RESUMO
1,2-trans methyl glycosides can be readily obtained from peracetylated sugars through their initial conversion into glycosyl iodide donors and subsequent exposure of these latter to a slight excess of sodium methoxide in methanol. Under these conditions a varied set of mono- and disaccharide precursors afforded the corresponding 1,2-trans glycosides with concomitant de-O-acetylation in satisfying yields (in the range 59-81%). A similar approach also proved effective when using GlcNAc glycosyl chloride as the donor.
Assuntos
Dissacarídeos , Glicosídeos , Configuração de Carboidratos , AcetilaçãoRESUMO
Trehalose, a disaccharide renowned for its ability to stabilize biomolecular architectures under strenuous conditions, finds extensive use in the cryopreservation of probiotics. A profound comprehension of its molecular-level interactions is of great significance. It is notable that current research in the realm of lipid-sugar interactions primarily employs single-component lipid bilayers, which are far from being representative of real cell membranes. Our investigation, however, utilizes molecular dynamics simulations to delve into the specifics of a realistic Escherichia coli membrane that encompasses a diverse array of lipid types, comprising fourteen distinct species, subject to varying hydration levels. The results of our study showcase that the reduction of hydration levels induces lipid ordering and the formation of gel phases, yet trehalose, by forming hydrogen bonds with lipid headgroups, serves to uphold fluidity and supplant the role of water. Moreover, our findings evince that augmented trehalose concentrations lead to a slowdown in lipid motion and contribute to the maintenance of fluidity by way of endowing a viscous matrix. It is noteworthy that our conclusions lend support to the notion that water replacement and vitrification, despite their seemingly disparate nature, need not be considered mutually exclusive in a real bacterial membrane.
Assuntos
Dessecação , Trealose , Trealose/química , Bicamadas Lipídicas/química , Dissacarídeos , Água/químicaRESUMO
Irritable bowel syndrome is a typical gastrointestinal disease that causes bloating, flatulence, abdominal pain, diarrhoea, constipation, or alteration of the last two in adults and children. A diet low in fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP) is one of the potential treatment strategies to reduce abdominal symptoms and increase the quality of life. The present narrative review aims to present a general overview of current studies that have evaluated the efficacy of a low-FODMAP diet against other diets in gastrointestinal symptoms, nutrient intake in adults and children, and lifestyle quality. The research was performed using seven searchable databases, which included the Cochrane Central Register of Controlled Trials (CENTRAL), Cochrane Database of Systematic Reviews (CDSR), Excerpta Medica Database (EMBASE), Medline, PubMed, Scopus, and Web of Science, up to March 2023. In conclusion, there is significant evidence that the follow-up of a low-FODMAP diet might be a feasible first-line therapeutic strategy to reduce stomach discomfort, pain, bloating, and quality of life for patients with irritable bowel syndrome.
Assuntos
Gastroenteropatias , Síndrome do Intestino Irritável , Humanos , Adulto , Criança , Dissacarídeos , Monossacarídeos , Qualidade de Vida , Dieta FODMAP , Revisões Sistemáticas como Assunto , Oligossacarídeos , Dieta , Gastroenteropatias/tratamento farmacológico , Flatulência , Dor Abdominal/etiologia , Dor Abdominal/tratamento farmacológico , Fermentação , Dieta com Restrição de CarboidratosRESUMO
BACKGROUND: "FODMAPs" (fermentable-oligo-, di-, monosaccharides, and polyols) are a group of fermentable carbohydrates and polyols largely diffused in food products. Despite their beneficial effects as prebiotics, people affected by irritable bowel syndrome manifest symptoms when eating these carbohydrates. A low-FODMAP diet seems to be the only possible therapy proposed for symptom management. Bakery products are a common source of FODMAPs, whose pattern and total amount can be affected by their processing. This work aims at studying some of the technological parameters that can influence the FODMAPs pattern in bakery products during the production process. METHODS: high-performance anion exchange chromatography coupled to a pulsed amperometric detector (HPAEC-PAD) was used as a highly selective system for carbohydrates evaluation analyses on flours, doughs, and crackers. These analyses were performed using two different columns, the CarboPac PA200 and CarboPac PA1, which are selective for oligosaccharide and simple sugar separation, respectively. RESULTS: emmer and hemp flours were selected to prepare doughs as they contained low oligosaccharide content. Two different mixes of ferments were used at different times of fermentation to evaluate the best conditions to achieve low-FODMAP crackers. CONCLUSION: the proposed approach allows carbohydrate evaluation during crackers processing and permits the selection of opportune conditions to obtain low-FODMAP products.
