Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome.

Fucosyl-oligosaccharides (FUS) provide many health benefits to breastfed infants, but they are almost completely absent from bovine milk, which is the basis of infant formula. Therefore, there is a growing interest in the development of enzymatic transfucosylation strategies for the production of FUS. In this work, the α-L-fucosidases Fuc2358 and Fuc5372, previously isolated from the intestinal bacterial metagenome of breastfed infants, were used to synthesize fucosyllactose (FL) by transfucosylation reactions using p-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as donor and lactose as acceptor. Fuc2358 efficiently synthesized the major fucosylated human milk oligosaccharide (HMO) 2'-fucosyllactose (2'FL) with a 35% yield. Fuc2358 also produced the non-HMO FL isomer 3'-fucosyllactose (3'FL) and traces of non-reducing 1-fucosyllactose (1FL). Fuc5372 showed a lower transfucosylation activity compared to Fuc2358, producing several FL isomers, including 2'FL, 3'FL, and 1FL, with a higher proportion of 3'FL. Site-directed mutagenesis using rational design was performed to increase FUS yields in both α-L-fucosidases, based on structural models and sequence identity analysis. Mutants Fuc2358-F184H, Fuc2358-K286R, and Fuc5372-R230K showed a significantly higher ratio between 2'FL yields and hydrolyzed pNP-Fuc than their respective wild-type enzymes after 4 h of transfucosylation. The results with the Fuc2358-F184W and Fuc5372-W151F mutants showed that the residues F184 of Fuc2358 and W151 of Fuc5372 could have an effect on transfucosylation regioselectivity. Interestingly, phenylalanine increases the selectivity for α-1,2 linkages and tryptophan for α-1,3 linkages. These results give insight into the functionality of the active site amino acids in the transfucosylation activity of the GH29 α-L-fucosidases Fuc2358 and Fuc5372. KEY POINTS: Two α-L-fucosidases from infant gut bacterial microbiomes can fucosylate glycans Transfucosylation efficacy improved by tailored point-mutations in the active site F184 of Fuc2358 and W151 of Fuc5372 seem to steer transglycosylation regioselectivity.

Improved Enzymatic Production of the Fucosylated Human Milk Oligosaccharide LNFP II with GH29B α-1,3/4-l-Fucosidases.

Five GH29B α-1,3/4-l-fucosidases (EC 3.2.1.111) were investigated for their ability to catalyze the formation of the human milk oligosaccharide lacto-N-fucopentaose II (LNFP II) from lacto-N-tetraose (LNT) and 3-fucosyllactose (3FL) via transglycosylation. We studied the effect of pH on transfucosylation and hydrolysis and explored the impact of specific mutations using molecular dynamics simulations. LNFP II yields of 91 and 65% were obtained for the wild-type SpGH29C and CpAfc2 enzymes, respectively, being the highest LNFP II transglycosylation yields reported to date. BbAfcB and BiAfcB are highly hydrolytic enzymes. The results indicate that the effects of pH and buffer systems are enzyme-dependent yet relevant to consider when designing transglycosylation reactions. Replacing Thr284 in BiAfcB with Val resulted in increased transglycosylation yields, while the opposite replacement of Val258 in SpGH29C and Val289 CpAfc2 with Thr decreased the transfucosylation, confirming a role of Thr and Val in controlling the flexibility of the acid/base loop in the enzymes, which in turn affects transglycosylation. The substitution of an Ala residue with His almost abolished secondary hydrolysis in CpAfc2 and BbAfcB. The results are directly applicable in the enhancement of transglycosylation and may have significant implications for manufacturing of LNFP II as a new infant formula ingredient.

Biochemical exploration of family GH119 reveals a single α-amylase specificity and confirms shared catalytic machinery with GH57 enzymes.

