Galactosyl Residues Research Articles

Structural elucidation of a Acanthopanax senticosus polysaccharide CQ-1 and its hepatoprotective activity via gut health regulation and antioxidative defense

Acanthopanax senticosus has proven health benefits, particularly for liver damage. The objective of this study was to elucidate the protective effects and the underlying mechanisms of action of A. senticosus against metabolic dysfunction-associated fatty liver disease (MAFLD). A novel homogeneous water-soluble polysaccharide, CQ-1, was successfully isolated and purified from A. senticosus root. The main chain structure of CQ-1 was identified as →2)-α-L-Rha-(1 → 4)-α-D-GalAp-(1 → 6)-β-D-Galp-(1→. Additionally, branched chains comprising an arabinosyl residue, galactosyl residue, and galacturonic acidic residue were identified as being attached to →2,4)-α-L-Rha-(1→, →3,6)-β-D-Galp-(1→, and →3,4)-α-D-GalAp-(1→, respectively. CQ-1 exhibited antioxidant and prebiotic activities in vitro. CQ-1 increased antioxidant capacity and reduced serum pro-inflammatory cytokines in mice. Additionally, CQ-1 has been shown to enhance the diversity and composition of the gut microbiota, thereby facilitating the restoration of gut function. These include improving intestinal barrier function and increasing short-chain fatty acid levels in mice. Our study has shown that CQ-1 has a hepatoprotective effect in MAFLD mice, and we have proposed that CQ-1 may be a promising strategy for the treatment of MAFLD.

Purification and characterization of a thermostable Galium aparine β-galactosidase: A competent agent with enhanced cytotoxic activity against MCF-7 cell line

β-galactosidases (EC 3.2.1.23) are capable of catalysing two distinct types of reactions: transgalactosylation and hydrolysis. It is very important for the senescence of cancer cells. It is a lysosomal exoglycosidase involved in the catabolism of glycoconjugates by sequential release of β-linked terminal galactosyl residues. It has profound significance in cancer cell senescence. It can be derived from various sources including plants, animals, bacteria, yeasts, and filamentous fungi. Purification and thermodynamic characterization of β-galactosidase from Galium aparine seeds, and in vitro efficacy assessment of a breast cancer cell line. The enzyme was purified by using ammonium sulphate precipitation, dialysis, DEAE cellulose, and Sephadex G-100 chromatography. The enzyme was purified to 6.4 fold with a yield of 14 % and specific activity of 11.6 U/mg of protein. SDS-PAGE and MALDI-TOF analysis revealed that β-galactosidase is a monomer with a molecular weight of 35 kDa. The optimum pH and temperature for the purified enzyme were 6.0 and 50 ºC. The enzyme had Km and Vmax values of 0.25 mM and 32 µmol/min, respectively. The thermodynamic parameters (ΔHº, ΔGº, ΔSº) for the irreversible denaturation of the enzyme were also investigated at different temperatures (50–75 ºC). Lactose in milk was successfully hydrolyzed using a purified enzyme at a rate of 0.0121 min−1 and 12.12 min. This is better than other studied β-galactosidases. These results suggest the use of β-galactosidase from G. aparine for producing delactosed milk for the lactose-intolerant population and prebiotic synthesis. pH and optimum temperature and its activation by Ca2+ shows that it is suitable for milk processing. As a result, the enzyme was stable even at higher temperatures and had the potential to undergo catalysis, making it economically viable, primarily in the food and pharmaceutical industries. The present study is aimed to purification and thermodynamic characterization of enzyme to assess in vitro efficacy of β-galactosidase on MCF-7 cell line to delineate its therapeutic efficacy.

Розповсюдженість, властивості та практичне значення α-галактозидази

The achievements of modern enzymology have greatly expanded the possibilities of practical use of enzymes, primarily in medicine and the food industry, which is due to their advantages over chemical catalysts: selectivity and stereospecificity of action, the possibility of achieving high rates of conversion of substrates under relatively mild technological conditions, harmlessness to the environment and humans. The microbial producers have proven themselves as a convenient and economical source of biotechnologically important enzyme preparations of hydrolytic action. One of such enzymes is α-galactosidase, which has the ability to cleave terminal α-linked galactosyl residues from a wide range of natural and synthetic compounds. The review presents data on the spread of the enzyme among different groups of microorganisms and provides a comparative description of their catalytic properties. A range of issues related to the physiological role of the enzyme in plant and animal organisms, localization and functional properties in bacteria and fungi are outlined. The place of α-galactosidase of microorganisms in the modern hierarchical classification of glycosidases is shown. The areas of possible use of the enzyme as an efficient tool for the modification of oligo- and polysaccharides in various industries are described.