Assuntos
Carboidratos , Síndrome do Intestino Irritável , Humanos , Oligossacarídeos , Monossacarídeos , Hexoses , Fermentação , DissacarídeosRESUMO
An efficient total synthesis of a conjugation-ready trisaccharide repeating unit of Staphylococcus aureus strain M is reported here. The main challenges involved in this synthesis are the procurement of rare sugars (d-FucNAc and d-GalNAcA) and installation of consecutive 1,2-cis-glycosidic linkages between them. Stereoselective 1,2-cis glycosylation with the linker acceptor was achieved with easily accessible benzylidene protected d-galactosamine thioglycoside by employing a DMF modulated preactivation glycosylation method. The consecutive 1,2-cis linkages were installed with the help of solvent participation. The carboxylic acid functionality was introduced via postglycosylation oxidation on the disaccharide moiety. The total synthesis of trisaccharide repeating unit was accomplished with the longest linear sequence of 24 steps in 4.5% overall yield.
Assuntos
Staphylococcus aureus , Trissacarídeos , Glicosilação , Dissacarídeos , OxirreduçãoRESUMO
Lung cancer is one of the most commonly diagnosed cancer types. Studying the molecular changes that occur in lung cancer is important to understand tumor formation and identify new therapeutic targets and early markers of the disease to decrease mortality. Glycosaminoglycan chains play important roles in various signaling events in the tumor microenvironment. Therefore, we have determined the quantity and sulfation characteristics of chondroitin sulfate and heparan sulfate in formalin-fixed paraffin-embedded human lung tissue samples belonging to different lung cancer types as well as tumor adjacent normal areas. Glycosaminoglycan disaccharide analysis was performed using HPLC-MS following on-surface lyase digestion. Significant changes were identified predominantly in the case of chondroitin sulfate; for example, the total amount was higher in tumor tissue compared to the adjacent normal tissue. We also observed differences in the degree of sulfation and relative proportions of individual chondroitin sulfate disaccharides between lung cancer types and adjacent normal tissue. Furthermore, the differences in the 6-O-/4-O-sulfation ratio of chondroitin sulfate were different between the lung cancer types. Our pilot study revealed that further investigation of the role of chondroitin sulfate chains and enzymes involved in their biosynthesis is an important aspect of lung cancer research.
Assuntos
Glicosaminoglicanos , Neoplasias Pulmonares , Humanos , Sulfatos de Condroitina , Projetos Piloto , Heparitina Sulfato , Dissacarídeos , Microambiente TumoralRESUMO
Chondroitinase ABC-type I (CSase ABC I), which can digest both chondroitin sulfate (CS) and dermatan sulfate (DS) in an endolytic manner, is an essential tool in structural and functional studies of CS/DS. Although a few CSase ABC I have been identified from bacteria, the substrate-degrading pattern and regulatory mechanisms of them have rarely been investigated. Herein, two CSase ABC I, IM3796 and IM1634, were identified from the intestinal metagenome of CS-fed mice. They show high sequence homology (query coverage: 88.00%, percent identity: 90.10%) except for an extra peptide (Met1-His109) at the N-terminus in IM1634, but their enzymatic properties are very different. IM3796 prefers to degrade 6-O-sulfated GalNAc residue-enriched CS into tetra- and disaccharides. In contrast, IM1634 exhibits nearly a thousand times more activity than IM3796 and can completely digest CS/DS with various sulfation patterns to produce disaccharides, unlike most CSase ABC I. Structure modeling showed that IM3796 did not contain an N-terminal domain composed of two ß-sheets, which is found in IM1634 and other CSase ABC I. Furthermore, deletion of the N-terminal domain (Met1-His109) from IM1634 caused the enzymatic properties of the variant IM1634-T109 to be similar to those of IM3796, and conversely, grafting this domain to IM3796 increased the similarity of the variant IM3796-A109 to IM1634. In conclusion, the comparative study of the new CSase ABC I provides two unique tools for CS/DS-related studies and applications and, more importantly, reveals the critical role of the N-terminal domain in regulating the substrate binding and degradation of these enzymes.
