Enzymatic Synthesis and Structural Confirmation of Novel Oligosaccharide,D-Fructofuranose-linked Chitin Oligosaccharide

Utilizing transglycosylation reaction catalyzed by β- N -acetylhexosaminidase of Stenotrophomonas maltophilia , β-D-fructofuranosyl-(2↔1)-α- N , N ´diacetylchitobioside (GlcNAc ₂ -Fru) was synthesized from N -acetylsucrosamine and N , N ´-diacetylchitobiose (GlcNAc ₂ ), and β-D-fructofuranosyl-(2↔1)-α- N , N ´, N ´´-triacetylchitotrioside (GlcNAc ₃ -Fru) was synthesized from GlcNAc ₂ -Fru and GlcNAc ₂ . Through purification by charcoal column chromatography, pure GlcNAc ₂ -Fru and GlcNAc ₃ -Fru were obtained in molar yields of 33.0 % and 11.7 % from GlcNAc ₂ , respectively. The structures of these oligosaccharides were confirmed by comparing instrumental analysis data of fragments obtained by enzymatic hydrolysis and acid hydrolysis of them with known data of these fragments.     

Paracoccin from Paracoccidioides brasiliensis; purification through affinity with chitin and identification of N‐acetyl‐β‐D‐glucosaminidase activity

The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, the most frequent systemic mycosis in Latin America. Our group has been working with paracoccin, a P. brasiliensis lectin with MM 70 kDa, which is purified by affinity with immobilized N‐acetylglucosamine (GlcNAc). Paracoccin has been described to play a role in fungal adhesion to extracellular matrix components and to induce high and persistent levels of TNFα and nitric oxide production by macrophages. In the cell wall, paracoccin colocalizes with the β‐1,4‐homopolymer of GlcNAc into the budding sites of the P. brasiliensis yeast cell. In this paper we present a protocol for the chitin‐affinity purification of paracoccin. This procedure provided higher yields than those achieved by means of the technique based on the affinity of this lectin with GlcNAc and had an impact on downstream assays. SDS–PAGE and Western blot analysis revealed similarities between the N‐acetylglucosamine‐ and chitin‐bound fractions, confirmed by MALDI–TOF–MS of trypsinic peptides. Western blot of two‐dimensional gel electrophoresis of the yeast extract showed a major spot with M_r 70 000 and pI approximately 5.63. Morevover, an N‐acetyl‐β‐D ‐glucosaminidase activity was reported for paracoccin, thereby providing new insights into the mechanisms that lead to cell wall remodelling and opening new perspectives for its structural characterization. Copyright © 2009 John Wiley & Sons, Ltd.     

Production of Lacto-N-biose I Using Crude Extracts of Bifidobacterial Cells

Lacto-N-biose I (LNB) is supposed to represent the bifidus factor in human milk oligosaccharides, and can be practically produced from sucrose and GlcNAc using four bifidobacterial enzymes, 1,3-β-galactosyl-N-acetylhexosamine phosphorylase, sucrose phosphorylase, UDP-glucose-hexose 1-phosphate uridylyltransferase, and UDP-glucose 4-epimerase, recombinantly produced by Escherichia coli. Here the production of LNB by the same enzymatic method without using genetically modified enzymes to consider the use of LNB for a food ingredient was reported. All four enzymes were produced as the intracellular enzymes of Bifidobacterium strains. The mixture of the crude extracts contained all four enzymes, with other enzymes interfering with the LNB production, namely, phosphoglucomutase, fructose 6-phosphate phosphoketolase, and glycogen phosphorylase. The first two interfering enzymes were selectively inactivated by heat treatment at 47 °C for 1 h in the presence of pancreatin, and glycogen phosphorylase was disabled by hydrolyzing its possible acceptor molecules using glucoamylase. Finally, 91 % of GlcNAc was converted into LNB in the 100-mL reaction mixture containing 300 mM GlcNAc.     

1,6-α-L-Fucosidases from Bifidobacterium longum subsp. infantis ATCC 15697 Involved in the Degradation of Core-fucosylated N -Glycan

