Eliminating the core fucose from the N-glycans of the Fc antibody segment by pathway engineering or enzymatic methods has been shown to enhance the potency of therapeutic antibodies, especially in the context of antibody-dependent cytotoxicity (ADCC). However, there is a significant challenge due to the limited defucosylation efficiency of commercially available α-l-fucosidases. In this study, we report a unique α-l-fucosidase (PnfucA) from the bacterium Prevotella nigrescens that has a low sequence identity compared with all other known α-l-fucosidases and is highly reactive toward a core disaccharide substrate with fucose α(1,3)-, α (1,4)-and α(1,6)-linked to GlcNAc, and is less reactive toward the Fuc-α(1,2)-Gal on the terminal trisaccharide of the oligosaccharide Globo H (Bb3). The kinetic properties of the enzyme, such as its Km and kcat, were determined and the optimized expression of PnfucA gave a yield exceeding 30 mg/L. The recombinant enzyme retained its full activity even after being incubated for 6 h at 37 °C. Moreover, it retained 92 and 87% of its activity after freezing and freeze-drying treatments, respectively, for over 28 days. In a representative glycoengineering of adalimumab (Humira), PnfucA showed remarkable hydrolytic efficiency in cleaving the α(1,6)-linked core fucose from FucGlcNAc on the antibody with a quantitative yield. This enabled the seamless incorporation of biantennary sialylglycans by Endo-S2 D184 M in a one-pot fashion to yield adalimumab in a homogeneous afucosylated glycoform with an improved binding affinity toward Fcγ receptor IIIa.

Mixed linkage (1,3;1,4)-β-d-glucan (MLG) is a well-recognized bioactive carbohydrate and dietary fibre with expanding applications in food industry. The MLG are small components of the cell wall of vegetative tissues of cereals synthetized by members of the Cellulose Synthase-Like genes (Csl). Within the family, the CslF6 has been the major contributor in wheat. It is of significant health and economic benefits to enhance MLG content in wheat, a staple grain with naturally low MLG levels. This study investigated the role of CslF6 gene in MLG synthesis and analysed total MLG contents, cell wall monosaccharide, glycosidic linkage composition, and profile of major comprising oligosaccharides of MLG in various wheat genotypes, their wild relatives (Aegilops caudata and Dasypyrum villosum), and hybrids between them. We observed a relationship between CslF6 gene expression and MLG accumulation across the different wheat lines. While Aegilops caudata and Dasypyrum villosum exhibited higher MLG content than other genotypes, hybrid breeding led to an increase in MLG content by 24.4% in durum wheat and 43.3% in T. aestivum. Variations in the ratios of major oligosaccharides released from MLG by lichenase treatment and in the compositions of cell wall monosaccharides and glycosidic linkages were also found. This study demonstrates that HPAEC-PAD and GC–MS-based glycomics are invaluable tools to assist breeders in selecting high MLG lines.

Barley (1,3;1,4)-β-d-glucanase is believed to have evolved from an ancestral monocotyledon (1,3)-β-d-glucanase, enabling the hydrolysis of (1,3;1,4)-β-d-glucans in the cell walls of leaves and germinating grains. In the present study, we investigated the substrate specificities of variants of the barley enzymes (1,3;1,4)-β-d-glucan endohydrolase [(1,3;1,4)-β-d-glucanase] isoenzyme EII (HvEII) and (1,3)-β-d-glucan endohydrolase [(1,3)-β-d-glucanase] isoenzyme GII (HvGII) obtained by protein segment hybridization and site-directed mutagenesis. Using protein segment hybridization, we obtained three variants of HvEII in which the substrate specificity was that of a (1,3)-β-d-glucanase and one variant that hydrolyzed both (1,3)-β-d-glucans and (1,3;1,4)-β-d-glucans; the wild-type enzyme hydrolyzed only (1,3;1,4)-β-d-glucans. Using substitutions of specific amino acid residues, we obtained one variant of HvEII that hydrolyzed both substrates. However, neither protein segment hybridization nor substitutions of specific amino acid residues gave variants of HvGII that could hydrolyze (1,3;1,4)-β-d-glucans; the wild-type enzyme hydrolyzed only (1,3)-β-d-glucans. Other HvEII and HvGII variants showed changes in specific activity and their ability to degrade the (1,3;1,4)-β-d-glucans or (1,3)-β-d-glucans to larger oligosaccharides. We also used molecular dynamics simulations to identify amino-acid residues or structural regions of wild-type HvEII and HvGII that interact with (1,3;1,4)-β-d-glucans and (1,3)-β-d-glucans, respectively, and may be responsible for the substrate specificities of the two enzymes.

