Glucuronan lyases from family PL7 use a Tyr/Tyr syn ß-elimination catalytic mechanism for glucuronan breakdown.

TpPL7A and TpPL7B, members of CAZy family PL7, act as ß-glucuronan lyases. TpPL7A diverges by lacking the catalytic histidine, identified as the Brønsted base in PL7 alginate lyases. Our research, including TpPL7A's crystal structure, and mutagenesis studies, reveals a shared syn-ß-elimination mechanism with a single tyrosine serving as both base and acid catalyst. This mechanism may extend to subfamily PL7_4 glucuronan lyases.

Investigation of the structural and immunomodulatory properties of alkali-soluble ß-glucans from Pleurotus eryngii fruiting bodies.

Fungal ß-glucans have received a lot of interest due to their proinflammatory activity towards cells of the innate immune system. Although commonly described as (1➔3)-ß-glucans with varying degree of (1➔6)-branching, the fungal ß-glucans constitute a diverse polysaccharide class. In this study, the alkali-soluble ß-glucans from the edible mushroom Pleurotus eryngii were extracted and characterized by GC, GC-MS and 2D NMR analyses. The extracts contain several structurally different polysaccharides, including a (1➔3)-ß-d-glucan with single glucose units attached at O-6, and a (1➔6)-ß-d-glucan, possibly branched at O-3. The immunomodulatory activities of the P. eryngii extracts were assessed by investigating their ability to bind to the receptor dectin-1, and their ability to induce production of the proinflammatory cytokines TNF-α, IL-6 and IL-1ß in LPS-differentiated THP-1 cells. Although the samples were able to bind to the dectin-1a receptor, they did not induce production of significant levels of cytokines in the THP-1 cells. Positive controls of yeast-derived (1➔3)-ß-d-glucans with branches at O-6 induced cytokine production in the cells. Thus, it appears that the P. eryngii ß-glucans are unable to induce production of proinflammatory cytokines in LPS-differentiated THP-1 cells, despite being able to activate the human dectin-1a receptor.

Functional characterization of a lytic polysaccharide monooxygenase from Schizophyllum commune that degrades non-crystalline substrates.

Lytic polysaccharide monooxygenases (LPMOs) are mono-copper enzymes that use O2 or H2O2 to oxidatively cleave glycosidic bonds. LPMOs are prevalent in nature, and the functional variation among these enzymes is a topic of great interest. We present the functional characterization of one of the 22 putative AA9-type LPMOs from the fungus Schizophyllum commune, ScLPMO9A. The enzyme, expressed in Escherichia coli, showed C4-oxidative cleavage of amorphous cellulose and soluble cello-oligosaccharides. Activity on xyloglucan, mixed-linkage ß-glucan, and glucomannan was also observed, and product profiles differed compared to the well-studied C4-oxidizing NcLPMO9C from Neurospora crassa. While NcLPMO9C is also active on more crystalline forms of cellulose, ScLPMO9A is not. Differences between the two enzymes were also revealed by nuclear magnetic resonance (NMR) titration studies showing that, in contrast to NcLPMO9C, ScLPMO9A has higher affinity for linear substrates compared to branched substrates. Studies of H2O2-fueled degradation of amorphous cellulose showed that ScLPMO9A catalyzes a fast and specific peroxygenase reaction that is at least two orders of magnitude faster than the apparent monooxygenase reaction. Together, these results show that ScLPMO9A is an efficient LPMO with a broad substrate range, which, rather than acting on cellulose, has evolved to act on amorphous and soluble glucans.

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.

The effect of linker conformation on performance and stability of a two-domain lytic polysaccharide monooxygenase.

A considerable number of lytic polysaccharide monooxygenases (LPMOs) and other carbohydrate-active enzymes are modular, with catalytic domains being tethered to additional domains, such as carbohydrate-binding modules, by flexible linkers. While such linkers may affect the structure, function, and stability of the enzyme, their roles remain largely enigmatic, as do the reasons for natural variation in length and sequence. Here, we have explored linker functionality using the two-domain cellulose-active ScLPMO10C from Streptomyces coelicolor as a model system. In addition to investigating the WT enzyme, we engineered three linker variants to address the impact of both length and sequence and characterized these using small-angle X-ray scattering, NMR, molecular dynamics simulations, and functional assays. The resulting data revealed that, in the case of ScLPMO10C, linker length is the main determinant of linker conformation and enzyme performance. Both the WT and a serine-rich variant, which have the same linker length, demonstrated better performance compared with those with either a shorter linker or a longer linker. A highlight of our findings was the substantial thermostability observed in the serine-rich variant. Importantly, the linker affects thermal unfolding behavior and enzyme stability. In particular, unfolding studies show that the two domains unfold independently when mixed, whereas the full-length enzyme shows one cooperative unfolding transition, meaning that the impact of linkers in biomass-processing enzymes is more complex than mere structural tethering.

