Amounts Of N-acetylglucosamine Research Articles

Polysaccharide Sequence Influences the Specificity and Catalytic Activity of Glucuronyl C5-Epimerase.

Heparin is a widely used biotherapeutic produced from animal tissues. However, it might be possible to produce a bioengineered version using a multienzyme process, relying on the isolation of the E. coli K5 capsule heparosan and its chemical conversion to N-sulfoheparosan, NSH. Glucuronyl C5-epimerase, the first enzyme that acts on NSH, catalyzes the reversible conversion of glucuronic acid (GlcA) to iduronic acid (IdoA). Using full-length NSH, containing different amounts of N-acetylglucosamine (GlcNAc) residues, we demonstrate that C5-epimerase specificity relates to polysaccharide sequence, particularly the location of GlcNAc residues within the chain. We leveraged the deuterium exchange and the novel β-glucuronidase heparanase BP, which cleaves at the GlcA residue. Liquid chromatography-mass spectrometry and gel permeation chromatography of partial/complete heparanase BP digestion products from various NSH substrates treated with C5-epimerase provide information on C5-epimerase activity and action pattern. This study provides insight into optimizing the large-scale production of bioengineered heparin.

A new method for determining the mycelial weight of the koji-mold Aspergillus oryzae by measuring its glycosylceramide content.

At present, the quantitation of the mycelial weight of the industrially important non-pathogenic fungus Aspergillus oryzae, which is used for manufacturing koji, is performed by quantitating N-acetylglucosamine. However, since N-acetylglucosamine is a cell wall component, the extraction procedure is costly and tedious, and its quantitative performance is poor. Here, we report a novel method for the quantitation of A. oryzae mycelial weight. The amount of glycosylceramide significantly correlated with both the mycelial weight of A. oryzae and the amount of N-acetylglucosamine, an established index of the mycelial weight of A. oryzae in koji. This new method is simple and efficient and can be used in the brewing and food industries to determine the mycelial weight of A. oryzae.

Characterization of Extraceluller Chitinase Produced by <i>Bacillus licheniformis</i> JP2 from Penen Hot Springs, North Sumatera

The research was about characterization of extraceluller chitinase produced by Bacillus licheniformis JP2 from Penen Hot Springs, North Sumatera. Precipitation of crude chitinase was performed with different levels of ammonium sulphate. Chitinase activity was determined colorimetrically by detecting the amount of N-acetylglucosamine (GlcNAc) released from colloidal chitin substrate with N-acetylglucosamine. Characterization of chitinase activity was measured at different pH, different temperature, Km and Vmax values. The optimum activity of chitinase was produced 4 days after incubation with chitinase activity of 0.0063 U/ml. Optimum chitinase activity was higher after 50% ammonium sulphate precipitation with activity of 0.0087 U/ml followed by decreasing in activity supernatant with activity of 0.0012 U/ml. Chitinase activity increased at pH 6 and at 60°C by 0.05066 U/ml with specific activity 3.8113 U/mg after dialysis. Km and Vmax values were 0.321 mg/ml and 71.429 µg, respectively.

Chitinase and cellulase activity from Bacillus thuringiensis strains

The present study aimed to analyze the production of chitinase and cellulase enzymes by strains of Bacillus thuringiensis toxic to Spodoptera frugiperda and Anthonomus grandis larvae. In order to evaluate the relationship between cellular growth and the chitinase and cellulase production, in vitro assays were carried through with bacteria cultures grown for 16h, 24h, 48h and 72h. Chitinase and cellulase activity was determined by a colorimetric method. The amount of N-acetylglucosamine (GlcNAc) or its equivalent was measured by development of color in acid medium. All strains presented enzymatic production after 16h of cellular growth until 72h. However, a Kruskal-Wallis test detected no significant differences among the chitinase and cellulase activity during the cellular growth. According to these results, was not possible to associate chitinase and cellulose activity with the different level of toxicity of Bt strains against S. frugiperda and A. grandis larvae.