The exopolysaccharides of the Gram-positive bacterium Romboutsia ilealis have recently been shown to include (1,3;1,4)-β-D-glucans. In the present study, we examined another Clostridia bacterium Clostridium ventriculi that has long been considered to contain abundant amounts of cellulose in its exopolysaccharides. We treated alcohol insoluble residues of C. ventriculi that include the exopolysaccharides with the enzyme lichenase that specifically hydrolyses (1,3;1,4)-β-D-glucans, and examined the oligosaccharides released. This showed the presence of (1,3;1,4)-β-D-glucans, which may have previously been mistaken for cellulose. Through genomic analysis, we identified the two family 2 glycosyltransferase genes CvGT2–1 and CvGT2–2 as possible genes encoding (1,3;1,4)-β-D-glucan synthases. Gain-of-function experiments in the yeast Saccharomyces cerevisiae demonstrated that both of these genes do indeed encode (1,3;1,4)-β-D-glucan synthases.

The intake of dietary fibers is related with important benefits for human health. We produced two different arabinoxylan fibers with (FAX) and without ferulic acid linked (AX), 12.5 and 0.1 mg g- 1 of ferulic acid respectively, by subcritical water extraction of wheat bran. Both FAX and AX fibers were used as supplement in bread production, while non-supplemented bread was used as control. Through an enzymatic deconstruction process we investigated the effect of bread making on the fibers, the preservation of their molecular structure (A/ X ratio of 0.13 and Mw of 105 Da) and the interaction with other macromolecules in the bread. By mimicking the upper track digestion, we could confirm the non-digestability of the fibers and we used them for the fermentation with B. ovatus and B. adolescentis. The presence of AX fibers during fermentation showed specific substrate adaptation by the probiotic bacteria in correlation with its potential prebiotic effect.

Within the intricate microbiological realm, exopolysaccharides (EPS) emerge as a significant class of high molecular weight polysaccharides, serving pivotal roles in bacterial survival, virulence, communication, and defense against environmental adversities. The versatile nature of bacterial EPS extends beyond mere biological functions, reaching into medications, cosmetics, functional food, and sustainable industries. Although EPS is a vital and much-exploited class of polysaccharide, their biosynthesis remain less explored or understood. This thesis delves into the exploration of enzymes integral to EPS synthesis, focusing specifically on the characterization of putative membrane-bound enzymes from the so-called glycosyltransferase family 2 (GT2). The initial investigations of the research are centered on (1,3;1,4)-β-D-glucans, polymers with mixed linkage backbones that are widely distributed across various biological systems. Despite their prevalence, collective understanding of the biosynthesis of these polymers in the bacterial domain, particularly in gram-positive strains, remains limited. Through extensive research, distinct genes encoding (1,3;1,4)-β-D-glucan synthases were identified in two gram-positive bacteria: Romboutsia ilealis and Clostridium ventriculi. A gain-of-function approach was employed and provided conclusive evidence of the synthase activity of these identified genes. Subsequently, the thesis shifts focus to Chitinophaga pinensis, a gram-negative bacterium with roles in maintaining ecosystem balance through its proficiency in carbohydrate breakdown and recycling. The exploration led to the discovery of an uncommon GT2 β-glucan synthase with activity in curdlan synthesis. Several unusual features of the C. pinensis enzyme highlight the extensive diversity and nuances within the GT2 polysaccharide synthase family, particularly the fact that such catalysts sometimes have close connections with carbohydrate-degrading enzymes. Characterization of these putative GT2 proteins was verified by a variety of techniques, including gene expression in E. coli and yeast host systems, enzyme-coupled oligosaccharide profiling, and in vitro radiometric activity assays. To advance the investigation, bioinformatics tools such as protein alignment, phylogenetic analysis, and model structure analysis were employed.

Inom det komplexa mikrobiologiska området framträder exopolysackarider (EPS) som en betydande klass av högmolekylära polysackarider. De spelar centrala roller inom bakteriers överlevnad, virulens, kommunikation och försvar mot miljömässiga motgångar. Den mångsidiga naturen hos bakteriell EPS sträcker sig bortom enbart biologiska funktioner och når in i läkemedel, kosmetika, funktionell mat och hållbara industrier. Även om EPS är en vital och ofta utnyttjad klass av polysackarider, är deras biosyntes mindre utforskad eller förstådd. Denna avhandling fördjupar sig i utforskningen utav enzymer som är centrala för EPS-syntes, med särskilt fokus på karaktäriseringen av tänkta membranbundna enzymer från den så kallade glykosyltransferasfamiljen 2 (GT2). De inledande undersökningarna av forskningen kretsar kring (1,3;1,4)-β-D-glukaner, polymerer med blandade länkryggar som är allmänt fördelade över olika biologiska system. Trots deras förekomst är den kollektiva förståelsen för biosyntesen av dessa polymerer inom bakteriedomänen, särskilt i gram-positiva stammar, begränsad. Genom omfattande forskning identifierades specifika gener som kodar för (1,3;1,4)-β-D-glukansyntaser i två gram-positiva bakterier: Romboutsia ilealis och Clostridium ventriculi. Ett tillvägagångssätt för att öka funktionen användes och gav definitiva bevis för syntasaktiviteten hos dessa identifierade gener. Avhandlingen skiftar därefter fokus till Chitinophaga pinensis, en gram-negativ bakterie som spelar en roll i att upprätthålla ekosystembalansen genom sin skicklighet i nedbrytning och återvinning av kolhydrater. Utforskningen ledde till upptäckten av ett ovanlig GT2 β-glukansyntas med aktivitet inom curdlan-syntes. Flera ovanliga egenskaper hos C. pinensis-enzymet framhäver den omfattande mångfalden och nyanserna inom GT2 polysackaridsyntasfamiljen, särskilt det faktum att sådana katalysatorer ibland har nära kopplingar med kolhydratnedbrytande enzymer. Karaktärisering av dessa tänkta GT2-proteiner verifierades med hjälp av en mängd olika tekniker, inklusive genuttryck i E. coli och jästvärdssystem, enzymkopplad oligosackaridprofilering och in vitro radiometriska aktivitetsanalyser. För att fördjupa undersökningen användes bioinformatikverktyg såsom proteinjustering, fylogenetisk analys och modellstrukturanalys.