Assuntos
Condroitina ABC Liase , Sulfatos de Condroitina , Animais , Camundongos , Condroitina ABC Liase/química , Sulfatos de Condroitina/metabolismo , Dissacarídeos/química , Peptídeos , Bactérias/metabolismo , Dermatan Sulfato/químicaRESUMO
Trehalose is a disaccharide and is often foliar applied by farmers aiming at increasing stress resistance or crop production. However, the physiological effect of exogenously applied trehalose on crops remains obscure. Here, we explored the effect of foliar trehalose application on style length of solanaceous crops, Solanum melongena and S. lycopersicum. Trehalose application promotes pistil to stamen ratio by gaining style length. Another disaccharide consisting of two glucose molecules, maltose, showed the same effect on style length of S. lycopersicum, while monosaccharide glucose did not. Trehalose is found to affect style length through uptake via roots or interaction with rhizosphere but not through absorption by shoots in S. lycopersicum. Our study suggests that yield improvement of solanaceous crops by trehalose application under stressed conditions is brought about by suppression of the occurrence of short-styled flowers. This study suggests that trehalose holds potential to act as a plant biostimulant in preventing short-styled flowers in solanaceous crops.
Assuntos
Dissacarídeos , Trealose , Produtos Agrícolas , Glucose , FloresRESUMO
Rare sugars have recently attracted attention as potential sugar replacers. Understanding the biochemical and biological behavior of these sugars is of importance in (novel) food formulations and prevention of type 2 diabetes. In this study, we investigated whether rare sugars may positively affect intestinal and liver metabolism, as well as muscle insulin sensitivity, compared to conventional sugars. Rare disaccharide digestibility, hepatic metabolism of monosaccharides (respirometry) and the effects of sugars on skeletal muscle insulin sensitivity (impaired glucose uptake) were investigated in, respectively, Caco-2, HepG2 and L6 cells or a triple coculture model with these cells. Glucose and fructose, but not l-arabinose, acutely increased extracellular acidification rate (ECAR) responses in HepG2 cells and impaired glucose uptake in L6 cells following a 24 h exposure at 28 mM. Cellular bioenergetics and digestion experiments with Caco-2 cells indicate that especially trehalose (α1-1α), D-Glc-α1,2-D-Gal, D-Glc-α1,2-D-Rib and D-Glc-α1,3-L-Ara experience delayed digestion and reduced cellular impact compared to maltose (α1-4), without differences on insulin-stimulated glucose uptake in a short-term setup with a Caco-2/HepG2/L6 triple coculture. These results suggest a potential for l-arabinose and specific rare disaccharides to improve metabolic health; however, additional in vivo research with longer sugar exposures should confirm their beneficial impact on insulin sensitivity in humans.
Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Humanos , Diabetes Mellitus Tipo 2/metabolismo , Células CACO-2 , Arabinose/farmacologia , Arabinose/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Músculo Esquelético/metabolismo , Fígado/metabolismo , Dissacarídeos/farmacologiaRESUMO
The objectives of this narrative review are to update readers on the current state-of-the-art regarding diverse approaches for the treatment of pain, global symptoms, or adequate relief in irritable bowel syndrome (IBS). The article appraises medications, dietary interventions including low fermentable oligosaccharides, disaccharides, and monosaccharides and polyols (FODMAP) diet, fecal microbial transplantation (FMT), electrical approaches, and behavioral therapies including cognitive behavioral therapy (CBT), gut-directed hypnotherapy (GDH), mindfulness, and open-label placebo. Current evidence demonstrates only modest benefit in global IBS symptoms and pain relief. A future approach that identifies pathophysiological mechanisms of IBS through validated biomarkers has the potential to individualize treatment of patients rather than sequential therapeutic trial and error approaches.
Assuntos
Síndrome do Intestino Irritável , Humanos , Síndrome do Intestino Irritável/induzido quimicamente , Síndrome do Intestino Irritável/tratamento farmacológico , Fermentação , Dissacarídeos/efeitos adversos , Oligossacarídeos/efeitos adversos , Dieta , Dor Abdominal/terapia , Dor Abdominal/induzido quimicamenteRESUMO
The multienzyme cascade has received growing attention to obtain structurally defined glycans in vitro. However, due to poor enzyme stability and low compatibility between glycoenzymes, artificially designed multienzyme pathways to access glycans are often inefficient. Herein, based on the strategy "Modular-Enzymes Assembly by Spatial Segregation" (MASS), we developed a universal immobilization platform to assemble multiple glycoenzymes in compartmentalized MOF particles, inside and outside, significantly reducing the undesired interference and cross-inhibitions. By changing the enzyme modules, a series of glycosyl donor, disaccharides, oligosaccharides, and polysaccharides bearing cofactor regeneration were efficiently prepared. This bioreactor was further successfully applied to the reaction system with high substrate concentration to demonstrate its industrial potential. This robust multienzyme immobilization platform should serve to promote the enzymatic synthesis of more complex glycans.
Assuntos
Oligossacarídeos , Polissacarídeos , Polissacarídeos/metabolismo , Enzimas Imobilizadas/metabolismo , Dissacarídeos , Reatores BiológicosRESUMO
The lactate dehydrogenase from rabbit skeletal muscle (rbLDH) is a tetrameric enzyme, known to undergo dissociation when exposed to acidic pH conditions. Moreover, it should be mentioned that this dissociation translates into a pronounced loss of enzyme activity. Notably, among the compounds able to stabilize proteins and enzymes, the disaccharide trehalose represents an outperformer. In particular, trehalose was shown to efficiently counteract quite a number of physical and chemical agents inducing protein denaturation. However, no information is available on the effect, if any, exerted by trehalose against the dissociation of protein oligomers. Accordingly, we thought it of interest to investigate whether this disaccharide is competent in preventing the dissociation of rbLDH induced by acidic pH conditions. Further, we compared the action of trehalose with the effects triggered by maltose and cellobiose. Surprisingly, both these disaccharides enhanced the dissociation of rbLDH, with maltose being responsible for a major effect when compared to cellobiose. On the contrary, trehalose was effective in preventing enzyme dissociation, as revealed by activity assays and by Dynamic Light Scattering (DLS) experiments. Moreover, we detected a significant decrease of both K0.5 and Vmax when the rbLDH activity was tested (at pH 7.5 and 6.5) as a function of pyruvate concentration in the presence of trehalose. Further, we found that trehalose induces a remarkable increase of Vmax when the enzyme is exposed to pH 5. Overall, our observations suggest that trehalose triggers conformational rearrangements of tetrameric rbLDH mirrored by resistance to dissociation and peculiar catalytic features.
