Concentrations Of N-acetylglucosamine Research Articles

Production, purification and characterization of chitinase from Micromonospora sp. AR17

N‐acetylglucosamine (NAG) is the monomer product of chitin, which has been widely used as a bioactive com‐ pound in applications such as anti‐tumor, anti‐microbial, and antioxidant activities. In production, biological processes using enzymes are preferable to chemicals due to environmental issues. This study aims to determine the activity, purity level, and molecular weight of purified chitinase from Micromonospora sp. AR17 determines the concentration of NAG produced by purified chitinase that has been characterized. Chitinase was produced by fermentation in colloidal chitin broth at 40 °C, pH 7, for 7 days, while chitinase activity was checked every 24 h. The optimal fermentation time was used to produce chitinase for a further purification step. Enzyme purification was carried out by ultrafiltration, ammonium sulfate precipitation, ion exchange chromatography (Q Sepharose Fast Flow), and gel filtration (Sephacryl S‐300). The purified enzyme was then char‐ acterized for optimum time, pH, and temperature to produce NAG. The results suggested that the fourth day was the optimal time for chitinase production, with chitinase activity of 0.0040 U/mL and a NAG concentration of 7.62 µg/mL. The purifica‐ tion step successfully increased the purity by 6.82 times with chitinase‐specific activity at 1.4648 U/mg. Production of NAG with purified chitinase produced a NAG concentration of 32.472 µg/mL with an incubation time of 30 min at 40 °C and pH 7.

Immobilization of Providencia stuartii Cells in Pumice Stone and Its Application for N-Acetylglucosamine Production.

SUMMARYResearch backgroundShrimp shells contain chitin that can be further processed into N-acetylglucosamine, which has been extensively used to treat joint damage. Providencia stuartii has a strong chitinolytic activity and may be utilized in the form of immobilized cells in repeated fermentation. Pumice is a porous and rigid stone that offers superior mechanical strength, making it suitable for immobilization.Experimental approachIn the research submerged fermentation with different pumice stone sizes and pumice stone/growth medium ratios (m/V) was carried out for 4 days at 37 °C and pH=7.0. The optimum pumice stone size and pumice stone/growth medium ratio (m/V) were used to determine the optimum fermentation cycle for the production of N-acetylglucosamine using immobilized P. stuartii.Results and conclusionsPumice stones of 1.0 cm×1.0 cm×1.0 cm and pumice stone/growth medium ratio of 1:5 were found to be the optimum conditions for successful immobilization of (90.0±1.6) % cells and production of (331.4±7.3) g/L N-acetylglucosamine. The highest N-acetylglucosamine concentration of (323.0±2.5) g/L was obtained in the first fermentation cycle, which then decreased and remained stable throughout the last three cycles.Novelty and scientific contributionP. stuartii, a strong chitinolytic bacterium previously isolated from rotten shrimp shells, was used for the first time in immobilized form to produce N-acetylglucosamine. The findings in this research showed the potential use of P. stuartii cells immobilized in pumice stone for continuous production of N-acetylglucosamine in repeated fermentation.

Immobilization of Providencia stuartii Cells in Papaya Trunk Wood for N-acetylglucosamine Production from Pennaeus vannamei Shrimp Shells

Highlight Research AbstractChitin is a natural compound found abundantly in shrimp shells. Chitin can be degraded to produce N-acetylglucosamine, which has wide applications in the food and pharmaceutical fields. Fermentation using chitinolytic microorganisms can be used to produce N-acetylglucosamine from shrimp shells’ chitin. One of the strong chitinolytic bacteria that was isolated from previous research was Providencia stuartii. To provide better stability and efficiency in fermentation, P. stuartii cells were immobilized using entrapment method in papaya trunk wood. The aims of this research were to determine the optimum papaya trunk wood size, ratio of papaya trunk wood and growth medium, as well as the optimum fermentation cycle to produce N-acetylglucosamine from P. vannamei shrimp shells using submerged fermentation method. The research used experimental method with treatment of different sizes of papaya trunk wood (1 x 1 x 1 cm3, 1.5 x 1.5 x 1.5 cm3, and 2 x 2 x 2 cm3), different ratio of papaya trunk wood and growth medium (1:10, 1:15 and 1:20), and 4 fermentation cycles. Results showed that papaya trunk wood with size of 1 x 1 x 1 cm3 and ratio (w/v) of 1:10 could immobilize 87.08±2.05% of P. stuartii cells and produce the highest N-acetylglucosamine concentration, which was 238177.78±3153.48 ppm. The highest N-acetylglucosamine production was obtained from first fermentation cycle and decreased over the last three cycles, but still produced high concentration of N-acetylglucosamine. Therefore, it is possible to perform continuous N-acetylglucosamine production from shrimp shells using P. stuartii cells immobilized in papaya trunk wood.

