Isomaltotriose

Structural insights into alpha-(1-->6)-linkage preference of GH97 glucodextranase from Flavobacterium johnsoniae.[Pubmed:38661728]

FEBS J. 2024 Apr 25.

Glycoside hydrolase family 97 (GH97) comprises enzymes like anomer-inverting alpha-glucoside hydrolases (i.e., glucoamylase) and anomer-retaining alpha-galactosidases. In a soil bacterium, Flavobacterium johnsoniae, we previously identified a GH97 enzyme (FjGH97A) within the branched dextran utilization locus. It functions as an alpha-glucoside hydrolase, targeting alpha-(1-->6)-glucosidic linkages in dextran and isomaltooligosaccharides (i.e., glucodextranase). FjGH97A exhibits a preference for alpha-(1-->6)-glucoside linkages over alpha-(1-->4)-linkages, while Bacteroides thetaiotaomicron glucoamylase SusB (with 69% sequence identity), which is involved in the starch utilization system, exhibits the highest specificity for alpha-(1-->4)-glucosidic linkages. Here, we examined the crystal structures of FjGH97A in complexes with glucose, panose, or Isomaltotriose, and analyzed the substrate preferences of its mutants to identify the amino acid residues that determine the substrate specificity for alpha-(1-->4)- and alpha-(1-->6)-glucosidic linkages. The overall structure of FjGH97A resembles other GH97 enzymes, with conserved catalytic residues similar to anomer-inverting GH97 enzymes. A comparison of active sites between FjGH97A and SusB revealed differences in amino acid residues at subsites +1 and +2 (specifically Ala195 and Ile378 in FjGH97A). Among the three mutants (A195S, I378F, and A195S-I378F), A195S and A195S-I378F exhibited increased activity toward alpha-(1-->4)-glucoside bonds compared to alpha-(1-->6)-glucoside bonds. This suggests that Ala195, located on the Gly184-Thr203 loop (named loop-N) conserved within the GH97 subgroup, including FjGH97A and SusB, holds significance in determining linkage specificity. The conservation of alanine in the active site of the GH97 enzymes, within the same gene cluster as the putative dextranase, indicates its crucial role in determining the specificity for alpha-(1-->6)-glucoside linkage.

Immobilization of Dextranase Obtained from the Marine Cellulosimicrobium sp. Y1 on Nanoparticles: Nano-TiO(2) Improving Hydrolysate Properties and Enhancing Reuse.[Pubmed:36985959]

Nanomaterials (Basel). 2023 Mar 15;13(6):1065.

Dextranase is widely used in sugar production, drug synthesis, material preparation, and biotechnology, among other fields. The immobilization of dextranase using nanomaterials in order to make it reusable, is a hot research topic. In this study, the immobilization of purified dextranase was performed using different nanomaterials. The best results were obtained when dextranase was immobilized on titanium dioxide (TiO(2)), and a particle size of 30 nm was achieved. The optimum immobilization conditions were pH 7.0, temperature 25 degrees C, time 1 h, and immobilization agent TiO(2). The immobilized materials were characterized using Fourier-transform infrared spectroscopy, X-ray diffractometry, and field emission gun scanning electron microscopy. The optimum temperature and pH of the immobilized dextranase were 30 degrees C and 7.5, respectively. The activity of the immobilized dextranase was >50% even after 7 times of reuse, and 58% of the enzyme was active even after 7 days of storage at 25 degrees C, indicating the reproducibility of the immobilized enzyme. The adsorption of dextranase by TiO(2) nanoparticles exhibited secondary reaction kinetics. Compared with free dextranase, the hydrolysates of the immobilized dextranase were significantly different, and consisted mainly of Isomaltotriose and isomaltotetraose. The highly polymerized isomaltotetraose levels could reach >78.69% of the product after 30 min of enzymatic digestion.

Chemical characterization and antineoplastic effect of oligosaccharides from Cabernet Franc red wine in mammary tumor model in mice.[Pubmed:36565967]

J Nutr Biochem. 2023 Mar;113:109253.