The correlation between human seminal plasma sialoproteins and ejaculate parameters

Sialic acids are negatively charged carbohydrates that are components of saccharide chains covalently linked to macromolecules. Sialylated glycoproteins are important for most biological processes, including reproduction, where they are associated with spermatogenesis, sperm motility, immune responses, and fertilization. Changes in the glycoprotein profile or sialylation in glycoproteins are likely to affect the quality of ejaculate. The aim of this study was to determine differences in the degree of sialylation between normozoospermic ejaculates and ejaculates with a pathological spermiogram using two lectins, Sambucus nigra (SNA) and Maackia amurensis (MAL II/MAA) recognizing α-2,6 or α-2,3 linkage of Sia to galactosyl residues. Our results show a close relationship between seminal plasma (SP) sialoproteins and the presence of anti-sperm antibodies in the ejaculate, apoptotic spermatozoa, and ejaculate quality. Using mass spectrometry, we identified SP sialoproteins such as, semenogelins, glycodelin, prolactin-inducible protein, lactotransferrin, and clusterin that are associated with spermatozoa and contribute to the modulation of the immune response and sperm apoptosis. Our findings suggest a correlation between the degree of SP glycoprotein sialylation and the existence of possible pathological states of spermatozoa and reproductive organs. Glycoproteins sialylation represents a potential parameter reflecting the overall quality of ejaculate and could potentially be utilised in diagnostics.

Jasmonates and Ethylene Shape Floridoside Synthesis during Carposporogenesis in the Red Seaweed Grateloupia imbricata.

Floridoside is a galactosyl-glycerol compound that acts to supply UDP-galactose and functions as an organic osmolyte in response to salinity in Rhodophyta. Significantly, the UDP-galactose pool is shared for sulfated cell wall galactan synthesis, and, in turn, affected by thallus development alongside carposporogenesis induced by volatile growth regulators, such as ethylene and methyl jasmonate, in the red seaweed Grateloupia imbricata. In this study, we monitored changes in the floridoside reservoir through gene expression controlling both the galactose pool and glyceride pool under different reproductive stages of G. imbricata and we considered changing salinity conditions. Floridoside synthesis was followed by expression analysis of galactose-1-phosphate uridyltransferase (GALT) as UDP-galactose is obtained from UDP-glucose and glucose-1P, and through α-galactosidase gene expression as degradation of floridoside occurs through the cleavage of galactosyl residues. Meanwhile, glycerol 3-phosphate is connected with the galactoglyceride biosynthetic pathway by glycerol 3-phosphate dehydrogenase (G3PD), monogalactosyl diacylglyceride synthase (MGDGS), and digalactosyl diacylglyceride synthase (DGDGS). The results of our study confirm that low GALT transcripts are correlated with thalli softness to locate reproductive structures, as well as constricting the synthesis of UDP-hexoses for galactan backbone synthesis in the presence of two volatile regulators and methionine. Meanwhile, α-galactosidase modulates expression according to cystocarp maturation, and we found high transcripts in late development stages, as occurred in the presence of methyljasmonate, compared to early stages in ethylene. Regarding the acylglyceride pool, the upregulation of G3PD, MGDGS, and DGDGS gene expression in G. imbricata treated with MEJA supports lipid remodeling, as high levels of transcripts for MGDGS and DGDGS provide membrane stability during late development stages of cystocarps. Similar behavior is assumed in three naturally collected thalli development stages-namely, fertile, fertilized, and fertile-under 65 psu salinity conditions. Low transcripts for α-galactosidase and high for G3PD are reported in infertile and fertilized thalli, which is the opposite to high transcripts for α-galactosidase and low for G3PD encountered in fertile thalli within visible cystocarps compared to each of their corresponding stages in 35 psu. No significant changes are reported for MGDGS and DGDGS. It is concluded that cystocarp and thallus development stages affect galactose and glycerides pools with interwoven effects on cell wall polysaccharides.

Conformational Preferences at the Glycosidic Linkage of Saccharides in Solution as Deduced from NMR Experiments and MD Simulations: Comparison to Crystal Structures.