Bifidobacterium longum subsp. infantis ATCC 15697 possesses five α-L-fucosidases, which have been previously characterized toward fucosylated human milk oligosaccharides containing α1,2/3/4-linked fucose [Sela et al.: Appl. Environ. Microbiol., 78, 795-803 (2012)]. In this study, two glycoside hydrolase family 29 α-L-fucosidases out of five (Blon_0426 and Blon_0248) were found to be 1,6-α-L-fucosidases acting on core α1,6-fucose on the N-glycan of glycoproteins. These enzymes readily hydrolyzed p-nitrophenyl-α-L-fucoside and Fucα1-6GlcNAc, but hardly hydrolyzed Fucα1-6(GlcNAcβ1-4)GlcNAc, suggesting that they de-fucosylate Fucα1-6GlcNAcβ1-Asn-peptides/proteins generated by the action of endo-β- N-acetylglucosaminidase. We demonstrated that Blon_0426 can de-fucosylate Fucα1-6GlcNAc-IgG prepared from Rituximab using Endo-CoM from Cordyceps militaris. To generate homogenous non-fucosylated N-glycan-containing IgG with high antibody-dependent cellular cytotoxicity (ADCC) activity, the resulting GlcNAc-IgG has a potential to be a good acceptor substrate for the glycosynthase mutant of Endo-M from Mucor hiemalis. Collectively, our results strongly suggest that Blon_0426 and Blon_0248 are useful for glycoprotein glycan remodeling.     

Identification and characterization of Clostridium paraputrificum M-21, a chitinolytic, mesophilic and hydrogen-producing bacterium

A strictly anaerobic, mesophilic and chitinolytic bacterial strain, M-21, was isolated from a soil sample collected from Mie University campus and identified as Clostridium paraputrificum based on morphological and physiological characteristics, and 16S rRNA sequence analysis. C. paraputrificum M-21 utilized chitin and N-acetyl- -glucosamine (GlcNAc), a constituent monosaccharide of chitin, to produce a large amount of gas along with acetic acid and propionic acid as major fermentation products. Hydrogen and carbon dioxide accounted for 65% and 35% of the gas evolved, respectively. The conditions for 1 l batch culture of C. paraputrificum, including pH of the medium, incubation temperature and agitation speed, were optimized for hydrogen production with GlcNAc as the carbon source. The bacterium grew rapidly on GlcNAc with a doubling time of around 30 min, and produced hydrogen gas with a yield of 1.9 mol H₂/mol GlcNAc under the following cultivation conditions: initial medium pH of 6.5, incubation temperature of 45°C, agitation speed of 250 rpm, and working volume of 50% of the fermentor. The dry cell weight harvested from this culture was 2.0 g/l.     

维氏气单胞菌来源几丁质酶的克隆表达及应用

丰年虾是一种小型甲壳类动物,孵化后的幼虫是水产动物的优质饵料,残留的卵壳则作为废弃物处理.然而丰年虾卵壳含有丰富的蛋白质和几丁质,具有极大的应用潜力.为了从丰年虾卵壳中制备几丁寡糖,选择了水解产物分布宽泛和含有几丁质结合域的维氏气单胞菌来源的几丁质酶ChiB565,将其在毕赤酵母Pichia pastoris中进行重组...     

Molecular Design and Synthesis of a Novel Substrate for Assaying Lysozyme Activity

A novel substrate {Galβ1,4GlcNAcβ1,4GlcNAc-β-pNP [Gal(GlcNAc)₂-β-pNP]} for assaying lysozyme activity has been designed using docking simulations and enzymatic synthesis via β-1,4-galactosyltransferase-mediated transglycosylation from UDP-Gal as the donor to (GlcNAc)₂-β-pNP as the acceptor. Hydrolysis of the synthesized Gal(GlcNAc)₂-β-pNP and related compounds using hen egg-white lysozyme (HEWL) demonstrated that the substrate was specifically cleaved to Gal(GlcNAc)₂ and p-nitrophenol (pNP). A combination of kinetic studies and docking simulation was further conducted to elucidate the mode of substrate binding. The results demonstrate that Gal(GlcNAc)₂-β-pNP selectively binds to a subsite of lysozyme to liberate the Gal(GlcNAc)₂ and pNP products. The work therefore describes a new colorimetric method for quantifying lysozyme on the basis of the determination of pNP liberated from the substrate.     

Neuroprotective effects of N-acetylglucosamine against hydrogen peroxide-induced apoptosis in human neuronal SK-N-SH cells by inhibiting the activation of caspase-3, PARP, and p38

Neuroprotective effects of N-acetylglucosamine (GlcNAc), a monosaccharide derivative of glucose, against H₂O₂-induced neurotoxicity and its underlying mechanism in human SK-N-SH neuroblastoma cells were investigated. Pretreatment of GlcNAc prior to exposure of cells to H₂O₂ stress significantly reduced the H₂O₂-mediated neuronal cell death and apoptosis. The GlcNAc dose-dependently decreased the level of intracellular reactive oxygen species (ROS) in H₂O₂-treated cells and also effectively inhibited H₂O₂-induced apoptotic features such as DNA fragmentation, caspase-3, and poly ADP-ribose polymerase (PARP) cleavages, and p38 phosphorylation. These results suggested that GlcNAc might potentially serve as agents for prevention of neurodegenerative diseases caused by oxidative stresses and this effect may be associated with the suppression of caspase-3, PARP, and p38 activation.     

