR assays. BGL-3 was purified from T. amestolkiae cultures in one step, and biochemically characterized. The enzyme showed high thermal stability, and very high efficiency on pNPG (Km of 0.14 mM and Vmax of 381.1 U/mg), cellobiose (Km of 0.48 mM and Vmax of 447.1 U/mg), and other cello-oligosaccharides. Surprisingly, it also showed remarkable ability to hydrolyze laminarin, a ß-1,3-glucan present in algae. The recombinant enzyme, obtained in the yeast Pichia pastoris, exhibited kinetic and physicochemical properties similar to those found for the native protein. Enzyme efficiency was examined in wheat straw saccharification processes, in which BGL-3 worked better supplementing Celluclast 1.5L than the commercial cellulase cocktail N-50010. Besides, BGL-3 hydrolyzed laminarin more efficiently than a commercial laminarinase. CONCLUSIONS: A very efficient 1,4-ß-glucosidase, which also showed activity over 1,3-ß-glucose bonds, has been produced, purified, and characterized. This is the first report of such versatility in a 1,4-ß-glucosidase. The application of this enzyme for saccharification of wheat straw and laminarin and its comparison with commercial enzymes suggest that it could be an interesting tool for the production of 2G and 3G biofuels.

Enzymatic Synthesis of a Novel Neuroprotective Hydroxytyrosyl Glycoside.

The eco-friendly synthesis of non-natural glycosides from different phenolic antioxidants was carried out using a fungal ß-xylosidase to evaluate changes in their bioactivities. Xylosides from hydroquinone and catechol were successfully formed, although the best results were obtained for hydroxytyrosol, the main antioxidant from olive oil. The formation of the new products was followed by thin-layer chromatography, liquid chromatography, and mass spectrometry. The hydroxytyrosyl xyloside was analyzed in more detail, to maximize its production and evaluate the effect of glycosylation on some hydroxytyrosol properties. The synthesis was optimized up to the highest production reported for a hydroxytyrosyl glycoside. The structure of this compound was solved by two-dimensional nuclear magnetic resonance and identified as 3,4-dihydroxyphenyl-ethyl-O-ß-d-xylopyranoside. Evaluation of its biological effect showed an enhancement of both its neuroprotective capacity and its ability to ameliorate intracellular levels of reactive oxygen species.