Glycans are central to information content and regulation in biological systems. These carbohydrate molecules are active either as oligo- or polysaccharides, often in the form of glycoconjugates. The monosaccharide entities are joined by glycosidic linkages and stereochemical arrangements are of utmost importance in determining conformation and flexibility of saccharides. The conformational preferences and population distributions at the glycosidic torsion angles φ and ψ have been investigated for O-methyl glycosides of three disaccharides where the substitution takes place at a secondary alcohol, viz., in α-l-Fucp-(1→3)-β-d-Glcp-OMe, α-l-Fucp-(1→3)-α-d-Galp-OMe and α-d-Glcp-(1→4)-α-d-Galp-OMe, corresponding to disaccharide structural elements present in bacterial polysaccharides. Stereochemical differences at or adjacent to the glycosidic linkage were explored by solution state NMR spectroscopy using one-dimensional 1 H,1 H-NOESY NMR experiments to obtain transglycosidic proton-proton distances and one- and two-dimensional heteronuclear NMR experiments to obtain 3 JCH transglycosidic coupling constants related to torsion angles φ and ψ. Computed effective proton-proton distances from molecular dynamics (MD) simulations showed excellent agreement to experimentally derived distances for the α-(1→3)-linked disaccharides and revealed that for the bimodal distribution at the ψ torsion angle for the α-(1→4)-linked disaccharide experiment and simulation were at variance with each other, calling for further force field developments. The MD simulations disclosed a highly intricate inter-residue hydrogen bonding pattern for the α-(1→4)-linked disaccharide, including a nonconventional hydrogen bond between H5' in the glucosyl residue and O3 in the galactosyl residue, supported by a large downfield 1 H NMR chemical shift displacement compared to α-d-Glcp-OMe. Comparison of population distributions of the glycosidic torsion angles φ and ψ in the disaccharide entities to those of corresponding crystal structures highlighted the potential importance of solvation on the preferred conformation.

Structure Flexibility of Alpha-galactosidase from a Marine Psychrophilic Yeast, Glaciozyma antarctica PI12

Factors that contribute to maintaining the flexibility or stability of an enzyme structure may depend on the composition of each amino acid with different characteristics, providing a purpose and bonding features within the structure. Based on this assumption, a study using homology modeling and a comparative study to observe different structure behaviors of an enzyme at an extremely low temperature (psychrophile) against temperate (mesophile) and high temperature (thermophile) was performed. The subject, a-galactosidase from Glaciozyma antarctica as a marine psychrophilic candidate was chosen against a-galactosidase from Trichoderma reesei (mesophile) and Ramsonia emersonii (thermophile). This enzyme catalyzes the hydrolysis of a-1-6 linked terminal galactosyl residues which can be found in a wide range of the organism. The ability of G. antarctica to grow in extremely cold temperatures rendered the question that the enzyme must have special characteristics to adapt to the cold condition. Based on the homology modeling and molecular dynamics study, a comparison of the structure of G. antarctica a-galactosidase enzymes with its homolog from the mesophilic and thermophilic fungi showed that G. antarctica a-galactosidase enzyme confers its flexibility by the increased number of small amino acids with reduced charges, more loops, a fewer number of hydrogen and disulfide bonds in its structure. Furthermore, a-galactosidase has potential for commercialization in bleach paper and the baking industry also a treatment for bloating and Fabry disease.

Effect of Dexamethasone on the Expression of the α2,3 and α2,6 Sialic Acids in Epithelial Cell Lines.

N-acetylneuraminic acid linked to galactose by α2,6 and α2,3 linkages (Siaα2,6 and Siaα2,3) is expressed on glycoconjugates of animal tissues, where it performs multiple biological functions. In addition, these types of sialic acid residues are the main targets for the binding and entry of influenza viruses. Here we used fluorochrome-conjugated Sambuccus nigra, Maackia amurensis, and peanut lectins for the simultaneous detection of Siaα2,3 and Siaα2,6 and galactosyl residues by two-color flow cytometry on A549 cells, a human pneumocyte cell line used for in vitro studies of the infection by influenza viruses, as well as on Vero and MDCK cell lines. The dexamethasone (DEX) glucocorticoid (GC), a widely used anti-inflammatory compound, completely abrogated the expression of Siaα2,3 in A549 cells and decreased its expression in Vero and MDCK cells; in contrast, the expression of Siaα2,6 was increased in the three cell lines. These observations indicate that DEX can be used for the study of the mechanism of sialylation of cell membrane molecules. Importantly, DEX may change the tropism of avian and human/pig influenza viruses and other infectious agents to animal and human epithelial cells.