N-Acetylglucosamine Production by Repeated-Batch Fermentation Using Immobilized Semi-Purified Chitinase Enzyme on Agar

Chitinolytic mold, such as Mucor circinelloidesis can be utilized to produce chitinase enzyme for shrimp shell’s chitin hydrolysis into N-acetylglucosamine (NAG). For that purpose, entrapment of chitinase on agar as a carrier could be an alternative way to improve NAG production. This study aimed to investigate the stability of immobilized semi-purified chitinase on agar for multiple cycles fermentation to produce NAG. In this study, 0.6 mL of semi-purified chitinase enzyme was immobilized into 3% of agar matrices and tested for four fermentation cycles to obtain highest NAG concentration and good enzyme activity. The results indicate that the immobilized chitinase could be used for 6 hours fermentation or three fermentation cycles. The NAG concentration produced after three cycle were 1042.22 ± 16.20 ppm. Besides, the immobilized enzyme was considerably stable up to the third cycles with activity value of about 4.74 U/mL.Keywords: agar; immobilized;NAG; repeated fermentation

The influence of N-acetylglucosamine: Inducing Rhodosporidium paludigenum to enhance the inhibition of Penicillium expansum on pears

On the way to impel Rhodosporidium paludigenum to behave like a serviceable and beneficial biocontrol antagonist, it was found that adding different concentrations of N-acetylglucosamine (GlcNAc) during the culture can reduce the incidence of pears caused by Penicillium expansum. 0.1 % GlcNAc is an optimal concentration in the range of 0.001 %–2 %. R. paludigenum, cultivated and gathered from the medium with a final concentration of 0.1 % GlcNAc, declared the best impact on restraint on P. expansum on pears. This inhibitory effect is analogous to the consequence of spore germination experiments in vitro. The R. paludigenum gathered after GlcNAc induction, at the same time, displayed exceptional viability and vigour under different stress conditions, including sodium chloride (NaCl), calcofluor white (CFW), Congo red (CR) and sodium dodecyl sulfate (SDS). The enzyme activity and the relative gene expression levels of some antioxidant-related enzymes in R. paludigenum, including peroxisomal catalase (CAT), thioredoxin reductase (TrxR), glutathione peroxidase (GSH-PX), glutathione reductase (GR) and superoxide dismutase (SOD), were detected to be up-regulated in further testing. At the same time, the reactive oxygen species (ROS) remained at a low status.

Chitinase Activity ofSerratia marcescensPT-6 Cultured in Bioreactor with Various Aeration Rate

Chitin is a biopolymer composed of N-acetylglucosamine (NAG) monomers. Bacterial chitinase helps in bioconversion process of chitin from shrimp shell waste into NAG. This study aimed to determine the effect of aeration on chitinase activity, NAG concentration in medium, bacterial growth, and pH medium; to determine the optimum aeration on chitinase production ofSerratia marcescensPT-6. Chitinase was produced using a 1.5 L bioreactor at pH 7, 30°C, 200 rpm agitation speed, and variation of aeration rates (0.4, 0.6 and 0.8 vvm). Parameters examined included pH, bacterial growth (log CFU/ml), chitinase activity (U/ml), and NAG concentration in medium (µg/ml). Bacterial growth was measured by enumeration of bacterial colony in medium, while chitinase activity and NAG concentration were analyzed by colorimetric assay. Parameters was measured every 8 hours during 60 hours of incubation. Aeration rate showed a significant effect (P≤0.05) on chitinase activity and NAG concentration, but not significant (P≥0.05) on bacterial growth and pH medium. Optimum aeration rate on producing chitinase was 0.6 vvm with the highest chitinase activity of 0.0125 U/ml at 40 hours incubation. This study implies that optimization of bioreactor condition improved the production of chitinase byS. marcescensPT-6.