The present study characterized oligosaccharide compounds (Oligo) in Cabernet Franc red wine and investigated its antineoplastic effects against mammary tumor cells in vivo and in vitro, isolated or in combination with chemotherapy. The Oligo fraction was characterized by nuclear magnetic resonance spectroscopy and mass spectrometry. The complex mixture of Oligo showed high amounts of oligoxyloglucuronans, oligorhamnogalacturonans, oligoarabinogalactans, and oligoglucans, such as trehalose and Isomaltotriose. To investigate the antineoplastic effects of Oligo, Female Swiss mice were subcutaneously inoculated with Ehrlich tumor cells and then received vehicle (distilled water, p.o.), Oligo solution (9, 35, or 70 mg/kg, p.o.), or methotrexate (1.5 mg/kg, i.p.). The treatments were administered in a conventional (21-d) or chemopreventive (42-d) protocol. Oligo reduced the growth of Ehrlich tumors in both protocols and increased the effectiveness of methotrexate in controlling tumor growth. Oligo did not reduce the viability of MCF-7, MDA-MB-231, MDA-MB-436, and HB4a human breast cells that were cultured for 48 h, showing no cytotoxicity. Overall, Oligo exerted an in vivo antineoplastic effect and modulated immune blood cells, dependent on treatment time, and was not directly cytotoxic to tumor cells. Thus, Oligo may indirectly regulate tumor cell development and may be a promising drug for cancer therapy in combination with methotrexate.

Purification and characterization of cold-adapted and salt-tolerant dextranase from Cellulosimicrobium sp. THN1 and its potential application for treatment of dental plaque.[Pubmed:36439846]

Front Microbiol. 2022 Nov 11;13:1012957.

The cold-adapted and/or salt-tolerant enzymes from marine microorganisms were confirmed to be meritorious tools to enhance the efficiency of biocatalysis in industrial biotechnology. We purified and characterized a dextranase CeDex from the marine bacterium Cellulosimicrobium sp. THN1. CeDex acted in alkaline pHs (7.5-8.5) and a broad temperature range (10-50 degrees C) with sufficient pH stability and thermostability. Remarkably, CeDex retained approximately 40% of its maximal activities at 4 degrees C and increased its activity to 150% in 4 M NaCl, displaying prominently cold adaptation and salt tolerance. Moreover, CeDex was greatly stimulated by Mg(2+), Na(+), Ba(2+), Ca(2+) and Sr(2+), and sugarcane juice always contains K(+), Ca(2+), Mg(2+) and Na(+), so CeDex will be suitable for removing dextran in the sugar industry. The main hydrolysate of CeDex was Isomaltotriose, accompanied by isomaltotetraose, long-chain IOMs, and a small amount of isomaltose. The amino acid sequence of CeDex was identified from the THN1 genomic sequence by Nano LC-MS/MS and classified into the GH49 family. Notably, CeDex could prevent the formation of Streptococcus mutans biofilm and disassemble existing biofilms at 10 U/ml concentration and would have great potential to defeat biofilm-related dental caries.

Potential inhibitory activity of phytoconstituents against black fungus: In silico ADMET, molecular docking and MD simulation studies.[Pubmed:36193218]

Comput Toxicol. 2022 Nov;24:100247.

Mucormycosis or "black fungus" has been currently observed in India, as a secondary infection in COVID-19 infected patients in the post-COVID-stage. Fungus is an uncommon opportunistic infection that affects people who have a weak immune system. In this study, 158 antifungal phytochemicals were screened using molecular docking against glucoamylase enzyme of Rhizopus oryzae to identify potential inhibitors. The docking scores of the selected phytochemicals were compared with Isomaltotriose as a positive control. Most of the compounds showed lower binding energy values than Isomaltotriose (-6.4 kcal/mol). Computational studies also revealed the strongest binding affinity of the screened phytochemicals was Dioscin (-9.4 kcal/mol). Furthermore, the binding interactions of the top ten potential phytochemicals were elucidated and further analyzed. In-silico ADME and toxicity prediction were also evaluated using SwissADME and admetSAR online servers. Compounds Piscisoflavone C, 8-O-methylaverufin and Punicalagin exhibited positive results with the Lipinski filter and drug-likeness and showed mild to moderate of toxicity. Molecular dynamics (MD) simulation (at 300 K for 100 ns) was also employed to the docked ligand-target complex to explore the stability of ligand-target complex, improve docking results, and analyze the molecular mechanisms of protein-target interactions.