Production, purification, characterization, and applications of α-galactosidase from Bacillus flexus JS27 isolated from Manikaran hot springs

α-Galactosidase hydrolyzes the α-1,6-linkage present at the non-reducing end of the sugars and results in the release of galactosyl residue from oligosaccharides like melibiose, raffinose, stachyose, etc. In the present study we report, α-galactosidase from Bacillus flexus isolated from Manikaran hot springs (India). Maximum enzyme production was obtained in guar gum and soybean meal after 72 h at 150 rpm. While, the temperature/pH of production was optimized at 50 °C and 7.0, respectively. Isoenzymes (α-gal I and II) were obtained and characterized based on temperature/pH optima along with their stability profile. JS27 α-Gal II was purified with a final purification fold of 11.54. Native and SDS-PAGE were used to determine the molecular weight of the enzyme as 86 and 41 kDa, respectively, indicating its homodimeric form. JS27 α-Gal II showed optimum enzyme activity at 55 °C and pH 7 (10 min). The enzyme displayed Km value of 2.3809 mM and V max of 2.0 × 104 µmol/min/ml with pNPG as substrate. JS27 α-Gal II demonstrated substrate hydrolysis and simultaneous formation of transgalactosylation products (α-GOS) with numerous substrates (sugar/sugar alcohols, oligosaccharides, and complex carbohydrates) which were verified by TLC and HPLC analysis. α-GOS are significant functional food ingredients and can be explored as prebiotics.

Anti-Lea monoclonal antibody SPM 522 recognizes an extended Lea epitope

Insights into the differential binding characteristics of anti-Lea and anti-LeaLex monoclonal antibodies (mAbs) provide information to develop LeaLex-based cancer immunotherapeutics while avoiding anti-Lea autoimmune reactions. We characterized the epitope recognized by anti-Lea mAb SPM 522. We synthesized the Lea 6-aminohexyl glycoside and report experimental evidence of a minor conformation in solution. The Lea and three other 6-aminohexyl glycosides were conjugated to BSA and titration experiments with SPM 522 show that: 1. SPM 522 binds to LeaLex better than to Lea; 2. the non-reducing Lea galactosyl residue is essential to binding. Competitive ELISA experiments using a panel of tri- to pentasaccharide fragments of LeaLex as well as Lea analogues indicate that: 1. the Lea β-d-galactosyl α hydrophobic patch is crucial to binding; 2. the Lea fucosyl residue contributes to binding; 3. the Lexd-galactosyl residue also contributes to binding. These results indicate that anti-Lea mAb SPM 522 recognizes the Lea[1,3]-β-d-Gal tetrasaccharide. We propose that a major recognition element is the extended hydrophobic surface defined by the Lea-β-d-Gal residue extending to the α faces of the β-d-GlcNAc and β-d-Gal residues.

Structural characterization and protective effects of polysaccharide from Gracilaria lemaneiformis on LPS-induced injury in IEC-6 cells

This study was aimed to characterize Gracilaria lemaneiformis polysaccharides and evaluate their protective effects on Lipopolysaccharide-induced injury in IEC-6 cells. The G. lemaneiformis polysaccharide was degraded by UV/H2O2 treatment and purified to three fractions named GLP-1.0 M, GLP-1.4 M and GLP-1.6 M. The purified fractions were mainly composed of galactose, glucose and xylose. The structural analysis showed that GLP-1.6 M was a typical sulfated red alga polysaccharide containing the linear backbone of β-(1 → 3)- and α-(1 → 4)-linked galactosyl residues, anhydro-galactose units. In the Lipopolysaccharide-induced IEC-6 cells model, GLP-1.6 M exerted the strongest in vitro anti-inflammatory activity by inhibiting the release and expressions of tumor necrosis factor-α, interleukin-6 and interleukin-1β by 89.93%, 67.82% and 38.06%, respectively. Meanwhile, GLP-1.6 M enhanced the intestinal barrier function via up-regulating the expressions of tight junctions and mucin. Therefore, the purified polysaccharide from G. lemaneiformis could be a promising candidate for maintaining intestinal health in the food and pharmaceutical industries.