Analysis of the hyphal load during early stages of wood decay by basidiomycetes in the presence of the wood preservative fungicides CuSO4 and cyproconazole

AbstractThis study considers variations in hyphal load in decaying wood in the presence of the fungicides CuSO4and cyproconazole. Variations in the chitin content of hyphae following exposure to both fungicides have been detected. Increasing concentrations of CuSO4and cyproconazole in wood caused an increase in the amount ofN-acetyl glucosamine in the mycelia ofCoriolus versicolorandGloeophyllum trabeum, which may be associated with increased deposition of chitin. This may in turn be an expression of the formation of a thicker cell wall at increased fungicide concentrations. Low concentrations of both fungicides also caused an increase in the amount of mycelium produced byG. trabeum. However, the same concentrations were effective at preventing mass loss, indicating that the fungal mycelium was less effective at decaying wood, despite being present in relatively large amounts. In the case ofC. versicolor, this effect was not observed, as increasing concentrations of both fungicides caused an initial decrease in the amount of mycelium, followed by a recovery phase at intermediate chemical concentrations. Again, mass loss was greatly inhibited by the presence of low concentrations of both fungicides.

Glycoprotein Hypersecretion Alters the Cell Wall in Trichoderma reesei Strains Expressing the Saccharomyces cerevisiae Dolichylphosphate Mannose Synthase Gene

Expression of the Saccharomyces cerevisiae DPM1 gene (coding for dolichylphosphate mannose synthase) in Trichoderma reesei (Hypocrea jecorina) increases the intensity of protein glycosylation and secretion and causes ultrastructural changes in the fungal cell wall. In the present work, we undertook further biochemical and morphological characterization of the DPM1-expressing T. reesei strains. We established that the carbohydrate composition of the fungal cell wall was altered with an increased amount of N-acetylglucosamine, suggesting an increase in chitin content. Calcofluor white staining followed by fluorescence microscopy indicated changes in chitin distribution. Moreover, we also observed a decreased concentration of mannose and alkali-soluble beta-(1,6) glucan. A comparison of protein secretion from protoplasts with that from mycelia showed that the cell wall created a barrier for secretion in the DPM1 transformants. We also discuss the relationships between the observed changes in the cell wall, increased protein glycosylation, and the greater secretory capacity of T. reesei strains expressing the yeast DPM1 gene.

The chemistry of carcin oembryonic antigen

The structure of carcinoembryonic antigen is being determined as an approach to greater diagnostic specificity in the radioimmune assay. Examination by combined gas chromatography-mass spectrometry of derivatized saccharides from CEA has given a general outline of its carbohydrate structure. Sialic acid and fucose were completely destroyed, and galactose and mannose were partially destroyed by a single periodate treatment. Serial periodate oxidation (Smith degradation) destroyed additional amounts of galactose and mannose, as well as significant amounts of N-acetylglucosamine. Antigenic activity persisted, indicating that the residues destroyed played little, if any, part in the antigenicity of DEA.

Process optimization for antifungal chitinase production by Trichoderma harzianum

Single-parameter optimization was carried out for the production of chitinase using a soil isolate belonging to Trichoderma harzianum in solid-state fermentation. Maximum chitinase activity (3.18 U/gds) was obtained after 96 h of incubation at 30 °C when wheat bran moistened (65.7%) with salt solution was supplemented with colloidal chitin (1%, w/w) and yeast extract (2%, w/w) used as the substrate. The inoculum contains 4×10 7 spores of T. harzianum (TUBF 781). The growth of the fungus on wheat bran particle was visualized by a scanning electron microscope. Chitinase activity was measured as the amount of N-acetyl glucosamine (NAG) liberated in μmol/min under reaction conditions. The crude extract showed antifungal activity against a wide range of fungal strains belonging to Aspergillus, Rhizopus and Mucor sp., and was found significant against Aspergillus niger (NCIM 563).

