The evolutionary advantage of an aromatic clamp in plant family 3 glycoside exo-hydrolases.

In the barley ß-D-glucan glucohydrolase, a glycoside hydrolase family 3 (GH3) enzyme, the Trp286/Trp434 clamp ensures ß-D-glucosides binding, which is fundamental for substrate hydrolysis during plant growth and development. We employ mutagenesis, high-resolution X-ray crystallography, and multi-scale molecular modelling methods to examine the binding and conformational behaviour of isomeric ß-D-glucosides during substrate-product assisted processive catalysis that operates in GH3 hydrolases. Enzyme kinetics reveals that the W434H mutant retains broad specificity, while W434A behaves as a strict (1,3)-ß-D-glucosidase. Investigations of reactant movements on the nanoscale reveal that processivity is sensitive to mutation-specific alterations of the tryptophan clamp. While wild-type and W434H utilise a lateral cavity for glucose displacement and sliding of (1,3)-linked hydrolytic products through the catalytic site without dissociation, consistent with their high hydrolytic rates, W434A does not adopt processive catalysis. Phylogenomic analyses of GH3 hydrolases disclose the evolutionary advantage of the tryptophan clamp that confers broad specificity, high catalytic efficiency, and processivity.

Changes of structures and biosynthesis/hydrolysis-associated genes expression of glucans at different Volvariella volvacea maturity stages.

In the present study, effects of maturity stage on structural characteristics and biosynthesis/hydrolysis-associated genes expression of glucans from Volvariella volvacea fruit body were well investigated. Elongation and pileus expansion stages decreased total soluble carbohydrate and protein contents to 17.09 mg/g and 8.33 mg/g, and significantly accumulated the total amino acids contents to 32.37 mg/g. Yields of crude polysaccharides significantly increased to 8.12% at egg stage and decreased to 3.72% at pileus expansion stage. Purified VVP I-a and VVP I-b were proved to be α-glucans. The maturity process affected the monosaccharide compositions, decreased the molecular weights of VVP I-a and VVP I-b with decreased transcription levels of glucan biosynthesis-associated enzyme genes vvugp and vvgls and increased glucan hydrolysis-associated glucanase gene vvexg2 expression with no significant effects on backbone structures including glycosidic linkages and configurations. The findings would benefit for understanding change patterns of V. volvacea glucan structures and their biosynthesis/hydrolysis-associated genes expression at maturity stages.

Structure and inhibition of Cryptococcus neoformans sterylglucosidase to develop antifungal agents.

Pathogenic fungi exhibit a heavy burden on medical care and new therapies are needed. Here, we develop the fungal specific enzyme sterylglucosidase 1 (Sgl1) as a therapeutic target. Sgl1 converts the immunomodulatory glycolipid ergosterol 3ß-D-glucoside to ergosterol and glucose. Previously, we found that genetic deletion of Sgl1 in the pathogenic fungus Cryptococcus neoformans (Cn) results in ergosterol 3ß-D-glucoside accumulation, renders Cn non-pathogenic, and immunizes mice against secondary infections by wild-type Cn, even in condition of CD4+ T cell deficiency. Here, we disclose two distinct chemical classes that inhibit Sgl1 function in vitro and in Cn cells. Pharmacological inhibition of Sgl1 phenocopies a growth defect of the Cn Δsgl1 mutant and prevents dissemination of wild-type Cn to the brain in a mouse model of infection. Crystal structures of Sgl1 alone and with inhibitors explain Sgl1's substrate specificity and enable the rational design of antifungal agents targeting Sgl1.

Techniques for Mitigating the Effects of Smoke Taint While Maintaining Quality in Wine Production: A Review.