Determination of Optimal Fermentation Condition for N-acetylglucosamine Production Using Mucor circinelloides Extracellular Chitinase

This research aimed to determine the best fermentation condition, consists of pH, temperature, fermentation time and substrate concentration, in N-acetylglucosamine production from shrimp shells using crude extracellular chitinase obtained from Mucor circinelloides mould. The method used was experimental method with fermentation treatment of different pH (5, 6, 7, 8 and 9) and temperature (30, 40, 50, 60, 70 and 80°C). The optimal pH and temperature of fermentation obtained was used to determine the maximum substrate concentration (0.5, 1, 1.5 and 2%) and fermentation time (2, 4, 6 and 24 hours) to produce the highest concentration of N-acetylglucosamine. The optimal pH for fermentation was 8, with chitinase activity of 4.38±0.06 U/ml, while the optimal temperature was 50°C with enzyme activity of 5.42±0.06 U/ml. Substrate concentration and fermentation time affected the N-acetylglucosamine production. The optimal fermentation condition was obtained with substrate concentration of 1.5% and fermentation time of 2 hours resulted to N-acetyl Glucosamine concentration of 2195.83±15.14 ppm.

Investigation of the mechanisms of Escherichia coli cells sterilization by the application of an electric field

The inactivation of microorganisms by an electric field was the purpose of this study. It was found that the application of an electric field had a lethal effect on Escherichia coli cells suspended in brain heart infusion broth. Experimental results indicated that the survival rate of E. coli decreased when an electric field with an intensity of 3A was applied during a time equal to 30 min. Mechanisms involved in the sterilization process were discussed. The concentration of proteins and N-acetylglucosamine (NAG) liberated by the cells during the experiment was recorded. The results indicated that the concentration of liberated proteins was 2.21 µg/ml after 5 min, and this concentration increased twofold after 45 min (4.10 µg/ml). The initial concentration of NAG was 0.084 µg/ml. It increased to 0.1 µg/ml after 5 min, to 0.2 µg/ml after 10 min and to 1.3 µg/ml after 30 min.

Chitinase activity of Pseudomonas stutzeri PT5 in different fermentation condition

This study aimed to determine the incubation condition of Pseudomonas stutzeri PT5 in producing chitin degrading enzyme in various pH and temperatures; to compare the production of chitin degrading enzyme in chitin medium supplemented with additional nitrogen, carbon and a mixture of nitrogen and carbon sources and to observe the production of chitin degrading enzyme in 250 mL-shake flasks and 2 L-fermentor. The parameters tested during production were chitinase activity (U·mL-1) of culture supernatant and N-acetylglucosamine concentration (μg·mL-1) in the medium. The results showed that Pseudomonas stutzeri PT5 was able to produce the highest chitinase activity at pH 6 and temperature of 37 °C (0.024 U·mL-1). The addition of 0.1 % of ammonium phosphate and 0.1 % of maltose, increased the chitinase activity of Pseudomonas stutzeri PT5 by 3.24 and 8.08 folds, respectively, compared to the control. The addition of 0.1 % ammonium phosphate and 0.1 % maltose mixture to chitin medium resulted in the shorter time of chitinase production compared to the addition of sole nutrition. The production of chitinase using 2 L-fermentor shows that the highest chitinase activity produced by Pseudomonas stutzeri PT5 was reached at 1-day incubation (0.0283 U·mL-1), which was shorter than in 250 mL-shake flasks.