While hundreds of starch- and glycogen-degrading enzymes have been characterized experimentally in historical families such as GH13, GH14, GH15, GH57 and GH126 of the CAZy database (www.cazy.org), the α-amylase from Bacillus circulans is the only enzyme that has been characterized in family GH119. Since glycosidase families have been shown to often group enzymes with different substrates or products, a single characterized enzyme in a family is insufficient to extrapolate enzyme function based solely on sequence similarity. Here we report the rational exploration of family GH119 through the biochemical characterization of five GH119 members. All enzymes shared single α-amylase specificity but display distinct product profile. We also report the first kinetic constants in family GH119 and the first experimental validation of previously predicted catalytic residues in family GH119, confirming that families GH119 and GH57 can be grouped in the novel clan GH-T of the CAZy database.

Characterization and structural study of a novel ß-N-acetylgalactosaminidase from Niabella aurantiaca.

We report here the identification, characterization and three-dimensional (3D) structure determination of NaNga, a newly identified ß-N-acetylgalactosaminidase from the Gram-negative soil bacterium Niabella aurantiaca DSM 17617. When recombinantly expressed in Escherichia coli, the enzyme selectively cleaved 4-nitrophenyl-N-acetyl-ß-d-galactosamine (pNP-ß-d-GalpNAc). The X-ray crystal structure of the protein was refined to 2.5 Å and consists of an N-terminal ß-sandwich domain and a (ß/α)8 barrel catalytic domain. Despite a mere 22% sequence identity, the 3D structure of NaNga is similar to those previously determined for family GH123 members, suggesting it also employs the same substrate-assisted catalytic mechanism. Inhibition by N-acetyl-galactosamine thiazoline (GalNAc-thiazoline) supports the suggested mechanism. A phylogenetic analysis of its proximal sequence space shows significant clustering of unknown sequences around NaNga with sufficient divergence with previously identified GH123 members to subdivide this family into distinct subfamilies. Although the actual biological substrate of our enzyme remains unknown, examination of the active site pocket suggests that it may be a ß-N-acetylgalactosaminide substituted by a monosaccharide at O-3. Analysis of the genomic context suggests, in turn, that this substituted ß-N-acetylgalactosaminide may be appended to a d-arabinan from an environmental Actinomycete.

Three alginate lyases provide a new gut Bacteroides ovatus isolate with the ability to grow on alginate.

Humans consume alginate in the form of seaweed, food hydrocolloids, and encapsulations, making the digestion of this mannuronic acid (M) and guluronic acid (G) polymer of key interest for human health. To increase knowledge on alginate degradation in the gut, a gene catalog from human feces was mined for potential alginate lyases (ALs). The predicted ALs were present in nine species of the Bacteroidetes phylum, of which two required supplementation of an endo-acting AL, expected to mimic cross-feeding in the gut. However, only a new isolate grew on alginate. Whole-genome sequencing of this alginate-utilizing isolate suggested that it is a new Bacteroides ovatus strain harboring a polysaccharide utilization locus (PUL) containing three ALs of families: PL6, PL17, and PL38. The BoPL6 degraded polyG to oligosaccharides of DP 1-3, and BoPL17 released 4,5-unsaturated monouronate from polyM. BoPL38 degraded both alginates, polyM, polyG, and polyMG, in endo-mode; hence, it was assumed to deliver oligosaccharide substrates for BoPL6 and BoPL17, corresponding well with synergistic action on alginate. BoPL17 and BoPL38 crystal structures, determined at 1.61 and 2.11 Å, respectively, showed (α/α)6-barrel + anti-parallel ß-sheet and (α/α)7-barrel folds, distinctive for these PL families. BoPL17 had a more open active site than the two homologous structures. BoPL38 was very similar to the structure of an uncharacterized PL38, albeit with a different triad of residues possibly interacting with substrate in the presumed active site tunnel. Altogether, the study provides unique functional and structural insights into alginate-degrading lyases of a PUL in a human gut bacterium.IMPORTANCEHuman ingestion of sustainable biopolymers calls for insight into their utilization in our gut. Seaweed is one such resource with alginate, a major cell wall component, used as a food hydrocolloid and for encapsulation of pharmaceuticals and probiotics. Knowledge is sparse on the molecular basis for alginate utilization in the gut. We identified a new Bacteroides ovatus strain from human feces that grew on alginate and encoded three alginate lyases in a gene cluster. BoPL6 and BoPL17 show complementary specificity toward guluronate (G) and mannuronate (M) residues, releasing unsaturated oligosaccharides and monouronic acids. BoPL38 produces oligosaccharides degraded by BoPL6 and BoPL17 from both alginates, G-, M-, and MG-substrates. Enzymatic and structural characterization discloses the mode of action and synergistic degradation of alginate by these alginate lyases. Other bacteria were cross-feeding on alginate oligosaccharides produced by an endo-acting alginate lyase. Hence, there is an interdependent community in our guts that can utilize alginate.