Smoke taint has become a prominent issue for the global wine industry as climate change continues to impact the length and extremity of fire seasons around the world. Although the issue has prompted a surge in research on the subject in recent years, no singular solution has yet been identified that is capable of maintaining the quality of wine made from smoke-affected grapes. In this review, we summarize the main research on smoke taint, the key discoveries, as well as the prevailing uncertainties. We also examine methods for mitigating smoke taint in the vineyard, in the winery, and post production. We assess the effectiveness of remediation methods (proposed and actual) based on available research. Our findings are in agreement with previous studies, suggesting that the most viable remedies for smoke taint are still the commercially available activated carbon fining and reverse osmosis treatments, but that the quality of the final treated wines is fundamentally dependent on the initial severity of the taint. In this review, suggestions for future studies are introduced for improving our understanding of methods that have thus far only been preliminarily investigated. We select regions that have already been subjected to severe wildfires, and therefore subjected to smoke taint (particularly Australia and California) as a case study to inform other wine-producing countries that will likely be impacted in the future and suggest specific data collection and policy implementation actions that should be taken, even in countries that have not yet been impacted by smoke taint. Ultimately, we streamline the available information on the topic of smoke taint, apply it to a global perspective that considers the various stakeholders involved, and provide a launching point for further research on the topic.

Effects and Mechanisms of Alkali Recycling and Ozone Recycling on Enzymatic Conversion in Alkali Combined with Ozone Pretreatment of Corn Stover.

In order to minimize waste liquor, save resources, and reduce costs, the effects of alkali recycling and ozone recycling on enzymatic conversion in alkali combined with ozone pretreatment of corn stover and the mechanism were studied. The results showed that as the number of cycles of alkali/ozone filtrate increased, the enzymatic conversion and the loss of reducing sugars showed a downward trend. It was indicated that the ability of alkali to damage lignocellulosic decreased with an increasing number of alkali circulation and the accumulation of lignin degradation products generated during ozonolysis inhibited enzymatic conversion. When the ozone filtrate was recovered and used for hydrolysis directly, the enzymatic conversion rates were basically the same compared with the first self-circulation of ozone filtrate, and no sewage was discharged. In conclusion, the optimal circulating pretreatment was four times alkali circulation and ozone filtrate was used as an enzymolysis liquid directly, and the conversion rates of cellulose and hemicellulose were 85.96% and 34.26%, respectively, saving 44% alkali consumption at the same time. This paper provided the theoretical basis for the development of lignocellulose pretreatment technology with low cost, high efficiency, and high conversion rate.

Structural and functional modification of kudzu starch using α-amylase and transglucosidase.

The large agglomeration of starch paste in hot water, and fast retrogradation tendency and low transparency of starch gel restrict widespread application of kudzu starch. To improve the above defects, kudzu starch was modified with sequentially α-amylase (AA) and transglucosidase (TG), the latter for varying times. The results indicated that, compared to kudzu starch, amylose content and molecular weight of AA/TG-treated starches reduced by 20.07% and 69.50%, respectively. The proportion of A chain increased by 68.68%, whereas B1, B2 and B3 chains decreased by 14.28%, 48.29% and 23.44%, respectively. The degree of branching dramatically increased by 128.3%. After AA→TG treatment, the changes of starch structure enhanced the functional properties of kudzu starch. The solubility, paste clarity and gelatinization temperature increased, whereas the relative crystallinity, viscosity, storage and loss moduli decreased. Overall, the AA→TG modification would be desirable to improve the functional properties of kudzu starch to expand more large-scale application.

Generation of short-chained granular corn starch by maltogenic α-amylase and transglucosidase treatment.

We describe a method for permitting efficient modification by transglucosidase (TGA), from glycoside hydrolase family 31 (GH31), sequentially after the pre-treatment by maltogenic α-amylases (MA) from GH13. TGA treatment without MA pre-treatment had negligible effects on native starch, while TGA treatment with MA pre-treatment resulted in porous granules and increased permeability to enzymes. MA→TGA treatments lead to decreased molecular size of amylopectin molecules, increased α-1,6 branching, and increased amounts of amylopectin chains with the degree of polymerization (DP)<10 and decreased amounts of DP 10-28 after debranching. Wide-angle X-ray scattering (WAXS) data showed a general decrease in crystallinity except for a long term (20 h) TGA post-treatment which increased the relative crystallinity back to normal. MA→TGA treatment significantly lowered the starch retrogradation of starch and retarded the increase of storage- and loss moduli during storage. This work demonstrates the potential of sequential addition of starch active enzymes to obtain granular starch with improved functionality.

Forty years of study on the thermostable ß-glycosidase from S. solfataricus: Production, biochemical characterization and biotechnological applications.