A glycan receptor kinase facilitates intracellular accommodation of arbuscular mycorrhiza and symbiotic rhizobia in the legume Lotus japonicus.

Receptors that distinguish the multitude of microbes surrounding plants in the environment enable dynamic responses to the biotic and abiotic conditions encountered. In this study, we identify and characterise a glycan receptor kinase, EPR3a, closely related to the exopolysaccharide receptor EPR3. Epr3a is up-regulated in roots colonised by arbuscular mycorrhizal (AM) fungi and is able to bind glucans with a branching pattern characteristic of surface-exposed fungal glucans. Expression studies with cellular resolution show localised activation of the Epr3a promoter in cortical root cells containing arbuscules. Fungal infection and intracellular arbuscule formation are reduced in epr3a mutants. In vitro, the EPR3a ectodomain binds cell wall glucans in affinity gel electrophoresis assays. In microscale thermophoresis (MST) assays, rhizobial exopolysaccharide binding is detected with affinities comparable to those observed for EPR3, and both EPR3a and EPR3 bind a well-defined ß-1,3/ß-1,6 decasaccharide derived from exopolysaccharides of endophytic and pathogenic fungi. Both EPR3a and EPR3 function in the intracellular accommodation of microbes. However, contrasting expression patterns and divergent ligand affinities result in distinct functions in AM colonisation and rhizobial infection in Lotus japonicus. The presence of Epr3a and Epr3 genes in both eudicot and monocot plant genomes suggest a conserved function of these receptor kinases in glycan perception.

Following the Fate of Lytic Polysaccharide Monooxygenases under Oxidative Conditions by NMR Spectroscopy.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of polysaccharides, such as cellulose and chitin. LPMO catalysis requires a reductant, such as ascorbic acid, and hydrogen peroxide, which can be generated in situ in the presence of molecular oxygen and various electron donors. While it is known that reduced LPMOs are prone to autocatalytic oxidative damage due to off-pathway reactions with the oxygen co-substrate, little is known about the structural consequences of such damage. Here, we present atomic-level insights into how the structure of the chitin-active SmLPMO10A is affected by oxidative damage using NMR and circular dichroism spectroscopy. Incubation with ascorbic acid could lead to rearrangements of aromatic residues, followed by more profound structural changes near the copper-active site and loss of activity. Longer incubation times induced changes in larger parts of the structure, indicative of progressing oxidative damage. Incubation with ascorbic acid in the presence of chitin led to similar changes in the observable (i.e., not substrate-bound) fraction of the enzyme. Upon subsequent addition of H2O2, which drastically speeds up chitin hydrolysis, NMR signals corresponding to seemingly intact SmLPMO10A reappeared, indicating dissociation of catalytically competent LPMO. Activity assays confirmed that SmLPMO10A retained catalytic activity when pre-incubated with chitin before being subjected to conditions that induce oxidative damage. Overall, this study provides structural insights into the process of oxidative damage of SmLPMO10A and demonstrates the protective effect of the substrate.

CRISPR-Cas9 driven structural elucidation of the heteroexopolysaccharides from Paenibacillus polymyxa DSM 365.

Paenibacillus polymyxa is a Gram-positive soil bacterium known for producing a wide range of exopolysaccharides. However, due to the biopolymer's complexity, structural elucidation has so far been inconclusive. Combinatorial knock-outs of glycosyltransferases were generated in order to separate distinct polysaccharides produced by P. polymyxa. Using a complementary analytical approach consisting of carbohydrate fingerprints, sequence analysis, methylation analysis as well as NMR spectroscopy, the structure of the repeating units of two additional heteroexopolysaccharides termed paenan I and paenan III were elucidated. Results for paenan I identified a trisaccharide backbone consisting of 1➔4-ß-d-Glc, 1➔4-ß-d-Man and a 1,3,4-branching ß-d-Gal residue with a sidechain comprising of a terminal ß-d-Gal3,4-Pyr and 1➔3-ß-d-Glc. For paenan III, results indicated a backbone consisting of 1➔3-ß-d-Glc, 1,3,4-linked α-d-Man and 1,3,4-linked α-d-GlcA. NMR analysis indicated monomeric ß-d-Glc and α-d-Man sidechains for the branching Man and GlcA residues respectively.