Structural and functional characterization of the novel endo-α(1,4)-fucoidanase Mef1 from the marine bacterium Muricauda eckloniae.

Fucoidanases (EC 3.2.1.-) catalyze the hydrolysis of glycosidic bonds between fucose residues in fucoidans. Fucoidans are a compositionally and structurally diverse class of fucose-containing sulfated polysaccharides that are primarily found in brown seaweeds. Here, the structural characterization of a novel endo-α(1,4)-fucoidanase, Mef1, from the marine bacterium Muricauda eckloniae is presented, showing sequence similarity to members of glycoside hydrolase family 107. Using carbohydrate polyacrylamide gel electrophoresis and nuclear magnetic resonance analyses, it is shown that the fucoidanase Mef1 catalyzes the cleavage of α(1,4)-linkages between fucose residues sulfated on C2 in the structure [-3)-α-L-Fucp2S-(1,4)-α-L-Fucp2S-(1-]n in fucoidan from Fucus evanescens. Kinetic analysis of Mef1 activity by Fourier transform infrared spectroscopy revealed that the specific Mef1 fucoidanase activity (Uf) on F. evanescens fucoidan was 0.1 × 10-3 Uf µM-1. By crystal structure determination of Mef1 at 1.8 Šresolution, a single-domain organization comprising a (ß/α)8-barrel domain was determined. The active site was in an extended, positively charged groove that is likely to be designed to accommodate the binding of the negatively charged, sulfated fucoidan substrate. The active site of Mef1 comprises the amino acids His270 and Asp187, providing acid/base and nucleophile groups, respectively, for the hydrolysis of glycosidic bonds in the fucoidan backbone. Electron densities were identified for two possible Ca2+ ions in the enzyme, one of which is partially exposed to the active-site groove, while the other is very tightly coordinated. A water wire was discovered leading from the exterior of the Mef1 enzyme into the active site, passing the tightly coordinated Ca2+ site.

Exploration of the Transglycosylation Activity of Barley Limit Dextrinase for Production of Novel Glycoconjugates.

A few α-glucan debranching enzymes (DBEs) of the large glycoside hydrolase family 13 (GH13), also known as the α-amylase family, have been shown to catalyze transglycosylation as well as hydrolysis. However, little is known about their acceptor and donor preferences. Here, a DBE from barley, limit dextrinase (HvLD), is used as a case study. Its transglycosylation activity is studied using two approaches; (i) natural substrates as donors and different p-nitrophenyl (pNP) sugars as well as different small glycosides as acceptors, and (ii) α-maltosyl and α-maltotriosyl fluorides as donors with linear maltooligosaccharides, cyclodextrins, and GH inhibitors as acceptors. HvLD showed a clear preference for pNP maltoside both as acceptor/donor and acceptor with the natural substrate pullulan or a pullulan fragment as donor. Maltose was the best acceptor with α-maltosyl fluoride as donor. The findings highlight the importance of the subsite +2 of HvLD for activity and selectivity when maltooligosaccharides function as acceptors. However, remarkably, HvLD is not very selective when it comes to aglycone moiety; different aromatic ring-containing molecules besides pNP could function as acceptors. The transglycosylation activity of HvLD can provide glycoconjugate compounds with novel glycosylation patterns from natural donors such as pullulan, although the reaction would benefit from optimization.

Soluble, Diferuloylated Corn Bran Glucuronoarabinoxylans Modulate the Human Gut Microbiota In Vitro.

