Biochemical characterization and antifungal activity of a recombinant ß-1,3-glucanase FlGluA from Flavobacterium sp. NAU1659.

ß-1,3-glucanases can degrade ß-1,3-glucoside bonds in ß-glucan which is the main cell-wall component of most of fungi, and have the crucial application potential in plant protection and food processing. Herein, a ß-1,3-glucanase FlGluA from Flavobacterium sp. NAU1659 composed of 333 amino acids with a predicted molecular mass of 36.6 kDa was expressed in Escherichia coli BL21, purified and characterized. The deduced amino acid sequence of FlGluA showed the high identity with the ß-1,3-glucanase belonging to glycoside hydrolase (GH) family 16. Enzymological characterization indicated FlGluA had the highest activity on zymosan A, with a specific activity of 3.87 U/mg, followed by curdlan (1.16 U/mg) and pachymaran (0.88 U/mg). It exhibited optimal catalytic activity at the pH 5.0 and 40 °C, and was stable when placed at 4 °C for 12 h in the range of pH 3.0-8.0 or at a temperature below 50 °C for 3 h. Its catalytic activity was enhanced by approximately 36 % in the presence of 1 mM Cr3+. The detection of thin-layer chromatography and mass spectrometry showed FlGluA hydrolyzed zymosan A mainly to glucose and disaccharide, and trace amounts of tetrasaccharide and pentasaccharide, however, it had no action on laminaribiose, indicating its endo-ß-1,3-glucanase activity. The mycelium growth of F. oxysporum treated by FlGluA was inhibited, with approximately 37 % of inhibition rate, revealing the potential antifungal activity of the enzyme. These results revealed the hydrolytic properties and biocontrol activity of FlGluA, laying a crucial foundation for its potential application in agriculture and industry.

Structural insights into ß-1,3-glucan cleavage by a glycoside hydrolase family.

The fundamental and assorted roles of ß-1,3-glucans in nature are underpinned on diverse chemistry and molecular structures, demanding sophisticated and intricate enzymatic systems for their processing. In this work, the selectivity and modes of action of a glycoside hydrolase family active on ß-1,3-glucans were systematically investigated combining sequence similarity network, phylogeny, X-ray crystallography, enzyme kinetics, mutagenesis and molecular dynamics. This family exhibits a minimalist and versatile (α/ß)-barrel scaffold, which can harbor distinguishing exo or endo modes of action, including an ancillary-binding site for the anchoring of triple-helical ß-1,3-glucans. The substrate binding occurs via a hydrophobic knuckle complementary to the canonical curved conformation of ß-1,3-glucans or through a substrate conformational change imposed by the active-site topology of some fungal enzymes. Together, these findings expand our understanding of the enzymatic arsenal of bacteria and fungi for the breakdown and modification of ß-1,3-glucans, which can be exploited for biotechnological applications.

Conformation and cooperative order-disorder transition in aqueous solutions of ß-1,3-d-glucan with different degree of branching varied by the Smith degradation.

ß-1,3-d-glucan with different degrees of branching were obtained by selectively and gradually removing side chains from schizophyllan, a water-soluble triple helical polysaccharide, using the Smith degradation. Size exclusion chromatography combined with a multi-angle light scattering detection was performed in aqueous 0.1 M NaCl. The degree of branching decreased after the Smith degradation, while the molar mass distributions were almost unchanged. The molecular conformation of the Smith-degraded ß-1,3-d-glucan was analyzed on the basis of the molar mass dependency of the radius gyration, and found to be comparable to the original triple helix of schizophyllan. Differential scanning calorimetry in deuterium oxide-hexadeuterodimethylsulfoxide mixtures was performed to investigate the effects of the degree of branching on the cooperative order-disorder transition. Removal of side chains affects both the transition temperature and transition enthalpy. The ordered structure is formed by the residual side chains in the triplex unit, so that the linear cooperative system of the triplex is maintained after the Smith degradation.

Improved extraction of resveratrol and antioxidants from grape peel using heat and enzymatic treatments.