The aim of this paper is to make the point on the fortieth years study on the ß-glycosidase from Sulfolobus solfataricus. This enzyme represents one of the thermophilic biocatalysts, which is more extensively studied as witnessed by the numerous literature reports available since 1980. Comprehensive biochemical studies highlighted its broad substrate specificity for ß-d-galacto-, gluco-, and fuco-sides and also showed its remarkable exo-glucosidase and transglycosidase activities. The enzyme demonstrated to be active and stable over a wide range of temperature and pHs, withstanding to several drastic conditions comprising solvents and detergents. Over the years, a great deal of studies were focused on its homotetrameric tridimensional structure, elucidating several structural features involved in the enzyme stability, such as ion pairs and post-translational modifications. Several ß-glycosidase mutants were produced in the years in order to understand its peculiar behavior in extreme conditions and/or to improve its functional properties. The ß-glycosidase overproduction was also afforded reporting numerous studies dealing with its production in the mesophilic host Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, and Lactococcus lactis. Relevant applications in food, beverages, bioenergy, pharmaceuticals, and nutraceutical fields of this enzyme, both in free and immobilized forms, highlighted its biotechnological relevance.

Entrapment of enzyme aggregates in chitosan beads for aroma release in white wines.

Glycosidases are enzymes involved in the cascade reactions leading to the release of aromatic compounds in white wines. However, the use of commercial soluble glycosidases is facing difficulties due to their fast inactivation, poor reaction control, low efficiency of enzyme use, and the presence of catalyst residues in the product. Co-immobilization as cross-linked enzyme aggregates (combi-CLEAs) is a sound alternative allowing the immobilization of enzymes in their own protein matrix, yielding highly stable and active biocatalysts. Notwithstanding, their micrometer sized particles limit their application in industrial processes. To overcome this, combi-CLEAs of ß-D-glucosidase (ßG) and α-L-arabinofuranosidase (ARA) were entrapped in polymeric chitosan beads. The effect of crosslinking reagents and crosslinking time on the specific activity and stability of combi-CLEAs was studied, and the best conditions for the entrapment of the combi-CLEAs in polymeric chitosan beads were determined varying the concentration of the chitosan solution and the pH of the gelation agent solution. The resulting biocatalyst beads (average diameter 1.24 mm), retained full activity after 91 days of incubation under winemaking conditions, having specific activities of 0.91 and 0.88 international units of activity per gram for ßG and ARA, respectively. Such characteristics make them suitable for aroma enhancement in wines.

Optimization of Catechin and Proanthocyanidin Recovery from Grape Seeds Using Microwave-Assisted Extraction.

Grape seed extract (GSE) is a rich source of condensed flavonoid tannins, also called proanthocyanidins (PACs). The high molecular weight of polymeric PAC limits their biological activity due to poor bioavailability. The present study was undertaken to explore the potential applicability of microwave-assisted extraction (MAE) to convert GSE-PAC into monomeric catechins. A central composite design (CCD) was used to optimize the processing conditions for the MAE. The maximum total yield of monomeric catechins (catechin, epicatechin, and epicatechin gallate) and PAC were 8.2 mg/g dry weight (DW) and 56.4 mg catechin equivalence (CE)/g DW, respectively. The optimized MAE condition was 94% ethanol, 170 °C temperature, and a duration of 55 min. Compared to the results for PACs extracted via conventional extraction (Con) (94% ethanol; shaking at 25 °C for 55 min), MAE yielded 3.9-fold more monomeric catechins and 5.5-fold more PACs. The MAE showed higher antioxidant capacity and α-glucosidase inhibitory activity than that of the conventional extract, suggesting the potential use of the MAE products of grape seeds as a functional food ingredient and nutraceutical.

Structures of ß-glycosidase LXYL-P1-2 reveals the product binding state of GH3 family and a specific pocket for Taxol recognition.

LXYL-P1-2 is one of the few xylosidases that efficiently catalyze the reaction from 7-ß-xylosyl-10-deacetyltaxol (XDT) to 10-deacetyltaxol (DT), and is a potential enzyme used in Taxol industrial production. Here we report the crystal structure of LXYL-P1-2 and its XDT binding complex. These structures reveal an enzyme/product complex with the sugar conformation different from the enzyme/substrate complex reported previously in GH3 enzymes, even in the whole glycohydrolases family. In addition, the DT binding pocket is identified as the structural basis for the substrate specificity. Further structure analysis reveals common features in LXYL-P1-2 and Taxol binding protein tubulin, which might provide useful information for designing new Taxol carrier proteins for drug delivery.