Assuntos
Maltose , Trealose , Animais , Coelhos , Trealose/química , Maltose/química , Maltose/metabolismo , Celobiose , L-Lactato Desidrogenase/metabolismo , Dissacarídeos/farmacologia , Dissacarídeos/metabolismo , Concentração de Íons de HidrogênioRESUMO
BACKGROUND: Alterations in gastrointestinal (GI) microbial composition have been reported in patients with systemic sclerosis (SSc). However, it is unclear to what degree these alterations and/or dietary changes contribute to the SSc-GI phenotype. OBJECTIVES: Our study aimed to 1) evaluate the relationship between GI microbial composition and SSc-GI symptoms, and 2) compare GI symptoms and GI microbial composition between SSc patients adhering to a low versus non-low fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP) diet. METHODS: Adult SSc patients were consecutively recruited to provide stool specimens for bacterial 16S rRNA gene sequencing. Patients completed the UCLA Scleroderma Clinical Trial Consortium Gastrointestinal Tract Instrument (GIT 2.0) and the Diet History Questionnaire (DHQ) II and were classified as adhering to a low or non-low FODMAP diet. GI microbial differences were assessed using three metrics of alpha diversity (species richness, evenness, and phylogenetic diversity), as well as beta diversity (overall microbial composition). Differential abundance analysis was performed to identify specific genera associated with SSc-GI phenotype and low versus non-low FODMAP diet. RESULTS: Of the 66 total SSc patients included, the majority were women (n = 56) with a mean disease duration of 9.6 years. Thirty-five participants completed the DHQ II. Increased severity of GI symptoms (total GIT 2.0 score) was associated with decreased species diversity and differences in GI microbial composition. Specifically, pathobiont genera (e.g., Klebsiella and Enterococcus) were significantly more abundant in patients with increased GI symptom severity. When comparing low (N = 19) versus non-low (N = 16) FODMAP groups, there were no significant differences in GI symptom severity or in alpha and beta diversity. Compared with the low FODMAP group, the non-low FODMAP group had greater abundance of the pathobiont Enterococcus. CONCLUSION: SSc patients reporting more severe GI symptoms exhibited GI microbial dysbiosis characterized by less species diversity and alterations in microbial composition. A low FODMAP diet was not associated with significant alterations in GI microbial composition or reduced SSc-GI symptoms; however, randomized controlled trials are needed to evaluate the impact of specific diets on GI symptoms in SSc.
Assuntos
Gastroenteropatias , Microbiota , Escleroderma Sistêmico , Humanos , Masculino , Feminino , RNA Ribossômico 16S , Filogenia , Dieta , Dissacarídeos , Oligossacarídeos , Monossacarídeos , Gastroenteropatias/etiologia , Escleroderma Sistêmico/complicaçõesRESUMO
Glucose aversion in the German cockroach, Blattella germanica (L.), results in behavioral resistance to insecticidal baits. Glucose-averse (GA) cockroaches reject foods containing glucose, even in relatively low concentrations, which protects the cockroaches from ingesting lethal amounts of toxic baits. Horizontal transfer of baits and the resulting secondary mortality have been documented in German cockroaches, including in insecticide resistant strains. However, the effects of the GA trait on secondary mortality have not been investigated. We hypothesized that ingestion of insecticide baits that contain glucose or glucose-containing disaccharides would result in behaviorally relevant glucose levels in the feces, possibly deterring coprophagy by GA nymphs. We fed adult female cockroaches hydramethylnon baits rich in either glucose, fructose, sucrose, or maltose and compared secondary mortality of GA and wild-type (WT) nymphs via coprophagy. When adult females were fed baits containing glucose, sucrose, or maltose and their feces offered to nymphs, secondary mortality was significantly lower in GA nymphs than in WT nymphs. However, survival of GA and WT nymphs was similar on feces generated by adult females fed fructose bait. Analysis of feces indicated that disaccharides in baits were hydrolyzed into glucose, some of which was excreted in the feces of females that ingested the bait. Based on these results, we caution that baits containing glucose or glucose-containing oligosaccharides may impede cockroach interventions; while GA adults and large nymphs avoid ingesting such baits, first instars reject the glucose-containing feces of any WT cockroaches that consumed the bait.