BACKGROUND: Resveratrol, an extensively recognized phytochemical that belongs to the stilbene family, is abundant in grape peel which is discarded as a by-product during grape juice processing. RESULTS: In this study, we established that pre-heating grape peel above 75 °C significantly improved the extractability of resveratrol and its glucoside piceid. In particular, thermal heating of grape peel at 95 °C for 10 min, followed by treatment with a mixture of exo-1,3-ß-glucanase and pectinases at 50 °C for 60 min, dramatically increased the conversion of piceid into resveratrol and the overall extractability of this phytochemical by 50%. Furthermore, thermal pre-treatment promoted a substantial increase in the total phenol, flavonoid, and anthocyanin concentrations in the grape peel extract. Ultimately, resveratrol-enriched grape peel extract significantly augmented the antioxidant response in vitro, possibly by attenuating the accumulation of intracellular reactive oxygen species via the Nrf2 signaling pathway. CONCLUSION: The method developed in this study for preparing grape peel extract introduces a potential low-cost green processing for the industrial fortification of food products with resveratrol and other health-beneficial antioxidants. © 2019 Society of Chemical Industry.

Properties of a family 56 carbohydrate-binding module and its role in the recognition and hydrolysis of ß-1,3-glucan.

BH0236 from Bacillus halodurans is a multimodular ß-1,3-glucanase comprising an N-terminal family 81 glycoside hydrolase catalytic module, an internal family 6 carbohydrate-binding module (CBM) that binds the nonreducing end of ß-1,3-glucan chains, and an uncharacterized C-terminal module classified into CBM family 56. Here, we determined that this latter CBM, BhCBM56, bound the soluble ß-1,3-glucan laminarin with a dissociation constant (Kd ) of ∼26 µm and displayed higher affinity for insoluble ß-1,3-glucans with Kd values of ∼2-10 µm but lacked affinity for ß-1,3-glucooligosaccharides. The X-ray crystal structure of BhCBM56 and NMR-derived chemical shift mapping of the binding site revealed a ß-sandwich fold, with the face of one ß-sheet possessing the ß-1,3-glucan-binding surface. On the basis of the functional and structural properties of BhCBM56, we propose that it binds a quaternary polysaccharide structure, most likely the triple helix adopted by polymerized ß-1,3-glucans. Consistent with the BhCBM56 and BhCBM6/56 binding profiles, deletion of the CBM56 from BH0236 decreased activity of the enzyme on the insoluble ß-1,3-glucan curdlan but not on soluble laminarin; additional deletion of the CBM6 also did not affect laminarin degradation but further decreased curdlan hydrolysis. The pseudo-atomic solution structure of BH0236 determined by small-angle X-ray scattering revealed structural insights into the nature of avid binding by the BhCBM6/56 pair and how the orientation of the active site in the catalytic module factors into recognition and degradation of ß-1,3-glucans. Our findings reinforce the notion that catalytic modules and their cognate CBMs have complementary specificities, including targeting of polysaccharide quaternary structure.

Molecular characterization and evolution of carnivorous sundew (Drosera rotundifolia L.) class V ß-1,3-glucanase.

MAIN CONCLUSION: A gene for ß-1,3-glucanase was isolated from carnivorous sundew. It is active in leaves and roots, but not in digestive glands. Analyses in transgenic tobacco suggest its function in germination. Ancestral plant ß-1,3-glucanases (EC 3.2.1.39) played a role in cell division and cell wall remodelling, but divergent evolution has extended their roles in plant defense against stresses to decomposition of prey in carnivorous plants. As available gene sequences from carnivorous plants are rare, we isolated a glucanase gene from roundleaf sundew (Drosera rotundifolia L.) by a genome walking approach. Computational predictions recognized typical gene features and protein motifs described for other plant ß-1,3-glucanases. Phylogenetic reconstructions suggest strong support for evolutionary relatedness to class V ß-1,3-glucanases, including homologs that are active in the traps of related carnivorous species. The gene is expressed in sundew vegetative tissues but not in flowers and digestive glands, and encodes for a functional enzyme when expressed in transgenic tobacco. Detailed analyses of the supposed promoter both in silico and in transgenic tobacco suggest that this glucanase plays a role in development. Specific spatiotemporal activity was observed during transgenic seed germination. Later during growth, the sundew promoter was active in marginal and sub-marginal areas of apical true leaf meristems of young tobacco plants. These results suggest that the isolated glucanase gene is regulated endogenously, possibly by auxin. This is the first report on a nuclear gene study from sundew.

Modulating the function of a ß-1,3-glucanosyltransferase to that of an endo-ß-1,3-glucanase by structure-based protein engineering.