Safety of the extension of use of galacto-oligosaccharides as a Novel food pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on the extension of use of galacto‐oligosaccharides (GOS) as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is a syrup containing ≥ 57% w/w GOS (w/w dry matter), consisting of different galactosyl residues linked to a terminal glucose by a β‐glycosidic bond and also containing lactose and the constituent monomers of lactose (galactose and glucose). The NF is already authorised and included in the Union list of NFs and is produced according to the same production process. This application is limited to an assessment of the extension of use as a food ingredient in dairy confectionary, cheese and processed cheese, butter and spreads. There is a 10–30% increase in total GOS intake from the requested extension of use compared to the currently authorised uses at the highest 95th percentile. It is noted that the total intake at the highest mean (8.7–22.0 g/day) is below the adequate intake (AI) of 25 g/day for dietary fibre set to ensure a normal laxation in adults, while the highest 95th percentile (27.2–41.6 g/day) is higher than the AI. When the maximum use as a food supplement is added to the highest 95th percentile combined intake from all proposed and authorised food categories a total intake up to 58 g GOS/day is estimated. This highest intake level would exceed the AI for dietary fibre; however, no tolerable upper intake level for dietary fibre has been set and only transient gastrointestinal symptoms may be related to high intake of fibre. The Panel concludes that the NF, that is composed of ≥ 57% GOS dry matter, lactose and related saccharides, is safe under the proposed extension of use.

Biosynthesis of Nondigestible Galactose-Containing Hetero-oligosaccharides by Lactobacillus plantarum WCFS1 MelA α-Galactosidase

This work describes the high capacity of MelA α-galactosidase from Lactobacillus plantarum WCFS1 to transfer galactosyl residues from melibiose to the C6-hydroxyl group of disaccharide-acceptors with β-linkages (lactulose, lactose, and cellobiose) or α-linkages (isomaltulose and isomaltose) to produce novel galactose-containing hetero-oligosaccharides (HOS). A comprehensive nuclear magnetic resonance characterization of the transfer products derived from melibiose:lactulose reaction mixtures revealed the biosynthesis of α-d-galactopyranosyl-(1 → 6)-β-d-galactopyranosyl-(1 → 4)-β-d-fructose as the main component as well as the presence of α-d-galactopyranosyl-(1 → 3)-β-d-galactopyranosyl-(1 → 4)-β-d-fructose and α-d-galactopyranosyl-(1 → 6)-α-d-galactopyranosyl-(1 → 6)-β-d-galactopyranosyl-(1 → 4)-β-d-fructose. Melibiose-derived α-galactooligosaccharides (α-GOS), manninotriose and verbascotetraose, were also simultaneously synthesized. An in vitro assessment of the intestinal digestibility of the novel biosynthesized HOS revealed a high resistance of α-galactosides derived from lactulose, lactose, cellobiose, and isomaltulose. According to the evidence gathered for conventional α-GOS and certain disaccharides used as acceptors in this work, these novel nondigestible α-galactosides could be potential candidates to selectively modulate the gut microbiota composition, among other applications, such as low-calorie food ingredients.

Safety of a change in the conditions of use of galacto-oligosaccharides as a novel food ingredient in food supplements pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on the change in the conditions of use of galacto‐oligosaccharides (GOS) as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is mainly composed of GOS, consisting of different galactosyl residues (two to nine) linked to a terminal glucose by a β‐glycosidic bond but also contains lactose and its monomers (galactose and glucose). The NF is a syrup containing ≥ 55% GOS (w/w dry matter) and is produced enzymatically by two β‐galactosidases. GOS produced by β‐galactosidases according to the same production process is already authorised and included in the EU Union list of novel foods. This application is limited to an assessment of the proposed increase of the use level as food supplement. The proposed change in the conditions of use increases the maximum level in food supplements from 0.333 kg GOS/kg food supplement (33.3%) to 0.450 kg GOS/kg food supplement (45.0%). Since it is recommended that individuals consume no more than 3 servings of 12 g/day, the maximum recommended daily intake would be no more than 16.2 g GOS. No new food uses or other increases to the already approved use levels are being proposed. The information provided on the proposed use levels and anticipated intake do not raise safety concerns. The Panel concludes that the proposed increase in the maximum level of galacto‐oligosaccharides as a NF in food supplements is safe under the proposed changes in conditions of use.

Hydrolysis of Lactose and Transglycosylation of Selected Sugar Alcohols by LacA β-Galactosidase from Lactobacillus plantarum WCFS1.