(1,3;1,4)-β-D-Glucans are widely distributed in the cell walls of grasses (family Poaceae) and closely related families, as well as some other vascular plants. Additionally, they have been found in other organisms, including fungi, lichens, brown algae, charophycean green algae, and the bacterium Sinorhizobium meliloti. Only three members of the Cellulose Synthase-Like (CSL) genes in the families CSLF, CSLH, and CSLJ are implicated in (1,3;1,4)-β-D-glucan biosynthesis in grasses. Little is known about the enzymes responsible for synthesizing (1,3;1,4)-β-D-glucans outside the grasses. In the present study, we report the presence of (1,3;1,4)-β-D-glucans in the exopolysaccharides of the Gram-positive bacterium Romboutsia ilealis CRIBT. We also report that RiGT2 is the candidate gene of R. ilealis that encodes (1,3;1,4)-β-D-glucan synthase. RiGT2 has conserved glycosyltransferase family 2 (GT2) motifs, including D, D, D, QXXRW, and a C-terminal PilZ domain that resembles the C-terminal domain of bacteria cellulose synthase, BcsA. Using a direct gain-of-function approach, we insert RiGT2 into Saccharomyces cerevisiae, and (1,3;1,4)-β-D-glucans are produced with structures similar to those of the (1,3;1,4)-β-D-glucans of the lichen Cetraria islandica. Phylogenetic analysis reveals that putative (1,3;1,4)-β-D-glucan synthase candidate genes in several other bacterial species support the finding of (1,3;1,4)-β-D-glucans in these species.

Partially acetylated chito-oligosaccharides (paCOSs) are bioactive compounds with potential medical applications. Their biological activities are largely dependent on their structural properties, in particular their degree of polymerization (DP) and the position of the acetyl groups along the glycan chain. The production of structurally defined paCOSs in a purified form is highly desirable to better understand the structure/bioactivity relationship of these oligosaccharides. Here, we describe a newly discovered chitinase from Paenibacillus pabuli (PpChi) and demonstrate by mass spectrometry that it essentially produces paCOSs with a DP of three and four that carry a single N-acetylation at their reducing end. We propose that this specific composition of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) residues, as in GlcN(n)GlcNAc1, is due to a subsite specificity toward GlcN residues at the −2, −3, and −4 positions of the partially acetylated chitosan substrates. In addition, the enzyme is stable, as evidenced by its long shelf life, and active over a large temperature range, which is of high interest for potential use in industrial processes. It exhibits a kcatof 67.2 s–1 on partially acetylated chitosan substrates. When PpChi was used in combination with a recently discovered fungal auxilary activity (AA11) oxidase, a sixfold increase in the release of oligosaccharides from the lobster shell was measured. PpChi represents an attractive biocatalyst for the green production of highly valuable paCOSs with a well-defined structure and the expansion of the relatively small library of chito-oligosaccharides currently available.

(1,3;1,4)-ß-D-Glucans, also named as mixed-linkage glucans, are unbranched non-cellulosic polysaccharides containing both (1,3)- and (1,4)-ß-linkages. The linkage ratio varies depending upon species origin and has a significant impact on the physicochemical properties of the (1,3;1,4)-ß-D-glucans. (1,3;1,4)-ß-D-Glucans were thought to be unique in the grasses family (Poaceae); however, evidence has shown that (1,3;1,4)-ß-D-glucans are also synthesized in other taxa, including horsetail fern Equisetum, algae, lichens, and fungi, and more recently, bacteria. The enzyme involved in (1,3;1,4)-ß-D-glucan biosynthesis has been well studied in grasses and cereal. However, how this enzyme is able to assemble the two different linkages remains a matter of debate. Additionally, the presence of (1,3;1,4)-ß-D-glucan across the species evolutionarily distant from Poaceae but absence in some evolutionarily closely related species suggest that the synthesis is either highly conserved or has arisen twice as a result of convergent evolution. Here, we compare the structure of (1,3;1,4)-ß-D-glucans present across various taxonomic groups and provide up-to-date information on how (1,3;1,4)-ß-D-glucans are synthesized and their functions.