María Fernández-Lobato

Although hydroxytyrosol (HT) exhibits anti-inflammatory, cardioprotective, neuroprotective, and anti-aging activities, it is unstable due to the oxidation of its reactive catechol moiety, causing browning and HT loss during storage. In this study, HT was extracted and purified from olive pomace (alperujo), the semi-solid by-product generated during olive oil production. To enhance its stability, HT was α-glucosylated using a sucrose phosphorylase. Notably, glucosylation proceeded rapidly and quantitatively, with modification occurring at the aromatic ring, yielding a nearly equimolar mixture of the novel compounds hydroxytyrosol 3-O-α-D-glucopyranoside and 4-O-α-D-glucopyranoside. Compared to HT, which is rather labile at pH 6.0 and 8.0, both α-glucosides exhibited remarkable stability across the entire pH range studied. α-Glucosylation of HT reduced its antioxidant capacity (TEAC of 1.24 for HT and 0.26 for the glucoside). Nevertheless, these glucosides exhibit improved stability and may function as prodrug forms, undergoing enzymatic hydrolysis in the intestine to release the active HT aglycone.

Chitosan is a polysaccharide-based biopolymer with unique properties and multiple applications in agricultural and medical industries. Chitin deacetylases (CDAs) can potentially deacetylate chitinous molecules, producing chitosans and partially acetylated chitooligosaccharides (COS). Here, we describe the expression and characterization of a CDA from the filamentous fungus Absidia coerulea, AcCDA1, active toward colloidal chitin, partially deacetylated chitin, chitosan with different degrees of acetylation, and COS. When incubated with COS with degrees of polymerization 3-6, the enzyme rapidly produced fully deacetylated COS, which is a relatively rare property among known CDAs. The mode of action of AcCDA1 toward penta-N-acetyl chitopentaose ((GlcNAc)5) was studied by mass spectrometry, using a novel approach for labeling the reducing N-acetyl-D-glucosamine unit of the COS with chitooligosaccharide oxidase (FgChitO) from Fusarium graminearum. Kinetic studies with COS revealed high enzyme efficiency with kcat/Km values reaching 18.8 mM-1s-1 for (GlcNAc)5. The enzyme removed 12 % of the acetyl groups in colloidal chitin whereas it drastically reduced the degree of acetylation of various chitosans. A comparative study with three other CDAs showed that AcCDA1 is highly efficient enzyme and revealed both similarities and differences. These findings show that various CDAs can achieve different deacetylation targets during the enzymatic processing of chitinous materials.

Enzymatic depolymerization of hyaluronic acid into well-defined oligosaccharides offers a sustainable alternative to chemical methods, providing precise control over product size and functionality. Yet, fungal enzymes capable of this transformation remain largely unexplored, despite fungi being prolific producers of carbohydrate-active enzymes through their complex secretomes. Here, we report the extracellular degradation of high molecular weight hyaluronic acid by the filamentous fungus Purpureocillium lilacinum . Hyaluronan depolymerization was monitored directly in culture supernatants, revealing the progressive accumulation of hyaluronan fragments and oligosaccharides with varying degrees of polymerization. Chromatographic time-resolved analyses showed the sequential appearance of long and short fragments, while mass spectrometric analysis demonstrated a complex product profile composed of even-numbered saturated and both even- and rare odd-numbered unsaturated species. This pattern differs from the uniform profiles typically generated by canonical hyaluronidases, suggesting a non-canonical degradation mechanism. Comparative cultivation of different Purpureocillium species showed that this activity is restricted to P. lilacinum among the species tested. To explore the genetic basis of this phenotype, a de novo transcriptome was assembled and functionally annotated. Although numerous carbohydrate-active enzymes were identified, none of the secreted candidates could be confidently assigned to known canonical hyaluronidase families. In particular, polysaccharide lyases associated with β-elimination mechanisms were not predicted to be secreted, and only a small fraction of glycoside hydrolases from families previously linked to hyaluronan degradation were extracellular. Together, our results establish P. lilacinum as a novel fungal system capable of extracellular hyaluronic acid degradation and support the existence of a non-canonical, potentially multienzymatic pathway. This work expands current knowledge of fungal glycosaminoglycan metabolism and highlights filamentous fungi as an underexplored source of hyaluronan-processing activities with biotechnological potential. Key points: • Purpureocillium lilacinum is a new hyaluronic acid degrader • Degradation yields diverse saturated and unsaturated hyaluronan oligosaccharides • Transcriptomics suggests a non-canonical, multienzymatic degradation system Graphical