Corn bran is exceptionally rich in substituted glucuronoarabinoxylan polysaccharides, which are monoferuloylated and cross-linked by diferulic acid moieties. Here, we assessed the potential prebiotic activity of three enzymatically solubilized corn bran glucuronoarabinoxylans: medium feruloylated (FGAX-M), laccase cross-linked FGAX-M (FGAX-H), and alkali-treated FGAX-M devoid of feruloyl substitutions (FGAX-B). We examined the influence of these soluble FGAX samples on the gut microbiome composition and functionality during in vitro simulated colon fermentations, determined by 16S rRNA gene amplicon sequencing and assessment of short-chain fatty acid (SCFAs) production. All FGAX samples induced changes in the relative composition of the microbiota and the SCFA levels after 24 h of in vitro fermentation. The changes induced by FGAX-M and FGAX-H tended to be more profound and more similar to the changes induced by inulin than changes conferred by FGAX-B. The microbiota changes induced by FGAX-M and FGAX-H correlated with an increase in the relative abundance of Anaerostipes and with increased butyric acid production, while the changes induced by the FGAX-B sample were less compelling. The results imply that solubilized, substituted diferuloylated corn bran glucuronoarabinoxylans may be potential prebiotic candidates and that both single feruloylations and diferuloyl cross-links influence the prebiotic potential of these arabinoxylan compounds.

Fungal feruloyl esterases can catalyze release of diferulic acids from complex arabinoxylan.

Feruloyl esterases (FAEs, EC 3.1.1.73) catalyze the hydrolytic cleavage of ester bonds between feruloyl and arabinosyl moieties in arabinoxylans. Recently, we discovered that two bacterial FAEs could catalyze release of diferulic acids (diFAs) from highly substituted, cross-linked corn bran arabinoxylan. Here, we show that several fungal FAEs, notably AnFae1 (Aspergillus niger), AoFae1 (A. oryzae), and MgFae1 (Magnaporthe oryzae (also known as M. grisae)) also catalyze liberation of diFAs from complex arabinoxylan. By comparing the enzyme kinetics of diFA release to feruloyl esterase activity of the enzymes on methyl- and arabinosyl-ferulate substrates we demonstrate that the diFA release activity cannot be predicted from the activity of the enzymes on these synthetic substrates. A detailed structure-function analysis, based on AlphaFold2 modeled enzyme structures and docking with the relevant di-feruloyl ligands, reveal how distinct differences in the active site topology and surroundings may explain the diFA releasing action of the enzymes. Interestingly, the analysis also unveils that the carbohydrate binding module of the MgFae1 may play a key role in the diFA releasing ability of this enzyme. The findings contribute further understanding of the function of FAEs in the deconstruction of complex arabinoxylans and provide new opportunities for enzyme assisted upgrading of complex bran arabinoxylans.

Fermented butter aroma for plant-based applications.

Plant-based dairy alternatives are gaining increasing interest, e.g. alternatives to yoghurt, cheese, and butter. In all these products butter flavor (diacetyl + acetoin) plays an important role. We previously have reported efficient butter flavor formation from low value dairy side streams using a dairy isolate of Lactococcus lactis deficient in lactate dehydrogenase. Here, we have tested the ability of this strain, RD1M5, to form butter flavor in plant milks based on oat and soy. We found that oat milk, with its high sugar content, supported more efficient production of butter aroma, when compared to soy milk. When supplemented with glucose, efficient butter aroma production was achieved in soy milk as well. We also carried out an extended adaptive laboratory evolution of the dairy strain in oat milk. After two months of adaptation, we obtained a strain with enhanced capacity for producing butter aroma. Despite of its high sugar content, RD1M5 and its adapted version only metabolized approximately 10% of the fermentable sugars available in the oat milk, which we found was due to amino acid starvation and partly starvation for vitamins. The study demonstrates that dairy cultures have great potential for use in plant-based fermentations.

Enzymatic Cleavage of Diferuloyl Cross-Links in Corn Bran Arabinoxylan by Two Bacterial Feruloyl Esterases.