Alkalic dextranase produced by marine bacterium Cellulosimicrobium sp. PX02 and its application.[Pubmed:34528722]

J Basic Microbiol. 2021 Nov;61(11):1002-1015.

The enzyme dextranase is widely used in the sugar and food industries, as well as in the medical field. Most land-derived dextranases are produced by fungi and have the disadvantages of long production cycles, low tolerance to environmental conditions, and low safety. The use of marine bacteria to produce dextranases may overcome these problems. In this study, a dextranase-producing bacterium was isolated from the Rizhao seacoast of Shandong, China. The bacterium, denoted as PX02, was identified as Cellulosimicrobium sp. and its growing conditions and the production and properties of its dextranase were investigated. The dextranase had a molecular weight of approximately 40 kDa, maximum activity at 40 degrees C and pH 7.5, with a stability range of up to 45 degrees C and pH 7.0-9.0. High-performance liquid chromatography showed that the dextranase hydrolyzed dextranT20 to Isomaltotriose, maltopentaose, and isomaltooligosaccharides. Hydrolysis by dextranase produced excellent antioxidant effects, suggesting its potential use in the health food industry. Investigation of the action of the dextranase on Streptococcus mutans biofilm and scanning electron microscopy showed that it to be effective both for removing and inhibiting the formation of biofilms, suggesting its potential application in the dental industry.

Separation of labeled isomeric oligosaccharides by hydrophilic interaction liquid chromatography - the role of organic solvent in manipulating separation selectivity of the amide stationary phase.[Pubmed:34147834]

J Chromatogr A. 2021 Aug 16;1651:462303.

The advantages of using mixtures of organic solvents for the separation of labeled oligosaccharides on the amide stationary phase under hydrophilic interaction liquid chromatography conditions are presented. The effect of the type of buffer as well as solvent or their mixtures on retention of uracil, saccharide labeling reagents (2-aminobenzoic acid, 2-aminobenzamide, ethyl 4-aminobenzoate, procainamide), and corresponding labeled saccharides were evaluated. The successful isocratic separation of labeled isomeric trisaccharides (maltotriose, panose, and Isomaltotriose) was achieved in the mobile phase consisting of a 90% (v/v) mixture of organic solvents (methanol/acetonitrile 60:40) and 10% (v/v) 30 mM ammonium formate, pH 3.3. Changing the volume ratio between methanol/acetonitrile from 60:40 to 50:50 (v/v) allowed to obtain the separation of di-, tri-, and tetrasaccharides labeled by ethyl 4-aminobenzoate in less than 10.5 min.

NMR analysis and molecular dynamics conformation of alpha-1,6-linear and alpha-1,3-branched isomaltose oligomers as mimetics of alpha-1,6-linked dextran.[Pubmed:33813322]

Carbohydr Res. 2021 May;503:108296.

The conformational preferences of several alpha-1,6-linear and alpha-1,3-branched isomalto-oligosaccharides were investigated by NMR and MD-simulations. Right-handed helical structure contributed to the solution geometry in Isomaltotriose and isomaltotetraose with one nearly complete helix turn and stabilizing intramolecular hydrogen bonds in the latter by MD-simulation. Decreased helix contribution was observed in alpha-1,3-glucopyranosyl- and alpha-1,3-isomaltosyl-branched saccharide chains. Especially the latter modification was predicted to cause a more compact structure consistent with literature rheology measurements as well as with published dextranase-resistant alpha-1,3-branched oligosaccharides. The findings presented here are significant because they shed further light on the conformational preference of isomalto-oligosaccharides and provide possible help for the design of dextran-based drug delivery systems or for the targeted degradation of capsular polysaccharides by dextranases in multi-drug resistant bacteria.

Resolving Power and Collision Cross Section Measurement Accuracy of a Prototype High-Resolution Ion Mobility Platform Incorporating Structures for Lossless Ion Manipulation.[Pubmed:33734709]

J Am Soc Mass Spectrom. 2021 Apr 7;32(4):1126-1137.