Here we report the first β-fructofuranosidase from the Trichoderma genus producing fructooligosaccharides (FOS). The novel enzyme from Trichoderma atroviride (TaINV) here characterized was heterologously expressed, purified, and biochemically analyzed. TaINV exhibited hydrolytic activity mainly toward sucrose and other substrates containing β-(2 → 1) linkages, with minor activity toward β-(2 → 6) bonds. In addition to hydrolysis, it catalyzed the synthesis of FOS of all three structural series ( ¹ F-FOS, ⁶ F-FOS, and ⁶ G-FOS). At the maximal production point, TaINV synthesized 252 g/L of total FOS, representing 50.3% (w/w) of the total sugars in the reaction mixture, with 1-kestose as the major product, representing ~ 85% of the total products synthesized. Structural analysis based on AlphaFold-predicted TaINV model and comparative superimposition with GH32-substrate complexes revealed conserved catalytic motifs and residues located in positions associated with substrate binding and specificity in characterized GH32 enzymes. Site-directed mutagenesis confirmed the essential role of the catalytic triad (Asp63, Asp201, Glu277) and identified additional residues shaping transfructosylation specificity. Variants including substitutions W60Y and N62S increased total FOS production, reaching 62.7% and 57.4% (w/w) of total sugars, respectively, which are comparable to yields obtained with commercial enzymes. Overall, TaINV represents a distinct intracellular fungal β-fructofuranosidase with strong transfructosylation capacity and preference for short-chain FOS. These findings expand the current knowledge of GH32 enzyme diversity and highlight TaINV as a promising biocatalyst for the efficient production of low-degree polymerization FOS with potential prebiotic applications. Key points: • Novel Trichoderma β-fructofuranosidase with high transfructosylation activity. • Catalytic residues defining FOS synthesis identified by structure–function analysis. • Engineered variant boosting FOS yield to 62% of total sugars. Graphical

Endochitinase Chit33 has shown great potential in converting chitin, a recalcitrant waste, into bioactive chitooligosaccharides (COS). This study evaluates how cellulose-binding domain (CBD) and chitin-binding domain (ChBD) affect the hydrolytic activity and product specificity of Chit33. Recombinant proteins were produced and isolated with a simple yeast extracellular medium concentration. The domain functionality was proved using chitin and cellulose supports. ChBD provided more stable immobilization than CBD but reduced the Chit33 activity. CBD enhanced the enzyme activity on both colloidal (α-/β-allomorphs) and crystalline chitin, doubling it on α-chitin, although not on their deacetylated forms. Besides, CBD increased the COS production from the colloidal forms of α-/β-chitin (by 30% and 85%, respectively) and expanded the product diversity from 1 to 9 N-acetylglucosamine units. In contrast, Chit33-ChBD predominantly yielded chitin tetrasaccharides. These findings highlight the importance of selecting appropriate binding domains to tailor product specificity, as polymerization and acetylation degrees directly impact the COS biological properties.