Corn bran is an abundant coprocessing stream of corn-starch processing, rich in highly substituted, diferuloyl-cross-linked glucurono-arabinoxylan. The diferuloyl cross-links make the glucurono-arabinoxylan recalcitrant to enzymatic conversion and constitute a hindrance for designing selective enzymatic upgrading of corn glucurono-arabinoxylan. Here, we show that two bacterial feruloyl esterases, wtsFae1A and wtsFae1B, each having a carbohydrate-binding module of family 48, are capable of cleaving the ester bonds of the cross-linkages and releasing 5-5', 8-5', 8-5' benzofuran, and 8-O-4' diferulate from soluble and insoluble corn bran glucurono-arabinoxylan. All four diferulic acids were released at similar efficiency, indicating nondiscriminatory enzymatic selectivity for the esterified dimer linkages, the only exception being that wtsFae1B had a surprisingly high propensity for releasing the dimers, especially 8-5' benzofuran diferulate, indicating a potential, unique catalytic selectivity. The data provide evidence of direct enzymatic release of diferulic acids from corn bran by newly discovered feruloyl esterases, i.e., a new enzyme activity. The findings yield new insight and create new opportunities for enzymatic opening of diferuloyl cross-linkages to pave the way for upgrading of recalcitrant arabinoxylans.

Discovery of novel secretome CAZymes from Penicillium sclerotigenum by bioinformatics and explorative proteomics analyses during sweet potato pectin digestion.

Novel selective enzymatic refining of sweet potato processing residues requires judicious enzyme selection and enzyme discovery. We prepared a pectinaceous cell wall polysaccharide fraction from sweet potato using an enzymatic a treatment to preserve the natural linkages and substitutions. Polysaccharide composition and linkage analysis data confirmed the pectinaceous polysaccharide fraction to be a rhamnogalacturonan I-rich fraction with a high content of arabinogalactan Type I. We hypothesized that the post-harvest tuber pathogenic fungus Penicillium sclerotigenum would harbor novel enzymes targeting selective sweet potato pectin modification. As part of the study, we also report the first genome sequence of P. sclerotigenum. We incubated the sweet potato pectinaceous fraction with P. sclerotigenum. Using proteomics accompanied by CUPP-bioinformatics analysis, we observed induced expression of 23 pectin-associated degradative enzymes. We also identified six abundantly secreted, induced proteins that do not correspond to known CAZymes, but which we suggest as novel enzymes involved in pectin degradation. For validation, the predicted CUPP grouping of putative CAZymes and the exo-proteome data obtained for P. sclerotigenum during growth on sweet potato pectin were compared with proteomics and transcriptomics data reported previously for pectin-associated CAZymes from Aspergillus niger strain NRRL3. The data infer that P. sclerotigenum has the capacity to express several novel enzymes that may provide novel opportunities for sweet potato pectin modification and valorization of sweet potato starch processing residues. In addition, the methodological approach employed represents an integrative systematic strategy for enzyme discovery.

Utilization of industrial citrus pectin side streams for enzymatic production of human milk oligosaccharides.

This study reports the enzymatic upgrading of fucosylated xyloglucan from depectinized citrus residues into 2'-fucosyllactose, a fucosylated human milk oligosaccharide. Alkaline and enzymatic xyloglucan extractions were compared. Of the original fucose present in the depectinized residues of lemon and orange, 35-36% and 48-51% were extracted as fucosylated xyloglucan by enzyme- or alkaline treatment, respectively. Furthermore, the enzymatically extracted xyloglucan structures had a narrower molecular weight distribution around 1 kDa, contrary to a more polydisperse distribution of the alkaline extracted xyloglucans, ranging from 1 to 500 kDa. The applicability of the fucosylated-xyloglucan extracts in transfucosylation reactions, was determined by use of a selected fungal fucosidase, resulting in yields of 10.2-11.4% enzymatic extracts, and 6.5-7.4% for alkaline extracts (orange and lemon respectively). The results demonstrate that depectinized citrus side streams are a useful source of fucosylated xyloglucan, preferably extracted by an enzyme catalyzed approach.

The Endo-α(1,3)-Fucoidanase Mef2 Releases Uniquely Branched Oligosaccharides from Saccharina latissima Fucoidans.