Molecular cloning and characterization of 58 kDa chitinase gene fromSerratia marcescens KCTC 2172

A chitinase gene (pCHi58) encoding a 58 kDa chitinase was isolated from theSerratia marcescens KCTC 2172 cosmid library. The chitinase gene consisted of a 1686 bp open reading frame that encoded 562 amino acids.Escherichia coil harboring the pChi58 gene secreted a 58 kDa chitinase into the culture supernatant. The 58 kDa chitinase was purified using a chitin affinity column and mono-S column. A nucleotide andN-terminal amino acid sequence analysis showed that the 58 kDa chitinase had a leader peptide consisting of 23 amino acids which was cleaved prior to the 24th alanine. The 58 KDa chitinase exhibited a 98% similarity to that ofS. marcescens QMB 1466 in its nuclotide sequence. The chitinolytic patterns of the 58 kDa chitinase released N,N′-diacetyl chitobiose (NAG2) as the major hydrolysis end-product with a trace amount ofN-acetylglucosamine. When a 4-methylumbellyferyl-N-acetylglucosamin monomer, dimmer, and tetramer were used as substrates, the 58 kDa chitinase did not digest the 4-Mu-NAG monomer (analogue of NAG2), thereby indicating that the 58 kDa chitinase was likely an endochitinase. The optimum reaction temperature and pH of the enzyme were 50°C and 5.0, respectively.

Carbohydrates of the Vestibular End Organs

The presence of carbohydrates in the glycocalyx of the vestibular end organs of the guinea pig was investigated at the ultrastructural level using lectin-gold solutions. The glycocalyx of both the sensory and the supporting cells has variable sugar components. The glycocalyx of the sensory cells including the ciliary interconnections contains N-acetyl-glucosamine, galactose and mannose. In contrast, the glycocalyx of the supporting cells had a lower amount of N-acetylglucosamine. The ciliary interconnections, which have been considered to be a part of the glycocalyx, have larger amounts of galactose and mannose than the surface glycocalyx. These findings indicate that the sugar components may be closely related to the functional significance of the inner-ear glycocalyx and that the functional properties of the glycocalyx may differ between the sensory and the supporting cells of the vestibular end organs.

Carbohydrate Composition of the α-Subunit of Human Choriogonadotropin (hCGα) and the Free α Molecules Produced in Pregnancy: Most Free α and Some Combined hCGα Molecules Are Fucosylated

The carbohydrate compositions of pregnancy-derived hCG alpha (dissociated from intact hCG) and free alpha-subunit were analyzed using a combination of chemical analysis, lectin affinity chromatography, and glycosidase sensitivity. For direct compositional analysis, parallel samples were hydrolyzed in trifluoroacetic acid and analyzed for sialic acid and neutral sugars without prior derivatization. Separation of the monosaccharides was achieved by HPLC on a Dionex CarboPac column eluted at high pH, and the resolved monosaccharides were quantified by pulsed amperometric detection. The amounts of sugar that were found relative to peptide indicated the presence of two N-linked oligosaccharides per molecule on both hCG alpha and free alpha. Free alpha contained 2.5-fold higher amounts of sialic acid and galactose as well as a higher amount of N-acetylglucosamine than did hCG alpha. Free alpha also contained a 6-fold higher amount of fucose than did hCG alpha (1.2 vs. 0.2 residues of fucose/molecule). Serial fractionation of intact hCG alpha and free alpha molecules by lectin affinity chromatography indicated that virtually all of the hCG alpha-subunits contained at least one Concanavalin-A (Con-A)-binding site, whereas as many as 32% of the free alpha molecules could not bind to Con-A. Chromatography on Lens culinaris (Lch) resulted in 12% binding of hCG alpha and approximately 72% binding of free alpha (80-85% of the Con-A-bound free alpha and 47-48% of the Con-A-nonbound free alpha bound to Lch). Endoglycosidase-H (endo-H) treatment of hCG alpha released a portion of the oligosaccharides. The endo-H-released material appeared to be a monoantennary hybrid based on DEAE-binding properties and carbohydrate composition. In contrast to hCG alpha, free alpha was completely resistant to endo-H treatment. Incubation of endo-H-resistant hCG alpha with glycopeptidase-A resulted in the release of two components, which could be separated into monoantennary and biantennary fractions on the basis of size and charge. The collective data suggest that hCG alpha contains primarily monoantennary hybrid oligosaccharide structures and relatively little fucose. In contrast, free alpha contains primarily multiantennary oligosaccharide structures, and most of the free alpha molecules contain at least one oligosaccharide with fucose attached to the asparagine-linked N-acetylglucosamine residue.