The production, biochemical characterization, and carbohydrate specificity of LacA β-galactosidase (locus lp_3469) belonging to the glycoside hydrolase family 42 from the probiotic organism Lactobacillus plantarum WCFS1 are addressed. The β-d-galactosidase activity was maximal in the pH range of 4.0-7.0 and at 30-37 °C. High hydrolysis capacity toward the β(1 → 4) linkages between galactose and glucose (lactose) or fructose (lactulose) was found. High efficiency toward galactosyl derivative formation was observed when lactose and glycerol, xylitol, or erythritol were used. Galactosyl derivatives of xylitol were characterized for the first time as 3-O-β-d-galactopyranosyl-xylitol and 1-O-β-d-galactopyranosyl-xylitol, displaying high preference of LacA β-galactosidase for the transfer of galactosyl residues from lactose to the C1 or C3 hydroxyl group of xylitol. These results indicate the feasibility of using LacA β-galactosidase for the synthesis of different galactosyl-polyols, which could be promising candidates for beneficial and appealing functional and technological applications such as novel prebiotics or hypocaloric sweeteners.

Characterization of three GH35 β-galactosidases, enzymes able to shave galactosyl residues linked to rhamnogalacturonan in pectin, from Penicillium chrysogenum 31B.

Three recombinant β-galactosidases (BGALs; PcBGAL35A, PcBGAL35B, and PcGALX35C) belonging to the glycoside hydrolase (GH) family 35 derived from Penicillium chrysogenum 31B were expressed using Pichia pastoris and characterized. PcBGAL35A showed a unique substrate specificity that has not been reported so far. Based on the results of enzymological tests and 1H-nuclear magnetic resonance, PcBGAL35A was found to hydrolyze β-1,4-galactosyl residues linked to L-rhamnose in rhamnogalacturonan-I (RG-I) of pectin, as well as p-nitrophenyl-β-D-galactopyranoside and β-D-galactosyl oligosaccharides. PcBGAL35B was determined to be a common BGAL through molecular phylogenetic tree and substrate specificity analysis. PcGALX35C was found to have similar catalytic capacities for the β-1,4-galactosyl oligomer and polymer. Furthermore, PcGALX35C hydrolyzed RG-I-linked β-1,4-galactosyl oligosaccharide side chains with a degree of polymerization of 2 or higher in pectin. The amino acid sequence similarity of PcBGAL35A was approximately 30% with most GH35 BGALs, whose enzymatic properties have been characterized. The amino acid sequence of PcBGAL35B was approximately 80% identical to those of BGALs from Penicillium sp. The amino acid sequence of PcGALX35C was classified into the same phylogenetic group as PcBGAL35A. Pfam analysis revealed that the three BGALs had five domains including a catalytic domain. Our findings suggest that PcBGAL35A and PcGALX35C are enzymes involved in the degradation of galactosylated RG-I in pectin. The enzymes characterized in this study may be applied for products that require pectin processing and for the structural analysis of pectin.

Purification and characterization of β-galactosidase from newly isolated Aspergillus terreus (KUBCF1306) and evaluating its efficacy on breast cancer cell line (MCF-7).

β-galactosidases (EC 3.2.1.23) are able to catalyze two different types of reactions, namely hydrolysis and transgalactosylation. It is a lysosomal exoglycosidase involved in the catabolism of glycoconjugates by sequential release of β-linked terminal galactosyl residues. It has profound significance in cancer cell senescence. It can be derived from microbial sources including bacteria, yeasts, and filamentous fungi. The enzyme was purified from the crude enzyme using ammonium sulfate precipitation, dialysis, ion exchange chromatography using DEAE cellulose, fast protein liquid chromatography and high performance liquid chromatography. The enzyme was purified with 10.78 -fold with specific activity of 62U/mg of protein and yield of 28.26%. Molecular weight of β -galactosidase as estimated by using SDS-PAGE was 42kDa. Kinetic parameters Km and Vmax for purified enzyme were 0.48 and 0.96 respectively. Further the characterization and kinetic studies of purified enzyme were carried out. The optimum pH and temperature for maximum β-galactosidase activity were found to be 6, 40°C, respectively. The present study is aimed to purification, characterization and in vitro efficacy assessment in breast cancer cell line. The β-galactosidase isolated from Aspergillus terreus was found to be effective in the proliferation of MCF-7 breast cancer cells in vitro. The present study is aimed to purification and characterization of enzyme to assess in vitro efficacy of β-galactosidase on MCF-7 cell line to delineate its therapeutic efficacy.