A glycoside hydrolase (GH) family 17 ß-1,3-glucanosyltransferase (RmBgt17A) from Rhizomucor miehei CAU432 (CGMCC No. 4967) shared very low sequence homology (∼20 % identity) with that of other ß-1,3-glucanases,despite their similar structural folds. Structural comparison and sequence alignment between RmBgt17A and GH family 17 ß-1,3-glucanases suggested important roles for three residues (Tyr102, Trp157, and Glu158) located in the substrate-binding cleft of RmBgt17A in transglycosylation activity. A series of site-directed mutagenesis studies indicated that a single Glu-to-Ala mutation (E158A) modulates the function of RmBgt17A to that of a ß-1,3-glucanase. Mutant E158A exhibited high hydrolytic activity (39.95 U/mg) toward reduced laminarin, 348.5-fold higher than the wild type. Optimal pH and temperature of the purified RmBgt17A-E158A were 4.5 and 55 °C, respectively. TLC analysis suggested that RmBgt17A-E158A is an endo-ß-1,3-glucanase. Our study provides novel insight into protein engineering of the substrate-binding cleft of glycoside hydrolases to modulate the function of transglycosylation and hydrolysis.

Biochemical and genetic characterization of ß-1,3 glucanase from a deep subseafloor Laceyella putida.

A ß-1,3-glucanase (LpGluA) of deep subseafloor Laceyella putida JAM FM3001 was purified to homogeneity from culture broth. The molecular mass of the enzyme was around 36 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). LpGluA hydrolyzed curdlan optimally at pH 4.2 and 80 °C. In spite of the high optimum temperature, LpGluA showed relatively low thermostability, which was stabilized by adding laminarin, xylan, colloidal chitin, pectin, and its related polysaccharides. The gene for LpGluA cloned by using degenerate primers was composed of 1236 bp encoding 411 amino acids. Production of both LpGluA and a chitinase (LpChiA; Shibasaki et al. Appl Microbiol Biotechnol 98, 7845-7853, 2014) was induced by adding N-acetylglucosamine (GluNAc) to a culture medium of strain JAM FM3001. Construction of expression vectors containing the gene for LpGluA and its flanking regions showed the existence of a putative repressor protein.

Enhancing the Antioxidant Ability of Trametes versicolor Polysaccharopeptides by an Enzymatic Hydrolysis Process.

Polysaccharopeptides (PSPs) are among the main bioactive constituents of Trametes versicolor (T. versicolor). The purpose of this research was to investigate the antioxidant activities of enzymatic hydrolysates obtained from T. versicolor polysaccharopeptides by 80 U/mL ß-1,3-glucanase (PSPs-EH80). The half-inhibitory concentration (IC50) of PSPs-EH80 in metal chelating assay, ABTS and DPPH radical scavenging test results were 0.83 mg/mL, 0.14 mg/mL and 0.52 mg/mL, respectively, which were lower than that of PSPs-EH 20 U/mL. The molecular weights of the PSPs-EH80 hydrolysates were 300, 190, 140 and 50 kDa, respectively, and the hydrolysis of polysaccharides by ß-1,3-glucanase did not change the original functional group. PSPs-EH80 reduced the reactive oxygen species (ROS) content at least twice that of treatment without PSPs-EH80. In addition, an oxidative damage test showed that PSPs-EH80 can improve HaCaT cell survival. According to our results, PSP demonstrates the potential of anti-oxidative damage; besides, enzyme hydrolysis can improve the ability of the PSP.

The ß-glucanase ZgLamA from Zobellia galactanivorans evolved a bent active site adapted for efficient degradation of algal laminarin.

Laminarinase is commonly used to describe ß-1,3-glucanases widespread throughout Archaea, bacteria, and several eukaryotic lineages. Some ß-1,3-glucanases have already been structurally and biochemically characterized, but very few from organisms that are in contact with genuine laminarin, the storage polysaccharide of brown algae. Here we report the heterologous expression and subsequent biochemical and structural characterization of ZgLamAGH16 from Zobellia galactanivorans, the first GH16 laminarinase from a marine bacterium associated with seaweeds. ZgLamAGH16 contains a unique additional loop, compared with other GH16 laminarinases, which is composed of 17 amino acids and gives a bent shape to the active site cleft of the enzyme. This particular topology is perfectly adapted to the U-shaped conformation of laminarin chains in solution and thus explains the predominant specificity of ZgLamAGH16 for this substrate. The three-dimensional structure of the enzyme and two enzyme-substrate complexes, one with laminaritetraose and the other with a trisaccharide of 1,3-1,4-ß-d-glucan, have been determined at 1.5, 1.35, and 1.13 Å resolution, respectively. The structural comparison of substrate recognition pattern between these complexes allows the proposition that ZgLamAGH16 likely diverged from an ancestral broad specificity GH16 ß-glucanase and evolved toward a bent active site topology adapted to efficient degradation of algal laminarin.