Assuntos
Blattellidae , Baratas , Inseticidas , Feminino , Animais , Glucose/farmacologia , Dissacarídeos/farmacologia , Maltose/farmacologia , Inseticidas/farmacologia , Sacarose , Ninfa , Frutose/farmacologiaRESUMO
The enzymatic degradation of seaweed polysaccharides is gaining interest for its potential in the production of functional oligosaccharides and fermentable sugars. Herein, a novel alginate lyase, AlyRm3, was cloned from a marine strain, Rhodothermus marinus DSM 4252. The AlyRm3 showed optimal activity (37,315.08 U/mg) at 70 °C and pH 8.0, with the sodium alginate used as a substrate. Noticeably, AlyRm3 was stable at 65 °C and also exhibited 30% of maximal activity at 90 °C. These results indicated that AlyRm3 is a thermophilic alginate lyase that efficiently degrades alginate at high industrial temperatures (>60 °C). The FPLC and ESI-MS analyses suggested that AlyRm3 primarily released disaccharides and trisaccharides from the alginate, polyM, and polyG in an endolytic manner. In the saccharification process of sodium alginate (0.5%, w/v), the AlyRm3 yielded numerous reducing sugars (1.73 g/L) after 2 h of reaction. These results indicated that AlyRm3 has a high enzymatic capacity for saccharifying the alginate, and could be used to saccharify the alginate biomass before the main fermentation process for biofuels. These properties make AlyRm3 a valuable candidate for both fundamental research and industrial applications.
Assuntos
Alginatos , Polissacarídeo-Liases , Alginatos/metabolismo , Polissacarídeo-Liases/metabolismo , Oligossacarídeos/metabolismo , Dissacarídeos , Concentração de Íons de Hidrogênio , Especificidade por Substrato , Proteínas de Bactérias/metabolismo , TemperaturaRESUMO
(1) Background: Irritable bowel syndrome (IBS) is a global public health problem, the pathogenesis of which has not been fully explored. Limiting fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP) can relieve symptoms in some patients with IBS. Studies have shown that normal microcirculation perfusion is necessary to maintain the primary function of the gastrointestinal system. Here, we hypothesized that IBS pathogenesis might be related to abnormalities in colonic microcirculation. A low-FODMAP diet could alleviate visceral hypersensitivity (VH) by improving colonic microcirculation; (2) Methods: C57BL/6 mice were raised to establish an IBS-like rodent model using water avoidance (WA) stress or SHAM-WA as a control, one hour per day for ten days. The mice in the WA group were administered different levels of the FODMAP diet: 2.1% regular FODMAP (WA-RF), 10% high FODMAP diet (WA-HF), 5% medium FODMAP diet (WA-MF), and 0% low FODMAP diet (WA-LF) for the following 14 days. The body weight and food consumption of the mice were recorded. Visceral sensitivity was measured as colorectal distention (CRD) using the abdominal withdrawal reflex (AWR) score. Colonic microcirculation was assessed using laser speckle contrast imaging (LCSI). Vascular endothelial-derived growth factor (VEGF) was detected using immunofluorescence staining; (3) Results: The threshold values of CRD pressure in the WA-RF, WA-HF, and WA-MF groups were significantly lower than those in the SHAM-WA group. Moreover, we observed that colonic microcirculation perfusion decreased, and the expression of VEGF protein increased in these three groups of mice. Interestingly, a low-FODMAP dietary intervention could reverse this situation. Specifically, a low-FODMAP diet increased colonic microcirculation perfusion, reduced VEGF protein expression in mice, and increased the threshold of VH. There was a significant positive correlation between colonic microcirculation and threshold for VH; (4) Conclusions: These results demonstrate that a low-FODMAP diet can alter VH by affecting colonic microcirculation. Changes in intestinal microcirculation may be related to VEGF expression.