Phenolic compounds extraction in enzymatic macerations of grape skins identified as low-level extractable total anthocyanin content.

Anthocyanins in wine principally depend on grape skin extractable anthocyanin content, that is, the amount of anthocyanins present in grape skin that are released to wine during the maceration stage. This amount of extractable anthocyanins is closely linked to the cell wall degradation of skin cells. Indeed, among other methodologies, the maceration in presence of different enzymes can be used to increase cell wall degradation, and therefore, the amount of anthocyanins extracted from grape skins to wine. Vitis vinifera L. cv. Tempranillo and Syrah red grapes have been identified as samples with low anthocyanin extraction potential by near infrared hyperspectral imaging. Grape skins have been macerated in the presence of cellulase, glucosidase, and pectinase. Then, color of the supernatants and phenolic compounds extracted from grape skins (total phenols, total flavanols, and total and individual anthocyanins) has been determined. Cellulase and glucosidase have shown a positive effect in the extraction of phenolic compounds from these grapes. Macerations carried out in the presence of cellulase have produced supernatants with a more intense color (lower lightness and higher chroma values), and a higher extraction of flavanols and anthocyanins than the respective control essays. However, pectinase treatments have produced the opposite effect, which could be partially explained by an eventual interaction between the cell wall polysaccharides liberated by pectinase and the phenolic compounds extracted. Synergy effects do not appear between cellulase and glucosidase. Moreover, the negative effect of the addition of pectinase might be due to the interactions between the cell wall material liberated by pectinase and the phenolic compounds extracted. PRACTICAL APPLICATION: In the present study, grape samples with a low anthocyanin extraction potential have been identified, and these samples have been macerated in the presence of different enzymes. The applied enzymes were three of the most common enzymes that are applied in the wine industry. Individual enzymes and mixtures have been applied to Syrah and Tempranillo grape skin samples and the results have been compared to control macerations. Knowledge in this topic will help the production of quality wines.

Molecular Mechanisms of Interactions between Monolayered Transition Metal Dichalcogenides and Biological Molecules.

Single layered two-dimensional (2D) materials such as transition metal dichalcogenides (TMDs) show great potential in many microelectronic or nanoelectronic applications. For example, because of extremely high sensitivity, TMD-based biosensors have become promising candidates for next-generation label-free detection. However, very few studies have been conducted on understanding the fundamental interactions between TMDs and other molecules including biological molecules, making the rational design of TMD-based sensors (including biosensors) difficult. This study focuses on the investigations of the fundamental interactions between proteins and two widely researched single-layered TMDs, MoS2, and WS2 using a combined study with linear vibrational spectroscopy attenuated total reflectance FTIR and nonlinear vibrational spectroscopy sum frequency generation vibrational spectroscopy, supplemented by molecular dynamics simulations. It was concluded that a large surface hydrophobic region in a relatively flat location on the protein surface is required for the protein to adsorb onto a monolayered MoS2 or WS2 surface with preferred orientation. No disulfide bond formation between cysteine groups on the protein and MoS2 or WS2 was found. The conclusions are general and can be used as guiding principles to engineer proteins to attach to TMDs. The approach adopted here is also applicable to study interactions between other 2D materials and biomolecules.

Insights into the pH-dependent catalytic mechanism of Sulfolobus solfataricus ß-glycosidase: A molecular dynamics study.