The α-glucosidase from Schwanniomyces occidentalis (GAM1p) was expressed in Komagataella phaffii to about 70 mg/L, and its transferase activity studied in detail. Several isomaltooligosaccharides (IMOS) were formed using 200 g/L maltose. The major production of IMOS (81.3 g/L) was obtained when 98% maltose was hydrolysed, of which 34.8 g/L corresponded to isomaltose, 26.9 g/L to isomaltotriose, and 19.6 g/L to panose. The addition of glucose shifted the IMOS synthesis towards products containing exclusively α(1 → 6)-linkages, increasing the production of isomaltose and isomaltotriose about 2–4 fold, enabling the formation of isomaltotetraose, and inhibiting that of panose to about 12 times. In addition, the potential of this enzyme to glycosylate 12 possible hydroxylated acceptors, including eight sugars and four phenolic compounds, was evaluated. Among them, only sucrose, xylose, and piceid (a monoglucosylated derivative of resveratrol) were glucosylated, and the main synthesised products were purified and characterised by MS and NMR. Theanderose, α(1 → 4)-D-glucosyl-xylose, and a mixture of piceid mono- and diglucoside were obtained with sucrose, xylose, and piceid as acceptors, respectively. Maximum production of theanderose reached 81.7 g/L and that of the glucosyl-xylose 26.5 g/L, whereas 3.4 g/L and only 1 g/L were produced of the piceid mono- and diglucoside respectively. Key points: • Overexpression of a yeast α-glucosidase producing novel molecules. • Yeast enzyme producing the heterooligosaccharides theanderose and glucosyl-xylose. • Glycosylation of the polyphenol piceid by a yeast α-glucosidase.

Background: Chitinases are widely distributed enzymes that perform the biotransformation of chitin, one of the most abundant polysaccharides on the biosphere, into useful value-added chitooligosaccharides (COS) with a wide variety of biotechnological applications in food, health, and agricultural fields. One of the most important group of enzymes involved in the degradation of chitin comprises the glycoside hydrolase family 18 (GH18), which harbours endo- and exo-enzymes that act synergistically to depolymerize chitin. The secretion of a chitinase activity from the ubiquitous yeast Mestchnikowia pulcherrima and their involvement in the post-harvest biological control of fungal pathogens was previously reported. Results: Three new chitinases from M. pulcherrima, MpChit35, MpChit38 and MpChit41, were molecularly characterized and extracellularly expressed in Pichia pastoris to about 91, 90 and 71 mU ml^− 1, respectively. The three enzymes hydrolysed colloidal chitin with optimal activity at 45 ºC and pH 4.0-4.5, increased 2-times their activities using 1 mM of Mn²⁺ and hydrolysed different types of commercial chitosan. The partial separation and characterization of the complex COS mixtures produced from the hydrolysis of chitin and chitosan were achieved by a new anionic chromatography HPAEC-PAD method and mass spectrometry assays. An overview of the predicted structures of these proteins and their catalytic modes of action were also presented. Depicted their high sequence and structural homology, MpChit35 acted as an exo-chitinase producing di-acetyl-chitobiose from chitin while MpChit38 and MpChit41 both acted as endo-chitinases producing tri-acetyl-chitotriose as main final product. Conclusions: Three new chitinases from the yeast M. pulcherrima were molecularly characterized and their enzymatic and structural characteristics analysed. These enzymes transformed chitinous materials to fully and partially acetylated COS through different modes of splitting, which make them interesting biocatalysts for deeper structural-function studies on the challenging enzymatic conversion of chitin.

Rhodotorula dairenensis β-fructofuranosidase is a highly glycosylated enzyme with broad substrate specificity that catalyzes the synthesis of 6-kestose and a mixture of the three series of fructooligosaccharides (FOS), fructosylating a variety of carbohydrates and other molecules as alditols. We report here its three-dimensional structure, showing the expected bimodular arrangement and also a unique long elongation at its N-terminus containing extensive O-glycosylation sites that form a peculiar arrangement with a protruding loop within the dimer. This region is not required for activity but could provide a molecular tool to target the dimeric protein to its receptor cellular compartment in the yeast. A truncated inactivated form was used to obtain complexes with fructose, sucrose and raffinose, and a Bis-Tris molecule was trapped, mimicking a putative acceptor substrate. The crystal structure of the complexes reveals the major traits of the active site, with Asn387 controlling the substrate binding mode. Relevant residues were selected for mutagenesis, the variants being biochemically characterized through their hydrolytic and transfructosylating activity. All changes decrease the hydrolytic efficiency against sucrose, proving their key role in the activity. Moreover, some of the generated variants exhibit redesigned transfructosylating specificity, which may be used for biotechnological purposes to produce novel fructosyl-derivatives.