Fucoidans are complex bioactive sulfated fucosyl-polysaccharides primarily found in brown macroalgae. Endo-fucoidanases catalyze the specific hydrolysis of α-L-fucosyl linkages in fucoidans and can be utilized to tailor-make fucoidan oligosaccharides and elucidate new structural details of fucoidans. In this study, an endo-α(1,3)-fucoidanase encoding gene, Mef2, from the marine bacterium Muricauda eckloniae, was cloned, and the Mef2 protein was functionally characterized. Based on the primary sequence, Mef2 was suggested to belong to the glycosyl hydrolase family 107 (GH107) in the Carbohydrate Active enZyme database (CAZy). The Mef2 fucoidanase showed maximal activity at pH 8 and 35 °C, although it could tolerate temperatures up to 50 °C. Ca2+ was shown to increase the melting temperature from 38 to 44 °C and was furthermore required for optimal activity of Mef2. The substrate specificity of Mef2 was investigated, and Fourier transform infrared spectroscopy (FTIR) was used to determine the enzymatic activity (Units per µM enzyme: Uf/µM) of Mef2 on two structurally different fucoidans, showing an activity of 1.2 × 10-3 Uf/µM and 3.6 × 10-3 Uf/µM on fucoidans from Fucus evanescens and Saccharina latissima, respectively. Interestingly, Mef2 was identified as the first described fucoidanase active on fucoidans from S. latissima. The fucoidan oligosaccharides released by Mef2 consisted of a backbone of α(1,3)-linked fucosyl residues with unique and novel α(1,4)-linked fucosyl branches, not previously identified in fucoidans from S. latissima.

The Endo-α(1,4) Specific Fucoidanase Fhf2 From Formosa haliotis Releases Highly Sulfated Fucoidan Oligosaccharides.

Fucoidanases are endo-fucoidanases (also known as endo-fucanases) that catalyze hydrolysis of α-glycosidic linkages in fucoidans, a family of sulfated fucose-rich polysaccharides primarily found in the cell walls of brown seaweeds. Fucoidanases are promising tools for producing bioactive fucoidan oligosaccharides for a range of biomedical applications. High sulfation degree has been linked to high bioactivity of fucoidans. In this study, a novel fucoidanase, Fhf2, was identified in the genome of the aerobic, Gram-negative marine bacterium Formosa haliotis. Fhf2 was found to share sequence similarity to known endo-α(1,4)-fucoidanases (EC 3.2.1.212) from glycoside hydrolase family 107. A C-terminal deletion mutant Fhf2∆484, devoid of 484 amino acids at the C-terminus, with a molecular weight of approximately 46 kDa, was constructed and found to be more stable than the full-length Fhf2 protein. Fhf2∆484 showed endo-fucoidanase activity on fucoidans from different seaweed species including Fucus evanescens, Fucus vesiculosus, Sargassum mcclurei, and Sargassum polycystum. The highest activity was observed on fucoidan from F. evanescens. The Fhf2∆484 enzyme was active at 20-45°C and at pH 6-9 and had optimal activity at 37°C and pH 8. Additionally, Fhf2∆484 was found to be calcium-dependent. NMR analysis showed that Fhf2∆484 catalyzed hydrolysis of α(1,4) linkages between L-fucosyl moieties sulfated on C2 (similar to Fhf1 from Formosa haliotis), but Fhf2∆484 in addition released oligosaccharides containing a substantial amount of 2,4-disulfated fucose residues. The data thus suggest that the Fhf2∆484 enzyme could be a valuable candidate for producing highly sulfated oligosaccharides applicable for fucoidan bioactivity investigations.

Discovery of a Novel Glucuronan Lyase System in Trichoderma parareesei.