Unravelling the carbohydrate specificity of MelA from Lactobacillus plantarum WCFS1: An α-galactosidase displaying regioselective transgalactosylation

This comprehensive work addresses, for the first time, the heterologous production, purification, biochemical characterization and carbohydrate specificity of MelA, a cold-active α-galactosidase belonging to the Glycoside Hydrolase family 36, from the probiotic organism Lactobacillus plantarum WCFS1. The hydrolytic activity of MelA α-galactosidase on a wide range of p-nitrophenyl glycoside derivatives and carbohydrates of different molecular-weights showed its high selectivity and efficiency towards the α(1 → 6) glycosidic bonds involving the anomeric carbon of galactose and the C6-hydroxyl group of galactose or glucose units. MelA α-galactosidase also presented a high regioselectivity, efficiency and diversity in accommodating donor and acceptor substrates for the synthesis of α-GOS through transgalactosylation reactions. The catalytic mechanism of MelA for the production of α-GOS was elucidated, revealing its great preference for the transfer of galactosyl residues to the C6-hydroxyl group of galactose units to elongate the chain of α-GOS having either a terminal sucrose (raffinose family oligosaccharides, RFOS) or a terminal glucose (melibiose, manninotriose and verbascotetraose). Our findings indicate the feasibility of using MelA α-galactosidase from Lactobacillus plantarum WCFS1 in the hydrolysis of RFOS and in the efficient and versatile synthesis of α-GOS with appealing functional properties in the context of food and nutraceutical applications.

Structural diversity and prebiotic potential of short chain β-manno-oligosaccharides generated from guar gum by endo-β-mannanase (ManB-1601)

Size exclusion chromatography of short chain β-manno-oligosaccharides (GG-β-MOS) produced after endo-mannanase (ManB-1601) hydrolysis of guar gum resulted in seven (P1–P7) peaks. Electron spray ionization mass-spectrometry (ESI-MS) revealed P3, P4, P5 and P6 peaks as pentasaccharide (DP5), tetrasaccharide (DP4), trisaccharide (DP3) and disaccharide (DP2), respectively. DP2 and DP3 GG-β-MOS were structurally characterized by NMR (1H and 13C), FTIR and XRD. DP2 GG-β-MOS was composed of two species (A) mannopyranose β-1,4 mannopyranose and (B) α-1,6-galactosyl-mannopyranose while, DP3 oligosaccharide showed presence of three species i.e. (A) α-d-galactosyl-β-d-mannobiose (galactosyl residue at reducing end), (B) α-d-galactosyl-β-d-mannobiose (galactosyl residue at non-reducing end) and (C) mannopyranose β-1,4 mannose β-1,4 mannopyranose. In batch fermentation, DP2 GG-β-MOS was preferred over DP3 by all Lactobacillus sp. except Lactobacillus casei var rhamnosus. DP2/DP3 and GG-β-MOS mixture inhibited the growth of enteropathogens in monoculture and co-culture fermentations, respectively. Fermentation of GG-β-MOS mixture by Lactobacillus sp. produced short chain fatty acids.

Analysis of cell wall synthesis and metabolism during early germination of Blumeria graminis f. sp. hordei conidial cells induced in vitro

As an obligate biotroph, Blumeria graminis f. sp. hordei (Bgh) cannot be grown in an axenic culture, and instead must be cultivated on its host species, Hordeum vulgare (barley). In this study an in vitro system utilizing n-hexacosanal, a constituent of the barley cuticle and known inducer of Bgh germination, was used to cultivate Bgh and differentiate conidia up to the appressorial germ tube stage for analysis. Transcriptomic and proteomic profiling of the appressorial germ tube stage revealed that there was a significant shift towards energy and protein production during the pre-penetrative phase of development, with an up-regulation of enzymes associated with cellular respiration and protein synthesis, modification and transport. Glycosidic linkage analysis of the cell wall polysaccharides demonstrated that during appressorial development an increase in 1,3- and 1,4-linked glucosyl residues and xylosyl residues was detected along with a significant decrease in galactosyl residues. The use of this in vitro cultivation method demonstrates that it is possible to analyse the pre-penetrative processes of Bgh development in the absence of a plant host.