Assuntos
Dissacarídeos , Síndrome do Intestino Irritável , Camundongos , Animais , Monossacarídeos , Água , Dieta FODMAP , Microcirculação , Fator A de Crescimento do Endotélio Vascular/metabolismo , Camundongos Endogâmicos C57BL , Fermentação , Oligossacarídeos , Dieta/métodos , Dieta com Restrição de Carboidratos/métodosRESUMO
Trehalase is an important enzyme in the metabolic cascades of many organisms, catalysing the hydrolysis of the disaccharide trehalose. Herein we describe the first examples of fluorometric nanoprobes for detection of trehalase, based on trehalose-functionalised quantum dots (QDs). QDs cross-linked with trehalose form aggregates, which are released upon enzymatic cleavage of the trehalose glycosidic bond proportionally to the enzyme concentration, offering a unique and efficient approach for specific sensing of this biologically important enzyme.
Assuntos
Pontos Quânticos , Trealose , Trealose/química , Trealase/química , Trealase/metabolismo , Dissacarídeos/metabolismoRESUMO
Carbohydrates are ubiquitous in nature but are among the least conserved biomolecules in life. These biopolymers pose a particular challenge to analytical chemists because of their high diversity and structural heterogeneity. In addition, they contain many isomerisms that complicate their structural characterization, notably by mass spectrometry. The tautomerism of the constitutive subunits is of particular interest. A given cyclized monosaccharide unit can take two forms: a most common 6-membered ring (pyranose, p) and a more flexible 5-membered ring (furanose, f). The tautomers impact the biological properties of polysaccharides, resulting in interesting properties of the derived oligosaccharides. From an analytical point of view, the impact of tautomerism on the gas-phase behavior of ions has scarcely been described in the literature. In this work, we study the behavior of Galf-containing oligosaccharides, ionized as [M+Li]+ species, under collisional dissociation (CID) conditions using high-resolution and multistage ion mobility (IMS) on a Cyclic IMS platform. In the first part of this work, we studied whether disaccharidic fragments released from Galf-containing (Gal)1(Man)2 trisaccharides (and their Galp counterpart) would match the corresponding disaccharide standards, andâdespite the fragments generally being a good matchâwe showed the possibility of Galf migrations and other unidentified alterations in the IMS profile. Next, we expanded on these unknown features using multistage IMS and molecular dynamics, unveiling the contributions of additional gas-phase conformers in the profile of fragments from a Galf-containing trisaccharide compared with the corresponding disaccharides.
Assuntos
Carboidratos , Oligossacarídeos , Humanos , Espectrometria de Massas/métodos , Oligossacarídeos/química , Polissacarídeos , Dissacarídeos/química , Trissacarídeos , Monossacarídeos , ÍonsRESUMO
Hyaluronate lyases (HA lyases) have been proved to distribute widely among microorganisms, with large potential in hyaluronan processing. Here, a highly active HA lyase HylC from Citrobacter freundii strain Cf1 is reported. HylC was expressed in Escherichia coli BL21(DE3) under the regulation of T7 promoter, and purified to electrophoretic homogeneity for enzymatic characterization, which suggested its suitable thermo- and pH stability under 45 °C and pH rang of 4-8, and high halotolerancy in 1.5 M NaCl. The enzyme exhibited the optimal activity under 37 °C and pH 5.5, and was activated by Ca2+, K+, Zn2+, Ni2+ and Li+. Analysis of degradation product proved it cleave HA in endolytic manner, releasing unsaturated disaccharides as final product. Then, through optimization of promoter and construction of dual promoter, expression level of HylC improved from 1.10 × 104 U/mL to 2.64 × 104 U/mL on shake-flask level. Finally, through batch fermentation, a highest activity of 2.65×105 U/mL was achieved in a 5-L fermenter. Taken together, this work demonstrates the potential of HylC and its recombinant strain in industrial applications. To our knowledge, the HA lyase production reported in this study was the highest level in literatures to date.