Partial Chemical and Physical Characterization of Two Extracellular Polysaccharides Produced by Marine, Periphytic Pseudomonas sp. Strain NCMB 2021

The marine bacterium Pseudomonas sp. strain NCMB 2021, which can attach to solid, and especially hydrophobic, surfaces, elaborates two different extracellular polysaccharides in batch cultures. One (polysaccharide A) was produced only during exponential growth and contained glucose, galactose, glucuronic acid, and galacturonic acid in a molar ratio of 1.00:0.81:0.42:0.32. It produced viscous solutions, formed gels at high concentrations, and precipitated with several multivalent cations. The other (polysaccharide B) was released at the end of the exponential phase and in the stationary phase. It contained equimolar amounts of N-acetylglucosamine, 2-keto-3-deoxyoctulosonic acid, an unidentified 6-deoxyhexose, and also O-acetyl groups. Despite its high molecular weight (10 to 10 as judged by gel filtration), the polysaccharide produced aqueous solutions with very low viscosities and was also soluble in 90% aqueous phenol, 80% methanol, and 80% ethanol.

The capsular (K51) antigen of Escherichia coli 01:K51:H−, an O-acetylated poly-N-acetylglucosamine phosphate

The capsular K51 antigen of E.coli was isolated from a liquid culture of E.coli 01:K51:H− by Cetavlon precipitation. After purification it was obtained in a yield of about 80 mg/l. The polymer consisted of equimolar amounts of N-acetylglucosamine and phosphate and contained about 1.6 O-acetyl groups per N-acetylglucosamine residue. After de-O-acetylation it was resistant to periodate oxidation. Mild acid hydrolysis yielded N-acetylglucosamine-3-phosphate. With the aid of 13C- and 31P-NMR spectroscopy it was ascertained that the K51 antigen is a poly-α-1.3-N-acetylglucosamine phosphate, in which most of the hydroxyl groups at C4 and C6 of the N-acetylglucosamine residue are acetylated.

Methanolysis products of asparagine-linked N-acetylglucosamine and a new method for determination of N- and O-glycosidic N-acetylglucosamine in glycoproteins that contain asparagine-linked carbohydrates

Amino acid analyzer and gas chromatographic analyses of methanolysates of model compounds of N-glycosidic and O-glycosidic N-acetylglucosamine shows that the main product of N-glycosidic N-acetylglucosamine is glucosamine and that of O-glycosidic N-acetylglucosamine is methyl α-glucosaminide. The amounts of N-acetylglucosamine in glycoproteins determined by methanolysis followed by gas-liquid chromatography represent the amount of O-glycosidic N-acetylglucosamine. A new method was developed to determine O-glycosidic and N-glycosidic N-acetylglucosamine in glycoproteins. Glucosamine and methyl α-glucosaminide in the methanolysates of glycoproteins are determined in an amino acid analyzer using the buffer system that is usually used for analysis of basic amino acids in the two-column system. The contents of O- and N-glycosidic N-acetylglucosamine of Taka-amylase A, ovalbumin, and egg white and yolk flavoproteins determined by the method agree reasonably well with the values calculated from the carbohydrate structures or compositions of the compounds. Therefore, this method can be used to determine the number of N-glycosidically linked carbohydrate chains together with the amount of O-glycosidic N-acetylglucosamine in glycoproteins.

