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.

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.

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.

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.