A production prototype structures for lossless ion manipulation ion mobility (SLIM IM) platform interfaced to a commercial high-resolution mass spectrometer (MS) is described. The SLIM IM implements the traveling wave ion mobility technique across a approximately 13m path length for high-resolution IM (HRIM) separations. The resolving power (CCS/DeltaCCS) of the SLIM IM stage was benchmarked across various parameters (traveling wave speeds, amplitudes, and waveforms), and results indicated that resolving powers in excess of 200 can be accessed for a broad range of masses. For several cases, resolving powers greater than 300 were achieved, notably under wave conditions where ions transition from a nonselective "surfing" motion to a mobility-selective ion drift, that corresponded to ion speeds approximately 30-70% of the traveling wave speed. The separation capabilities were evaluated on a series of isomeric and isobaric compounds that cannot be resolved by MS alone, including reversed-sequence peptides (SDGRG and GRGDS), triglyceride double-bond positional isomers (TG 3, 6, 9 and TG 6, 9, 12), trisaccharides (melezitose, raffinose, Isomaltotriose, and maltotriose), and ganglioside lipids (GD1b and GD1a). The SLIM IM platform resolved the corresponding isomeric mixtures, which were unresolvable using the standard resolution of a drift-tube instrument ( approximately 50). In general, the SLIM IM-MS platform is capable of resolving peaks separated by as little as approximately 0.6% without the need to target a specific separation window or drift time. Low CCS measurement biases <0.5% were obtained under high resolving power conditions. Importantly, all the analytes surveyed are able to access high-resolution conditions (>200), demonstrating that this instrument is well-suited for broadband HRIM separations important in global untargeted applications.

Products Released from Structurally Different Dextrans by Bacterial and Fungal Dextranases.[Pubmed:33530339]

Foods. 2021 Jan 26;10(2):244.

Dextran hydrolysis by dextranases is applied in the sugar industry and the medical sector, but it also has a high potential for use in structural analysis of dextrans. However, dextranases are produced by several organisms and thus differ in their properties. The aim of this study was to comparatively investigate the product patterns obtained from the incubation of linear as well as O3- and O4-branched dextrans with different dextranases. For this purpose, genes encoding for dextranases from Bacteroides thetaiotaomicron and Streptococcus salivarius were cloned and heterologously expressed in Escherichia coli. The two recombinant enzymes as well as two commercial dextranases from Chaetomium sp. and Penicillium sp. were subsequently used to hydrolyze structurally different dextrans. The hydrolysis products were investigated in detail by HPAEC-PAD. For dextranases from Chaetomium sp., Penicillium sp., and Bacteroides thetaiotaomicron, isomaltose was the end product of the hydrolysis from linear dextrans, whereas Penicillium sp. dextranase led to isomaltose and isomaltotetraose. In addition, the latter enzyme also catalyzed a disproportionation reaction when incubated with Isomaltotriose. For O3- and O4-branched dextrans, the fungal dextranases yielded significantly different oligosaccharide patterns than the bacterial enzymes. Overall, the product patterns can be adjusted by choosing the correct enzyme as well as a defined enzyme activity.

Distinguishing Carbohydrate Isomers with Rapid Hydrogen/Deuterium Exchange-Mass Spectrometry.[Pubmed:33124815]

J Am Soc Mass Spectrom. 2021 Jan 6;32(1):152-156.

Carbohydrates play key roles in facilitating cellular functions, yet characterizing their structures is analytically challenging due to the presence of epimers, regioisomers, and stereoisomers. In-electrospray-hydrogen/deuterium exchange-mass spectrometry (in-ESI HDX-MS) is a rapid HDX method that samples solvated carbohydrates with minimal instrument modification. When applied to proteins, HDX is often measured after multiple time points to sample the dynamics of structures. Herein, we alter the HDX reaction time by modifying the spray-solvent conductivity, which changes the initial size of ESI droplets, and thus, the droplet lifetimes. We show that this change in droplet lifetime alters the magnitude of HDX for carbohydrate-metal adducts. Furthermore, we illustrate how monitoring HDX at multiple time points enables three trisaccharide isomers (melezitose, maltotriose, and Isomaltotriose) to be distinguished. This work illustrates the feasibility of this method for characterizing solvated carbohydrates, including isomeric species which differ only by linkage.

In vitro digestibility of commercial and experimental isomalto-oligosaccharides.[Pubmed:32517953]

Food Res Int. 2020 Aug;134:109250.