Hydrogels based on methylated-alginates as a platform to investigate the effect of material properties on cell activity. The role of material compliance.

Alginate-based hydrogels with tunable mechanical properties are developed by chemical methylation of the polysaccharide backbone, which was performed either in homogeneous phase (in solution) or in heterogeneous phase (on hydrogels). Nuclear Magnetic Resonance (NMR) and Size Exclusion Chromatography (SEC-MALS) analyses of methylated alginates allow to identify the presence and location of methyl groups on the polysaccharide, and to investigate the influence of methylation on the stiffness of the polymer chains. The methylated polysaccharides are employed for the manufacturing of calcium-reticulated hydrogels for cell growth in 3D. The rheological characterization shows that the shear modulus of hydrogels is dependent on the amount of cross-linker used. Methylated alginates represent a platform to explore the effect of mechanical properties on cell activity. As an example, the effect of compliance is investigated using hydrogels displaying similar shear modulus. An osteosarcoma cell line (MG-63) was encapsulated in the alginate hydrogels and the effect of material compliance on cell proliferation and localization of YAP/TAZ protein complex is investigated by flow cytometry and immunohistochemistry, respectively. The results point out that an increase of material compliance leads to an increase of the proliferative rate of cells and correlates with the translocation of YAP/TAZ inside the cell nucleus.

Tuning alginate ß-lactoglobulin complex coacervation by modulating pH and temperature.

The use of biomolecules in food matrices and encapsulation systems is, as in other areas, moving towards greener solutions and a center piece here is the complex coacervation between natural anionic polysaccharides and proteins. Both alginate and ß-lactoglobulin (ß-Lg) are used in different sectors and have been shown to coacervate at pH < 5.2. Albeit with increased interest, complex coacervation has almost exclusively been studied from a macromolecular perspective, and described as an interaction based on charge-charge attraction. Here, we show that through changes in pH and temperature, alginate ß-Lg complex coacervation can be tuned to purpose. By detailed biophysical and chemical characterization of coacervation and coacervate particles, insights into the molecular interaction and effect of external factors are obtained. We find that carboxylate resonance stabilization causes a release of protons at pH < pKa,alginate and an uptake of protons at pH > pKa,alginate upon coacervation. Proton release and uptake were quantified at pH 2.65 and 4.00 by isothermal titration calorimetry to be 4 and 2 protons per ß-Lg molecule, respectively. By increasing the temperature to 65 °C, we discovered a secondary ß-Lg concentration dependent coacervation step, where the formed particles change into large assemblies driven by entropy. These findings bring new insights to complex coacervation and its applicability in microencapsulation and drug delivery.

Molecular insights into alginate ß-lactoglobulin A multivalencies-The foundation for their amorphous aggregates and coacervation.

For improved control of biomaterial property design, a better understanding of complex coacervation involving anionic polysaccharides and proteins is needed. Here, we address the initial steps in condensate formation of ß-lactoglobulin A (ß-LgA) with nine defined alginate oligosaccharides (AOSs) and describe their multivalent interactions in structural detail. Binding of AOSs containing four, five, or six uronic acid residues (UARs), either all mannuronate (M), all guluronate (G), or alternating M and G embodying the block structural components of alginates, was characterized by isothermal titration calorimetry, nuclear magnetic resonance spectroscopy (NMR), and molecular docking. ß-LgA was highly multivalent exhibiting binding stoichiometries decreasing from five to two AOSs with increasing degree of polymerization (DP) and similar affinities in the mid micromolar range. The different AOS binding sites on ß-LgA were identified by NMR chemical shift perturbation analyses and showed diverse compositions of charged, polar and hydrophobic residues. Distinct sites for the shorter AOSs merged to accommodate longer AOSs. The AOSs bound dynamically to ß-LgA, as concluded from saturation transfer difference and 1 H-ligand-targeted NMR analyses. Molecular docking using Glide within the Schrödinger suite 2016-1 revealed the orientation of AOSs to only vary slightly at the preferred ß-LgA binding site resulting in similar XP glide scores. The multivalency coupled with highly dynamic AOS binding with lack of confined conformations in the ß-LgA complexes may help explain the first steps toward disordered ß-LgA alginate coacervate structures.