Cloning and characterization of a β-N-acetylglucosaminidase (BmFDL) from silkworm Bombyx mori

In insects, β-N-acetylglucosaminidase (GlcNAcase) participates in critical physiological processes such as fertilization, metamorphosis, and glycoconjugate degradation. Insects produce glycoproteins carrying paucimannosidic-type N-glycans, the terminal GlcNAc residue of which is cleaved by a GlcNAc-linkage specific GlcNAcase, also known as the fused lobes (FDL) protein. To obtain information on the structure of GlcNAcases and insight into their contribution to physiological processes, we cloned Bombyx mori FDL (BmFDL) from silkworm larvae. The full-length cDNA (1.9 kb) encoded a protein of 633 amino acids with 42% amino acid sequence identity to Drosophila melanogaster FDL (DmFDL). Recombinant BmFDL cleaved only β-1,2-linked GlcNAc residues from the α-1,3 branch of biantennary N-glycan. This substrate specificity was similar to that of DmFDL. Microsomal FDL activity was inhibited by anti-BmFDL antibodies. Taken together, our results suggest that BmFDL is a N-glycan-processing GlcNAcase in B. mori.     

Purification and characterization of a 49-kDa chitinase from Streptomyces griseus HUT 6037

A 49-kDa chitinase (pI7.3) was purified to homogeneity from the culture supernatant of Streptomyces griseus HUT 6037 by ultrafiltration, DEAE-Sephadex A-50 and Sephadex G-100 column chromatographies, and chromatofocusing. The purified enzyme was stable up to 40 degrees C. The N-terminal amino acid sequence of the enzyme was highly homologous to the N-terminal region of the fibronectin type III-like domain of S. olivaceoviridis chitinase 01 belonging to family 18 glycosyl hydrolases. The 49-kDa chitinase hydrolyzed partially N-acetylated chitosan more easily than colloidal chitin. The hydrolyzate of 54% deacetylated chitosan by the enzyme was separated by CM-Sephadex C-25 column chromatography. The structures of the oligosaccharides obtained were determined by MALDI-TOF MS analysis combined with exo-glycosidase digestion. In addition to GlcNAc, (GlcNAc)2, and (GlcNAc)3, hetero-chitooligosaccharides with GlcNAc at the reducing end were detected. Thus, the specificity of the enzyme for the hydrolysis of the beta-1,4-glycosidic linkages in partially N-acetylated chitosan was similar to that of the family 18 chitinases.     

Expression of the cytoplasmic domain of NodC as an active form in Drosophila S2 cells

NodC, a membrane protein that catalyzes the synthesis of the chitin oligosaccharide chain, was successfully produced in a soluble form. The truncated NodC gene encoding only the cytoplasmic domain that deletes the hydrophobic N-terminus expressed both cytoplasmic and secreted proteins in Drosophila Schneider 2 cells. The expressed protein maintained the ability to synthesize chitin oligosaccharides, primarily (GlcNAc)4, similar to the native membrane-bound NodC. This evidence suggests that only the large hydrophilic loop of NodC is efficient for enzymatic activity. Moreover, immobilizing the soluble NodC to a solid phase has no effect on the enzymatic activity. This, anchoring NodC is not necessary for its activity.     

Oligosaccharide formation during hydrolysis of lactose with Saccharomyces lactis lactase (Maxilact®): Part 2—Oligosaccharide structures

The oligosaccharides produced during the hydrolysis of lactose with Saccharomyces lactis lactase were isolated and structurally analysed.Six oligosaccharides with two to four monosaccharide units were studied. All were linear and had one or two galactosyl residues linked β-(1 → 6)- to glucose, galactose or lactose. Thus, in addition to lactose hydrolysing activity, the enzyme has transglycosylation activity with high specificity for formation of β-(1 → 6)- galactosidic linkages.     

The cryoprotectant effect of shrimp chitin and shrimp chitin hydrolysate on denaturation and unfrozen water of lizardfish surimi during frozen storage

Shrimp chitin and shrimp chitin hydrolysate prepared from black tiger (Penaeus monodon), endeavour (Metapenaeus endeavouri) and giant freshwater (Macrobrachium rosenbergii) prawns were added to lizardfish surimi, and it was frozen and stored at −25 °C for 6 months to evaluate freeze-induced protein denaturation. Changes in total Ca-ATPase activity and the amount of unfrozen water in samples were periodically determined during frozen storage. The surimi containing shrimp chitin showed the same freeze-denaturaion rate (K_D) as the surimi without cryoprotectants (control), and the amount of unfrozen water was lower than that of control. However, surimi containing shrimp chitin hydrolysates showed K_D values equal to surimi containing sucrose or glucose but lower than the control, and the amount of unfrozen water was increased compared to control. The results revealed that shrimp chitin hydrolysates retarded the effects on freeze-induced denaturation by stabilizing the hydrated water molecules that surround the protein.     