Sulfolobus solfataricus ß-glycosidase (SS-ßGly) belongs to Glycosyl Hydrolase family1 (GH1) with broad substrate specificity. SS-ßGly catalyzes both hydrolysis and transglycosylation reactions. SS-ßGly is commonly used to synthesize variety of galacto-oligosaccharides. A comparison of SS-ßGly with bacterial and eukaryotic homologs, using DALI search, revealed unique inserts. Free enzyme molecular dynamics (MD) simulation was performed under two different pH conditions (pH 6.5 and 2.5) at a constant temperature of 65 °C using GROMACS. A probable active-site loop (residues 331-364) in SS-ßGly was identified. Dynamics of substrate binding cavity revealed that it was buried and inaccessible during most timeframes at pH 6.5 whereas open and accessible at pH 2.5. New cavities identified during both simulations may act as probable water channel or product egress path. Analyses of docked complexes of 3D structures obtained at every 1ns interval with compounds, involved in hydrolysis and tranglycosylation reactions, demonstrated that conformational states sampled by SS-ßGly during free enzyme dynamics mimic the stages in enzyme catalysis thereby providing a mechanistic perspective. Current study revealed that conformational changes were conducive for hydrolysis at pH 6.5 and multiple cycles of transglycosylation at pH 2.5. Probable role of salt-bridge interactions in determining the type of reaction mechanism was also explored.

Discovery of processive catalysis by an exo-hydrolase with a pocket-shaped active site.

Substrates associate and products dissociate from enzyme catalytic sites rapidly, which hampers investigations of their trajectories. The high-resolution structure of the native Hordeum exo-hydrolase HvExoI isolated from seedlings reveals that non-covalently trapped glucose forms a stable enzyme-product complex. Here, we report that the alkyl ß-D-glucoside and methyl 6-thio-ß-gentiobioside substrate analogues perfused in crystalline HvExoI bind across the catalytic site after they displace glucose, while methyl 2-thio-ß-sophoroside attaches nearby. Structural analyses and multi-scale molecular modelling of nanoscale reactant movements in HvExoI reveal that upon productive binding of incoming substrates, the glucose product modifies its binding patterns and evokes the formation of a transient lateral cavity, which serves as a conduit for glucose departure to allow for the next catalytic round. This path enables substrate-product assisted processive catalysis through multiple hydrolytic events without HvExoI losing contact with oligo- or polymeric substrates. We anticipate that such enzyme plasticity could be prevalent among exo-hydrolases.

Synergistic effects of branching enzyme and transglucosidase on the modification of potato starch granules.

Potato starch displayed high viscosity, low hydroscopicity and dispersity, and acid susceptibility leading to the limited application of potato starch. To expand the potato starch utility with appropriate processing characteristics, potato starch granules were modified with branching enzyme (BE) and transglucosidase (TG). The results indicated that the susceptibility of potato starch granules to TG was higher than BE. Moreover, the two enzymes showed the synergistic effect in enzymatic modification of potato starch granules. They cooperatively attacked the external and interior of potato starch granules. The crystal forms of potato starch changed from B to C-type after double enzyme treatments, and enzyme-treated starches exhibited homogeneous crystal distribution. Compared to BE or TG alone, the combined action of BE and TG increased significantly the ratio of α-1,6-glycosidic linkage and the amounts of short chains of potato starch, which led to the significant reduction in degree of crystallinity, viscosity, gelatinization temperature and enthalpy, and a remarkable increase in solubility. Especially, the physicochemical characteristics of modified starch largely depended on the treatment time of TG. Thus, through the combination of BE and TG, the appropriate treatment time of TG may be chosen to improve the physicochemical properties of potato starch in processed starch-based products.

New Acylglycosides Flavones from Fuzhuan Brick Tea and Simulation Analysis of Their Bioactive Effects.