Hyphal growth ofHypomyces chlorinus Tul.: An autoradiographic study

The incorporation of the chitin precursor N-acetyl-D-(1-3H) glucosamine byH. chlorinus has been studied by light and electron microscopic autoradiography. Light microscopic autoradiography showed that the incorporation occured preferentially at the hyphal apex. Autoradiograms from electron microscopy were quantitatively evaluated to determine the relative radioactivity incorporation between the cell wall and cytoplasm: this showed that (3H) incorporation took place mainly in the plasmalemma-wall complex. However a small amount of N-acetyl glucosamine can enter into the cytoplasmic space and is then transported by endomembranes (Golgi apparatus-vesicles) to the plasmalemma-cell wall interface before polymerization.

Determination of N-acetylglucosamine in human immunoglobulin M and its fragments

1. The process of the splitting out of N-acetylglucosamine in the hydrolysis of myelomic IgM and its (Fc)5 and Fab fragments with 3 N HCl at 100, 110, and 120°C has been investigated as a function of the time. 2. The maximum splitting out of N-acetylglucosamine from IgM and the (Fc)5 and Fab fragments takes place at 110°C in 4, 6, and 8 h, respectively. 3. The amounts of N-acetylglucosamine found in the preparations studied agree well with the primary structure of the oligosaccharide groups of IgM.

CHEMICAL EFFECTS OF AN ENZYME PREPARATION FROM Cl. tertium O(H) TO Diplococcus pneumoniae TYPE X IV POLYSACCHARIDE

1. An enzyme preparation partially purified from Clostridium tertium O(H) has an ability to destruct a common antigen of poeumococcus Type X IV in human blood group substance as well as to destroy a common antigen of blood group substance in Diplococcus pneumoniae Type X IV polysaccharide.2. After enzymic digestion, N-acetylglucosamine, glucose, and galactose were found as simple sugar in dialyzable portion in which amount of N-acetylglucosamine was larger than other two sugars. In addition, small amount of several oligosaccharides composed of such three sugars were liberated in the same portion.3 A determinant structure of pneumococcus Type X IV antigenicity in human blood group substance was presumed to be β-N-acetyl-D-glucosaminoyl-1→3-D-galactose and also that of the common antigenicity of human blood group substance in Dip. pneumoniae Type X IV polysaccharide, to be the same disaccharide or a trisaccharide in which β-D-glucosyl-1 →4 was linked to nonreducing end of the disaccharide.

水産動物のキチン分解酵素に関する研究-IV

In the previous paper1) in this series, some properties of chitinolytic enzyme present in the stomach of fish, Lateolabrax japonicus, were investigated by measuring the reducing sugar produced. In the present study, chitinase activity was observed by decreasing substrate (Precipitated chitin, P. Ch), because the amount of P. Ch present in the reaction mixture could be measured turbidimetrically using a Hitachi EPU type spectrophotometer at 430 mμ. The amount of N-acetylglucosamine (NGA) was measured by the method described by REISSIG et. al.2) Oligo-NGA was estimated from the amount of NGA produced by the addition of β-glucosidase. Results obtained are as follows: 1) The amount of P. Ch present in the reaction mixture can be measured turbidimetrically (Fig. 1). 2) In the early stage of incubation, a considerable decrease of P. Ch is observed in amounts, although little amount of NGA is formed (Table 1). Judging from the result, it might be assumed that the most of the product obtaind in the early stage of the reaction is oligo-NGA. 3) Most of these oligo-NGA may be higher oligo-NGA which is not attacked by β-gluco-sidase (Table 1). 4) Optimum pH value and temperature on the chitinolytic action were almost similar to those estimated from the previous results of determination of the reducing sugar produced1). (Fig. 6, Fig. 8). 5) It is ascertained that the chitinolytic enzyme of fish consists of two or more different kinds of enzyme.