Unaided efficient transglutaminase cross-linking of whey proteins strongly impacts the formation and structure of protein alginate particles.

There is a dogma within whey protein modification, which dictates the necessity of pretreatment to enzymatic cross-linking of ß-lactoglobulin (ß-Lg). Here microbial transglutaminase (MTG) cross-linked whey proteins and ß-Lg effectively in 50 mM NaHCO3, pH 8.5, without pretreatment. Cross-linked ß-Lg spanned 18 to >240 kDa, where 6 of 9 glutamines reacted with 8 of 15 lysines. The initial isopeptide bond formation caused loss of ß-Lg native structure with t1/2 = 3 h, while the polymerization occurred with t1/2 = 10 h. Further, cross-linking effects on protein carbohydrate interaction have been overlooked, leaving a gap in understanding of these complex food matrices. Complexation with alginate showed that ß-Lg cross-linking decreased onset of particle formation, hydrodynamic diameter, stoichiometry (ß-Lg/alginate) and dissociation constant. The complexation was favored at higher temperatures (40 °C), suggesting that hydrophobic interactions were important. Thus, ß-Lg was cross-linked without pretreatment and the resulting polymers gave rise to altered complexation with alginate.

Carbohydr Polym Special Issue Invited contribution: Click chemistry for block polysaccharides with dihydrazide and dioxyamine linkers - A review.

Engineered block polysaccharides is a relatively new class of biomacromolecules consisting of chemical assembly of separate block structures at the chain termini. In contrast to conventional, laterally substituted polysaccharide derivatives, the block arrangement allows for much higher preservation of inherent chain properties such as biodegradability and stimuli-responsive self-assembly, while at the same time inducing new macromolecular properties. Abundant, carbon neutral, and even recalcitrant biomass is an excellent source of blocks, opening for numerous new uses of biomass for a wide range of novel biomaterials. Among a limited range of methodologies available for block conjugation, bifunctional linkers allowing for oxyamine and hydrazide 'click' reactions have recently proven useful additions to the repertoire. This article focuses the chemistry and kinetics of these reactions. It also presents some new data with the aim to provide useful protocols and methods for general use towards new block polysaccharides.

Structural elucidation of the fucose containing polysaccharide of Paenibacillus polymyxa DSM 365.

Paenibacillus polymyxa is an avid producer of exopolysaccharides of industrial interest. However, due to the complexity of the polymer composition, structural elucidation of the polysaccharide remained unfeasible for a long time. By using a CRISPR-Cas9 mediated knock-out strategy, all single glycosyltransferases as well as the Wzy polymerases were individually deleted in the corresponding gene cluster for the first time. Thereby, it was observed that the main polymer fraction was completely suppressed (or deleted) and a pure minor fucose containing polysaccharide could be isolated, which was named paenan II. Applying this combinatorial approach, the monosaccharide composition, sequence and linkage pattern of this novel polymer was determined via HPLC-MS, GC-MS and NMR. Furthermore, we demonstrated that the knock-out of the glycosyltransferases PepQ, PepT, PepU and PepV as well as of the Wzy polymerase PepG led to the absence of paenan II, attributing those enzymes to the assembly of the repeating unit.

The impact of reductants on the catalytic efficiency of a lytic polysaccharide monooxygenase and the special role of dehydroascorbic acid.

Monocopper lytic polysaccharide monooxygenases (LPMOs) catalyse oxidative cleavage of glycosidic bonds in a reductant-dependent reaction. Recent studies indicate that LPMOs, rather than being O2 -dependent monooxygenases, are H2 O2 -dependent peroxygenases. Here, we describe SscLPMO10B, a novel LPMO from the phytopathogenic bacterium Streptomyces scabies and address links between this enzyme's catalytic rate and in situ hydrogen peroxide production in the presence of ascorbic acid, gallic acid and l-cysteine. Studies of Avicel degradation showed a clear correlation between the catalytic rate of SscLPMO10B and the rate of H2 O2 generation in the reaction mixture. We also assessed the impact of oxidised ascorbic acid, dehydroascorbic acid (DHA), on LPMO activity, since DHA, which is not considered a reductant, was recently reported to drive LPMO reactions. Kinetic studies, combined with NMR analysis, showed that DHA is unstable and converts into multiple derivatives, some of which are redox active and can fuel the LPMO reaction by reducing the active site copper and promoting H2 O2 production. These results show that the apparent monooxygenase activity observed in SscLPMO10B reactions without exogenously added H2 O2 reflects a peroxygenase reaction.