Glucuronan lyases (EC 4.2.2.14) catalyze depolymerization of linear ß-(1,4)-polyglucuronic acid (glucuronan). Only a few glucuronan lyases have been characterized until now, most of them originating from bacteria. Here we report the discovery, recombinant production, and functional characterization of the full complement of six glucuronan specific polysaccharide lyases in the necrotic mycoparasite Trichoderma parareesei. The enzymes belong to four different polysaccharide lyase families and have different reaction optima and glucuronan degradation profiles. Four of them showed endo-lytic action and two, TpPL8A and TpPL38A, displayed exo-lytic action. Nuclear magnetic resonance revealed that the monomeric end product from TpPL8A and TpPL38A underwent spontaneous rearrangements to tautomeric forms. Proteomic analysis of the secretomes from T. parareesei growing on pure glucuronan and lyophilized A. bisporus fruiting bodies, respectively, showed secretion of five of the glucuronan lyases and high-performance anion-exchange chromatography with pulsed amperometric detection analysis confirmed the presence of glucuronic acid in the A. bisporus fruiting bodies. By systematic genome annotation of more than 100 fungal genomes and subsequent phylogenetic analysis of the putative glucuronan lyases, we show that glucuronan lyases occur in several ecological and taxonomic groups in the fungal kingdom. Our findings suggest that a diverse repertoire of glucuronan lyases is a common trait among Hypocreales species with mycoparasitic and entomopathogenic lifestyles. IMPORTANCE This paper reports the discovery of a set of six complementary glucuronan lyase enzymes in the mycoparasite Trichoderma parareseei. Apart from the novelty of the discovery of these enzymes in T. parareesei, the key importance of the study is the finding that the majority of these lyases are induced when T. parareesei is inoculated on Basidiomycete cell walls that contain glucuronan. The study also reveals putative glucuronan lyase encoding genes in a wealth of other fungi that furthermore points at fungal cell wall glucuronan being a target C-source for many types of fungi. In a technical context, the findings may lead to controlled production of glucuronan oligomers for advanced pharmaceutical applications and pave the way for development of new fungal biocontrol agents.

A novel thermostable prokaryotic fucoidan active sulfatase PsFucS1 with an unusual quaternary hexameric structure.

Fucoidans are sulfated, fucose-rich marine polysaccharides primarily found in cell walls of brown seaweeds (macroalgae). Fucoidans are known to possess beneficial bioactivities depending on their structure and sulfation degree. Here, we report the first functional characterization and the first crystal structure of a prokaryotic sulfatase, PsFucS1, belonging to sulfatase subfamily S1_13, able to release sulfate from fucoidan oligosaccharides. PsFucS1 was identified in the genome of a Pseudoalteromonas sp. isolated from sea cucumber gut. PsFucS1 (57 kDa) is Ca2+ dependent and has an unusually high optimal temperature (68 °C) and thermostability. Further, the PsFucS1 displays a unique quaternary hexameric structure comprising a tight trimeric dimer complex. The structural data imply that this hexamer formation results from an uncommon interaction of each PsFucS1 monomer that is oriented perpendicular to the common dimer interface (~ 1500 Å2) that can be found in analogous sulfatases. The uncommon interaction involves interfacing (1246 Å2) through a bundle of α-helices in the N-terminal domain to form a trimeric ring structure. The high thermostability may be related to this unusual quaternary hexameric structure formation that is suggested to represent a novel protein thermostabilization mechanism.

High throughput in vitro characterization of pectins for pig(let) nutrition.

BACKGROUND: Fiber-rich feed components possess prebiotic potential to enhance pig health and are considered a potential solution to the high prevalence of post-weaning diarrhea in pig production under the phased suspension of antibiotics and zinc oxide use. METHODS: We screened the gut microbiota modulatory properties of pectin substrates prepared from sugar beet within the freshly weaned piglet gut microbiome using an in vitro colon model, the CoMiniGut. We focused on testing a variety (13) of sugar beet-derived pectin substrates with defined structures, as well as known prebiotics such as inulin, fructooligosaccharide (FOS) and galactooligosaccharide (GOS), to gain insights on the structure-function related properties of specific substrates on the weaner gut microbial composition as well as shortchain fatty acid production (SCFA). RESULTS: Sugar beet-derived pectin and rhamnogalacturonan-I selectively increased the relative abundance of Bacteroidetes, specifically Prevotella copri, Bacteroides ovatus, Bacteroides acidificiens, and an unclassified Bacteroides member. The degree of esterification impacted the relative abundance of these species and the SCFA production during the in vitro fermentations. Modified arabinans derived from sugar beet promoted the growth of Blautia, P. copri, Lachnospiraceae members and Limosilactobacillus mucosae and amongst all oligosaccharides tested yielded the highest amount of total SCFA produced after 24 h of fermentation. Sugar beet-derived substrates yielded higher total SCFA concentrations (especially acetic and propionic acid) relative to the known prebiotics inulin, FOS and GOS. CONCLUSION: Our results indicate that the molecular structures of pectin, that can be prepared form just one plant source (sugar beet) can selectively stimulate different GM members, highlighting the potential of utilizing pectin substrates as targeted GM modulatory ingredients.