Four novel acylglycosides flavones (AGFs) including two quercetin acylglycosides and two kaempferol acylglycosides were isolated from Fuzhuan brick tea (FBT) as follows: quercetin 3-O-[α-l-rhamnopyranosyl (1→3)] [2-O''-(E)-p-coumaroyl] [ß-d-glucopyranosyl (1→3)-α-l-rhamnopyranosyl (1→6)]-ß-d-galactoside was named as camelliquercetiside E (1), quercetin 3-O-[α-l-rhamnopyranosyl (1→3)] [2-O''-(E)-p-coumaroyl] [α-l-rhamnopyranosyl (1→6)]-ß-d-galactoside was named as camelliquercetiside F (2), kaempferol 3-O-[α-l-arabinopyranosyl (1→3)] [2-O''-(E)-p-coumaroyl] [ß-d-glucopyranosyl (1→3)-α-l-rhamnopyranosyl (1→6)]-ß-d-glucoside was named as camellikaempferoside D (3), kaempferol 3-O-[α-l-arabinopyranosyl (1→3)] [2-O''-(E)-p-coumaroyl] [α-l-rhamnopyranosyl (1→6)]-ß-d-glucoside was named as camellikaempferoside E (4). Chemical structures of AGFs were identified by time-of-flight mass (TOF-MS) and NMR spectrometers (¹H NMR, 13C NMR, ¹H-¹H COSY, HMBC and HSQC), and the MS² fragmentation pathway of AGFs was further investigated. The inhibitory abilities of AGFs and their proposed metabolites on α-glucosidase and HMG-CoA reductase were analyzed by molecular docking simulation, and the results suggested that inhibitory activities of AGFs were significantly affected by acyl structure, number of glycosyl and conformation, and part of them had strong inhibitory activities on α-glucosidase and HMG-CoA reductase, suggesting that AGFs and their metabolites might be important ingredients that participate in the regulation of hypoglycemic and hypolipidemic effects. The results provided new AGFs and research directions for the practical study of FBT health functions in future.

Structure of the GH9 glucosidase/glucosaminidase from Vibrio cholerae.

Glycoside hydrolase family 9 (GH9) of carbohydrate-processing enzymes primarily consists of inverting endoglucanases. A subgroup of GH9 enzymes are believed to act as exo-glucosidases or exo-glucosaminidases, with many being found in organisms of the family Vibrionaceae, where they are proposed to function within the chitin-catabolism pathway. Here, it is shown that the GH9 enzyme from the pathogen Vibrio cholerae (hereafter referred to as VC0615) is active on both chitosan-derived and ß-glucoside substrates. The structure of VC0615 at 3.17 Šresolution is reported from a crystal form with poor diffraction and lattice disorder. VC0615 was highly refractory to crystallization efforts, with crystals only appearing using a high protein concentration under conditions containing the precipitant poly-γ-glutamic acid (PGA). The structure is highly mobile within the crystal lattice, which is likely to reflect steric clashes between symmetry molecules which destabilize crystal packing. The overall tertiary structure of VC0615 is well resolved even at 3.17 Šresolution, which has allowed the structural basis for the exo-glucosidase/glucosaminidase activity of this enzyme to be investigated.

Non-aqueous homogenous biocatalytic conversion of polysaccharides in ionic liquids using chemically modified glucosidase.

The increasing requirement to produce platform chemicals and fuels from renewable sources means advances in biocatalysis are rapidly becoming a necessity. Biomass is widely used in nature as a source of energy and as chemical building blocks. However, recalcitrance towards traditional chemical processes and solvents provides a significant barrier to widespread utility. Here, by optimizing enzyme solubility in ionic liquids, we have discovered solvent-induced substrate promiscuity of glucosidase, demonstrating an unprecedented example of homogeneous enzyme bioprocessing of cellulose. Specifically, chemical modification of glucosidase for solubilization in ionic liquids can increase thermal stability to up to 137 °C, allowing for enzymatic activity 30 times greater than is possible in aqueous media. These results establish that through a synergistic combination of chemical biology (enzyme modification) and reaction engineering (solvent choice), the biocatalytic capability of enzymes can be intensified: a key step towards the full-scale deployment of industrial biocatalysis.

Synthesis, in vitro [Formula: see text]-glucosidase inhibitory activity, and in silico study of (E)-thiosemicarbazones and (E)-2-(2-(arylmethylene)hydrazinyl)-4-arylthiazole derivatives.

This study is focused on the identification of thiazole-based inhibitors for the [Formula: see text]-glucosidase enzyme. For that purpose, (E)-2-(2-(arylmethylene)hydrazinyl)-4-arylthiazole derivatives were synthesized in two steps and characterized by various spectroscopic techniques. All derivatives and intermediates were evaluated for their in vitro [Formula: see text]-glucosidase inhibitory activity. Thiosemicarbazones 20 and 35, and cyclized thiazole derivatives 2, 5-11, 13, 15, 21-24, 27-31, and 36-37 showed significant inhibitory potential in the range of [Formula: see text]-[Formula: see text] as compared to standard acarbose ([Formula: see text]). A molecular modeling study was carried out to understand the binding interactions of compounds with the active site of enzyme.