Structural and functional variation of chitin-binding domains of a lytic polysaccharide monooxygenase from Cellvibrio japonicus.

Among the extensive repertoire of carbohydrate-active enzymes, lytic polysaccharide monooxygenases (LPMOs) have a key role in recalcitrant biomass degradation. LPMOs are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides such as cellulose and chitin. Several LPMOs contain carbohydrate-binding modules (CBMs) that are known to promote LPMO efficiency. However, structural and functional properties of some CBMs remain unknown, and it is not clear why some LPMOs, like CjLPMO10A from the soil bacterium Cellvibrio japonicus, have multiple CBMs (CjCBM5 and CjCBM73). Here, we studied substrate binding by these two CBMs to shine light on their functional variation and determined the solution structures of both by NMR, which constitutes the first structure of a member of the CBM73 family. Chitin-binding experiments and molecular dynamics simulations showed that, while both CBMs bind crystalline chitin with Kd values in the micromolar range, CjCBM73 has higher affinity for chitin than CjCBM5. Furthermore, NMR titration experiments showed that CjCBM5 binds soluble chitohexaose, whereas no binding of CjCBM73 to this chitooligosaccharide was detected. These functional differences correlate with distinctly different arrangements of three conserved aromatic amino acids involved in substrate binding. In CjCBM5, these residues show a linear arrangement that seems compatible with the experimentally observed affinity for single chitin chains. On the other hand, the arrangement of these residues in CjCBM73 suggests a wider binding surface that may interact with several chitin chains. Taken together, these results provide insight into natural variation among related chitin-binding CBMs and the possible functional implications of such variation.

Structure-function analysis of a new PL17 oligoalginate lyase from the marine bacterium Zobellia galactanivorans DsijT.

Alginate is a major compound of brown macroalgae and as such an important carbon and energy source for heterotrophic marine bacteria. Despite the rather simple composition of alginate only comprising mannuronate and guluronate units, these bacteria feature complex alginolytic systems that can contain up to seven alginate lyases. This reflects the necessity of large enzyme systems for the complete degradation of the abundant substrate. Numerous alginate lyases have been characterized. They belong to different polysaccharide lyase (PL) families, but only one crystal structure of a family 17 (PL17) alginate lyase has been reported to date, namely Alg17c from the gammaproteobacterium Saccharophagus degradans. Biochemical and structural characterizations are helpful to link sequence profiles to function, evolution of functions and niche-specific characteristics. Here, we combined detailed biochemical and crystallographic analysis of AlyA3, a PL17 alginate lyase from the marine flavobacteria Zobellia galactanivorans DsijT, providing the first structure of a PL17 in the Bacteroidetes phylum. AlyA3 is exo-lytic and highly specific of mannuronate stretches. As part of an "alginate utilizing locus", its activity is complementary to that of other characterized alginate lyases from the same bacterium. Structural comparison with Alg17c highlights a common mode of action for exo-lytic cleavage of the substrate, strengthening our understanding of the PL17 catalytic mechanism. We show that unlike Alg17c, AlyA3 contains an inserted flexible loop at the entrance to the catalytic groove, likely involved in substrate recognition, processivity and turn over.

RPA2 winged-helix domain facilitates UNG-mediated removal of uracil from ssDNA; implications for repair of mutagenic uracil at the replication fork.