Specificities and Synergistic Actions of Novel PL8 and PL7 Alginate Lyases from the Marine Fungus Paradendryphiella salina.

Alginate is an anionic polysaccharide abundantly present in the cell walls of brown macroalgae. The enzymatic depolymerization is performed solely by alginate lyases (EC 4.2.2.x), categorized as polysaccharide lyases (PLs) belonging to 12 different PL families. Until now, the vast majority of the alginate lyases have been found in bacteria. We report here the first extensive characterization of four alginate lyases from a marine fungus, the ascomycete Paradendryphiella salina, a known saprophyte of seaweeds. We have identified four polysaccharide lyase encoding genes bioinformatically in P. salina, one PL8 (PsMan8A), and three PL7 alginate lyases (PsAlg7A, -B, and -C). PsMan8A was demonstrated to exert exo-action on polymannuronic acid, and no action on alginate, indicating that this enzyme is most likely an exo-acting polymannuronic acid specific lyase. This enzyme is the first alginate lyase assigned to PL8 and polymannuronic acid thus represents a new substrate specificity in this family. The PL7 lyases (PsAlg7A, -B, and -C) were found to be endo-acting alginate lyases with different activity optima, substrate affinities, and product profiles. PsAlg7A and PsMan8A showed a clear synergistic action for the complete depolymerization of polyM at pH 5. PsAlg7A depolymerized polyM to mainly DP5 and DP3 oligomers and PsMan8A to dimers and monosaccharides. PsAlg7B and PsAlg7C showed substrate affinities towards both polyM and polyG at pH 8, depolymerizing both substrates to DP9-DP2 oligomers. The findings elucidate how P. salina accomplishes alginate depolymerization and provide insight into an efficient synergistic cooperation that may provide a new foundation for enzyme selection for alginate degradation in seaweed bioprocessing.

Improved Transglycosylation by a Xyloglucan-Active α-l-Fucosidase from Fusarium graminearum.

Fusarium graminearum produces an α-l-fucosidase, FgFCO1, which so far appears to be the only known fungal GH29 α-l-fucosidase that catalyzes the release of fucose from fucosylated xyloglucan. In our quest to synthesize bioactive glycans by enzymatic catalysis, we observed that FgFCO1 is able to catalyze a transglycosylation reaction involving transfer of fucose from citrus peel xyloglucan to lactose to produce 2'-fucosyllactose, an important human milk oligosaccharide. In addition to achieving maximal yields, control of the regioselectivity is an important issue in exploiting such a transglycosylation ability successfully for glycan synthesis. In the present study, we aimed to improve the transglycosylation efficiency of FgFCO1 through protein engineering by transferring successful mutations from other GH29 α-l-fucosidases. We investigated several such mutation transfers by structural alignment, and report that transfer of the mutation F34I from BiAfcB originating from Bifidobacterium longum subsp. infantis to Y32I in FgFCO1 and mutation of D286, near the catalytic acid/base residue in FgFCO1, especially a D286M mutation, have a positive effect on FgFCO1 transfucosylation regioselectivity. We also found that enzymatic depolymerization of the xyloglucan substrate increases substrate accessibility and in turn transglycosylation (i.e., transfucosylation) efficiency. The data include analysis of the active site amino acids and the active site topology of FgFCO1 and show that transfer of point mutations across GH29 subfamilies is a rational strategy for targeted protein engineering of a xyloglucan-active fungal α-l-fucosidase.