Variations in bovine milk oligosaccharides during early and middle lactation stages analyzed by high-performance liquid chromatography-chip/mass spectrometry

Milk oligosaccharides (OS) are not only a source of nutrition for newborns, but also provide numerous important biological functions including the prevention of pathogen binding to the intestinal epithelium and serving as nutritive sources for beneficial bacteria. High-performance mass spectrometry and separation methods were used to evaluate changes of bovine milk oligosaccharides (bMO) in different lactation stages. Previously, 40 bMO were identified in bovine milk with many consisting of short oligomeric chains that were less complex than human milk oligosaccharides (hMO). The bMO are also significantly more anionic than hMO, with nearly 70% in measured abundances containing either N-acetylneuraminic acid or N-glycolylneuraminic acid, and no fucosylated OS. In this study, we examined factors that could affect the abundances of bMO including stage of lactation and breed. The total concentrations dropped rapidly in the first several days of lactation. Moreover, the anionic oligosaccharides (including N-glycolylneuraminic acid) decreased more rapidly compared with the neutral oligosaccharides.     

Immunomodulatory effects of feruloylated oligosaccharides from rice bran

Feruloylated oligosaccharides (FOs), the ferulic acid ester of oligosaccharides, can be released either by the enzymatic or mild acid hydrolysis of arabinoxylans present in cereal bran, and are usually considered as natural antioxidants. However, no related research is available to explain their immunomodulatory effects. This report elucidated their immunomodulatory effects through the variations of pro-inflammatory mediators in vitro. FOs were obtained from the mild acid hydrolysis of rice bran. We found that FOs (0.1–100 μg/ml) induced tumour necrosis factor alpha (TNF-α), IL-1β, IL-6, nitric oxide (NO) and PGE₂ production in unstimulated macrophages, RAW264.7 cells. Furthermore, pre- and post-treated FOs (0.1–100 μg/ml) dose-dependently suppressed TNF-α, IL-1β, IL-6 and NO production, and induced IL-10 production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells without exerting cytotoxicity. As a result anti-inflammatory and therapeutic activities were revealed. It is noteworthy that prostaglandin E₂ (PGE₂) production was significantly suppressed at an FO level of 100 μg/ml. The in vitro assessment of inflammatory mediators should be useful in further characterising the effects of FOs on immunomodulation. Moreover, it will create the economical value of rice bran, which has long been considered as conventional agricultural wastes.     

Comparison of the antipathogenic effect toward Staphylococcus aureus of N-linked and free oligosaccharides derived from human,bovine, and goat milk

N-linked oligosaccharides (N-glycans) derived from milk were recently found to be antipathogenic. This study compares the antimicrobial activity of N-linked glycans and free oligosaccharides from human, bovine, and goat milk against Staphylococcus aureus. Milk N-glycans showed a bactericidal/bacteriostatic effect on the pathogen when compared to free milk oligosaccharides, evidenced by the clear zone from the halo assay, with the order of human milk >goat milk >bovine milk. None of the free milk oligosaccharide samples were bactericidal/bacteriostatic, despite its positive results in growth curve and minimum inhibitory concentration (MIC) assays which are believed to be related to hyperosmosis. Both N-glycans and free milk oligosaccharides can reduce the adhesion of Staphylococcus aureus to Caco-2 cells, however, N-glycans worked significantly more effective than free milk oligosaccharides. Structural analysis of all free oligosaccharide and N-glycan samples showed the obvious interspecies differences, and the structure/function relationship of the respected N-glycans is of interest for future study. The significant bactericidal/bacteriostatic activity possessed by human, bovine, and goat milk N-linked glycans holds great potential as a novel substitute for antibiotics.     