Uracil occurs at replication forks via misincorporation of deoxyuridine monophosphate (dUMP) or via deamination of existing cytosines, which occurs 2-3 orders of magnitude faster in ssDNA than in dsDNA and is 100% miscoding. Tethering of UNG2 to proliferating cell nuclear antigen (PCNA) allows rapid post-replicative removal of misincorporated uracil, but potential 'pre-replicative' removal of deaminated cytosines in ssDNA has been questioned since this could mediate mutagenic translesion synthesis and induction of double-strand breaks. Here, we demonstrate that uracil-DNA glycosylase (UNG), but not SMUG1 efficiently excises uracil from replication protein A (RPA)-coated ssDNA and that this depends on functional interaction between the flexible winged-helix (WH) domain of RPA2 and the N-terminal RPA-binding helix in UNG. This functional interaction is promoted by mono-ubiquitination and diminished by cell-cycle regulated phosphorylations on UNG. Six other human proteins bind the RPA2-WH domain, all of which are involved in DNA repair and replication fork remodelling. Based on this and the recent discovery of the AP site crosslinking protein HMCES, we propose an integrated model in which templated repair of uracil and potentially other mutagenic base lesions in ssDNA at the replication fork, is orchestrated by RPA. The UNG:RPA2-WH interaction may also play a role in adaptive immunity by promoting efficient excision of AID-induced uracils in transcribed immunoglobulin loci.

Impact of Alginate Mannuronic-Guluronic Acid Contents and pH on Protein Binding Capacity and Complex Size.

Alginates, serving as hydrocolloids in the food and pharma industries, form particles at pH < 4.5 with positively charged proteins, such as ß-lactoglobulin (ß-Lg). Alginates are linear anionic polysaccharides composed of 1,4-linked ß-d-mannuronate (M) and α-l-guluronate (G) residues. The impact of M and G contents and pH is investigated to correlate with the formation and size of ß-Lg alginate complexes under relevant ionic strength. It is concluded, using three alginates of M/G ratios 0.6, 1.1, and 1.8 and similar molecular mass, that ß-Lg binding capacity is higher at pH 4.0 than at pH 2.65 and for high M content. By contrast, the largest particles are obtained at pH 2.65 and with high G content. At pH 4.0 and 2.65, the stoichiometry was 28-48 and 3-10 ß-Lg molecules bound per alginate, respectively, increasing with higher M content. The findings will contribute to the design of formation of the desired alginate-protein particles in the acidic pH range.

Alginate Degradation: Insights Obtained through Characterization of a Thermophilic Exolytic Alginate Lyase.

Enzymatic depolymerization of seaweed polysaccharides is gaining interest for the production of functional oligosaccharides and fermentable sugars. Herein, we describe a thermostable alginate lyase that belongs to polysaccharide lyase family 17 (PL17) and was derived from an Arctic Mid-Ocean Ridge (AMOR) metagenomics data set. This enzyme, AMOR_PL17A, is a thermostable exolytic oligoalginate lyase (EC 4.2.2.26), which can degrade alginate, poly-ß-d-mannuronate, and poly-α-l-guluronate within a broad range of pHs, temperatures, and salinity conditions. Site-directed mutagenesis showed that tyrosine Y251, previously suggested to act as a catalytic acid, indeed is essential for catalysis, whereas mutation of tyrosine Y446, previously proposed to act as a catalytic base, did not affect enzyme activity. The observed reaction products are protonated and deprotonated forms of the 4,5-unsaturated uronic acid monomer, Δ, two hydrates of DEH (4-deoxy-l-erythro-5-hexulosuronate), which are formed after ring opening, and, finally, two epimers of a 5-member hemiketal called 4-deoxy-d-manno-hexulofuranosidonate (DHF), formed through intramolecular cyclization of hydrated DEH. The detection and nuclear magnetic resonance (NMR) assignment of these hemiketals refine our current understanding of alginate degradation.IMPORTANCE The potential markets for seaweed-derived products and seaweed processing technologies are growing, yet commercial enzyme cocktails for complete conversion of seaweed to fermentable sugars are not available. Such an enzyme cocktail would require the catalytic properties of a variety of different enzymes, where fucoidanases, laminarinases, and cellulases together with endo- and exo-acting alginate lyases would be the key enzymes. Here, we present an exo-acting alginate lyase that efficiently produces monomeric sugars from alginate. Since it is only the second characterized exo-acting alginate lyase capable of degrading alginate at a high industrially relevant temperature (≥60°C), this enzyme may be of great biotechnological and industrial interest. In addition, in-depth NMR-based structural elucidation revealed previously undescribed rearrangement products of the unsaturated monomeric sugars generated from exo-acting lyases. The insight provided by the NMR assignment of these products facilitates future assessment of product formation by alginate lyases.