Isomalto-oligosaccharides (IMO) significantly contribute to the global oligosaccharide market. IMO are linear alpha-(1 --> 6) linked oligosaccharides with Isomaltotriose as the representative trisaccharide. Commercial IMO preparations ypically also contain panose-series oligosaccharides as a major component. In humans, IMO are partially digestible but the digestibility of specific components of commerical IMO preparations remains unknown. This study aimed to compare the in vitro digestibility of reference compounds, experimental alpha-gluco-oligosaccharides and commercial IMO. Experimental alpha-gluco-oligosaccharides were synthesized with the recombinant dextransucrase DsrM. Two in vitro digestion methods were used, a reference method matching the AOAC method for dietary fibre, and a protocol that uses brush border glycosyl hydrolases from the rat intestine. The alpha-gluco-oligosaccharides patterns after hydrolysis remain were analyzed by high performance anion exchange chromatography coupled to pulsed amperometric detection. Panose-series oligosaccharides were hydrolysed more rapidly by amylase and amyloglucosidase when compared to hydrolysis by rat intestinal enzymes. The rate of hydrolysis by rat intestinal enzymes decreased in the order panose > isomaltose, kojibiose or nigerose. Hydrolysis of panose-series oligosaccharides but not the hydrolysis of isomalto-oligosaccharides was dependent on the degree of polymerization. Qualitative analysis of oligosaccharides remaining after hydrolysis indicated that rat small intestinal enzymes hydrolyse their substrates from the non-reducing end. Taken together, results inform on the modification or optimization of current production processes for IMO to obtain tailored oligosaccharide preparations with reduced digestibility and an increased content of dietary fibre.

Phenolics and Carbohydrates in Buckwheat Honey Regulate the Human Intestinal Microbiota.[Pubmed:32184894]

Evid Based Complement Alternat Med. 2020 Feb 26;2020:6432942.

Intestinal microbiota plays an important role in human health. The aim of this paper is to determine the impact of the phenolics and carbohydrate in buckwheat honey on human intestinal microbiota. We investigated the phenolics and carbohydrate compositions of eight buckwheat honey samples using high-performance liquid chromatography and ion chromatography. The human intestinal microbes were cultured in a medium supplemented with eight buckwheat honey samples or the same concentration of fructooligosaccharides. The bacterial 16S rDNA V4 region sequence of DNA extraction was determined by the Illumina MiSeq platform. 12 phenolics and 4 oligosaccharides were identified in almost all buckwheat honey samples, namely, protocatechuic acid, 4-hydroxy benzoic acid, vanillin, gallic acid, p-coumaric acid, benzoic acid, isoferulic acid, methyl syringate, trans,trans-abscisic acid, cis,trans-abscisic acid, ferulic acid, 4-hydroxybenzaldehyde, kestose, isomaltose, Isomaltotriose, and panose. Most notably, this is the first study to reveal the presence of 4-hydroxybenzaldehyde in buckwheat honey. 4-Hydroxybenzaldehyde seems to be a land marker of buckwheat honey. Our results indicate that buckwheat honey can provide health benefits to the human gut by selectively supporting the growth of indigenous Bifidobacteria and restraining the pathogenic bacterium in the gut tract. We infer that buckwheat honey may be a type of natural intestinal-health products.

Digestibility of branched and linear alpha-gluco-oligosaccharides in vitro and in ileal-cannulated pigs.[Pubmed:31882112]

Food Res Int. 2020 Jan;127:108726.

Isomalto-oligosaccharides (IMOs) may promote health by modulating intestinal microbiota. We hypothesized that the proportion of alpha-(1 --> 6) linkages in IMOs determines their digestibility. Ileal-cannulated pigs were fed diets containing IMO, IMO-DP3 with a greater DP and more alpha-(1 --> 4) linkages, and digestible or resistant maltodextrins. Oligosaccharides were analysed by high-performance anion-exchange chromatography. Compared to IMO, IMO-DP3 contained more panose (18.6 vs. 10.3%) but less isomaltose (7.5 vs. 22.3%) and Isomaltotriose (6.1 vs. 12.6%). The apparent ileal digestibility of dry matter were 3% greater for IMO-DP3 and digestible maltodextrin than resistant maltodextrin; the digestibility of IMO was not different from other oligosaccharides. Ileal propionate, isovalerate, and total SCFA was greater for IMO-DP3 and digestible maltodextrin than IMO. In conclusion, IMO was less digestible than IMO-DP3. Structural properties of IMOs are important determinants of their functional properties within the porcine digestive tract.