Characterization of bifidobacteria as starters in fermented milk containing raffinose family of oligosaccharides from lupin as prebiotic

The characterization of six bifidobacteria strains used as starters in the manufacture of fermented milk, containing the raffinose family of oligosaccharides (RFOs) as prebiotics, was carried out by applying in vitro methodologies. Selection criteria were the specific growth rate (μ), generation time (T_g), titratable acidity in milk, and enzymatic activities. Strain growth and acidification rate were determined in milk with and without 2% RFOs. Different growth rates, acidification rates and enzymatic patterns were observed among tested bifidobacterial strains. Bifidobacteria with higher levels of α-galactosidase activity showed growth and acidification rate enhancement when RFOs were added compared with lactose as sole carbon source. Based on the results, Bifidobacterium lactis Bb-12 was selected for further investigation as it possessed the enzymes required to enhance growth and metabolic activity in the presence of RFOs from lupins.     

Chemical characterization of the oligosaccharides in Bactrian camel (Camelus bactrianus) milk and colostrum

Bactrian camel milk and colostrum are commonly used as foods in Mongolia, whose people believe that these products promote human health. It has been hypothesized that milk oligosaccharides are biologically significant components of human milk, acting as receptor analogs that inhibit the attachment of pathogenic microorganisms to the colonic mucosa, and as prebiotics, which stimulate the growth of bifidobacteria within the infant colon. To evaluate their biological significance, we studied the oligosaccharides present in samples of Bactrian camel milk and colostrum. Using ¹H-nuclear magnetic resonance spectroscopy, we identified and characterized the following oligosaccharides of camel colostrum: Gal(β1–4)[Fuc(α1–3)]Glc (3-fucosyllactose), Gal(β1–3)Gal(β1–4)Glc (3′-galactosyllactose), Gal(β1–6)Gal(β1–4)Glc (6′-galactosyllactose), Neu5Ac(α2–3)Gal(β1–4)Glc (3′-sialyllactose), Neu5Ac(α2–6)Gal(β1–4)Glc (6′-sialyllactose), Neu5Ac(α2–3)Gal(β1–3)Gal(β1–4)Glc (sialyl-3′-galactosyllactose), Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc (sialyllacto-N-tetraose c), Neu5Ac(α2–3)Gal(β1–3)[Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (sialyllacto-N-novopentaose a), Gal(β1–3)[Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (sialyllacto-N-novopentaose b); and Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3)[Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (monosialyllacto-N-neohexaose). The oligosaccharides in the mature camel milk were characterized as 3′-galactosyllactose, Gal(β1–3)[Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (lacto-N-novopentaose I), and 3′-sialyllactose.     

Purification and characterization of trypsin from the pyloric caeca of brownstripe red snapper (Lutjanus vitta)

Trypsin was purified from the pyloric caeca of brownstripe red snapper (Lutjanus vitta) by ammonium sulphate (40–60% saturation) precipitation, soybean trypsin inhibitor (SBTI)-Sepharose 4B column and DEAE-Sephacel column chromatography. Purified trypsin showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) and native-PAGE. A yield of 4.9% with the purification-fold of 20 was obtained. Trypsin had an apparent molecular weight of 23 kDa. SBTI and N-ρ-tosyl-l-lysine-chloromethylketone (TLCK) showed a strong inhibitory effect on the purified trypsin, while other protease inhibitors exhibited negligible inhibition. Trypsin had maximal activity at pH 8.5 and 60 °C for the hydrolysis of α-N-benzoyl-dl-arginine-ρ-nitroanilide (BAPNA). It was stable within the temperature range of 25–55 °C and pH range of 7.0–10.0. Purified trypsin had a Michaelis–Menten constant (K_m) and catalytic constant (k_cat) of 0.507 mM and 4.71 s⁻¹, respectively, when BAPNA was used as the substrate. For the hydrolysis of α-N-ρ-tosyl-l-arginine methyl ester (TAME), K_m and k_cat were 0.328 mM and 112 s⁻¹, respectively.     

Production of chitin oligosaccharides with different molecular weights and their antioxidant effect in RAW 264.7 cells

The purpose of this research is not only to produce two kinds of chitin oligosaccharides or N-acetyl chito-oligosaccharides (NA-COSs) with different molecular weights (MW) from crab chitin hydrolysis solution but also to determine their effect against oxidative stress in live cells. Two kinds of NA-COSs with MW 1–3 kDa (NA-COS 1–3 kDa) and below 1 kDa (NA-COS < 1 kDa) were obtained using an ultrafiltration membrane system. They exhibited an inhibitory effect against DNA and protein oxidation. In addition, in their presence, intracellular glutathione (GSH) level and direct intracellular radical scavenging effect were significantly increased in a time-dependent manner in mouse macrophages (RAW 264.7) and rendered inhibitory effect against cellular oxidative stress. In particular, NA-COS 1–3 kDa was more effective than NA-COS < 1 kDa in protein oxidation and production of intracellular free radicals in live cells. These results suggest that NA-COSs act as a potential scavenger against oxidative stress in cells.