Conditions Inducing Excessive O-GlcNAcylation Inhibit BMP2-Induced Osteogenic Differentiation of C2C12 Cells

Hyperglycemic conditions in diabetic patients can affect various cellular functions, including the modulation of osteogenic differentiation. However, the molecular mechanisms by which hyperglycemia affects osteogenic differentiation are yet to be clarified. This study aimed to investigate whether the aberrant increase in protein O-linked-β-N-acetylglucosamine glycosylation (O-GlcNAcylation) contributes to the suppression of osteogenic differentiation due to hyperglycemia. To induce osteogenic differentiation, C2C12 cells were cultured in the presence of recombinant human bone morphogenetic protein 2 (BMP2). Excessive protein O-GlcNAcylation was induced by treating C2C12 cells with high glucose, glucosamine, or N-acetylglucosamine concentrations or by O-GlcNAc transferase (OGT) overexpression. The effect of O-GlcNAcylation on osteoblast differentiation was then confirmed by examining the expression levels of osteogenic marker gene mRNAs, activity of alkaline phosphatase, and transcriptional activity of Runx2, a critical transcription factor for osteoblast differentiation and bone formation. Cell treatment with high glucose, glucosamine or N-acetylglucosamine increased O-GlcNAcylation of Runx2 and the total levels of O-GlcNAcylated proteins, which led to a decrease in the transcriptional activity of Runx2, expression levels of osteogenic marker genes (Runx2, osterix, alkaline phosphatase, and type I collagen), and activity of alkaline phosphatase. These inhibitory effects were rescued by lowering protein O-GlcNAcylation levels by adding STO45849, an OGT inhibitor, or by overexpressing β-N-acetylglucosaminidase. Our findings suggest that excessive protein O-GlcNAcylation contributes to high glucose-suppressed osteogenic differentiation.

A Putative Polypeptide N-Acetylgalactosaminyltransferase/Williams-Beuren Syndrome Chromosome Region 17 (WBSCR17) Regulates Lamellipodium Formation and Macropinocytosis

We previously identified a novel polypeptide N-acetylgalactosaminyltransferase (GalNAc-T) gene, which is designated Williams-Beuren syndrome chromosome region 17 (WBSCR17) because it is located in the chromosomal flanking region of the Williams-Beuren syndrome deletion. Recent genome-scale analysis of HEK293T cells treated with a high concentration of N-acetylglucosamine (GlcNAc) demonstrated that WBSCR17 was one of the up-regulated genes possibly involved in endocytosis (Lau, K. S., Khan, S., and Dennis, J. W. (2008) Genome-scale identification of UDP-GlcNAc-dependent pathways. Proteomics 8, 3294-3302). To assess its roles, we first expressed recombinant WBSCR17 in COS7 cells and demonstrated that it was N-glycosylated and localized mainly in the Golgi apparatus, as is the case for the other GalNAc-Ts. Assay of recombinant WBSCR17 expressed in insect cells showed very low activity toward typical mucin peptide substrates. We then suppressed the expression of endogenous WBSCR17 in HEK293T cells using siRNAs and observed phenotypic changes of the knockdown cells with reduced lamellipodium formation, altered O-glycan profiles, and unusual accumulation of glycoconjugates in the late endosomes/lysosomes. Analyses of endocytic pathways revealed that macropinocytosis, but neither clathrin- nor caveolin-dependent endocytosis, was elevated in the knockdown cells. This was further supported by the findings that the overexpression of recombinant WBSCR17 stimulated lamellipodium formation, altered O-glycosylation, and inhibited macropinocytosis. WBSCR17 therefore plays important roles in lamellipodium formation and the regulation of macropinocytosis as well as lysosomes. Our study suggests that a subset of O-glycosylation produced by WBSCR17 controls dynamic membrane trafficking, probably between the cell surface and the late endosomes through macropinocytosis, in response to the nutrient concentration as exemplified by environmental GlcNAc.

Oral glucosamine increases expression of transforming growth factor β1 (TGFβ1) and connective tissue growth factor (CTGF) mRNA in rat cartilage and kidney: Implications for human efficacy and toxicity

Glucosamine is used for alleviating pain in osteoarthritis. Clinical trials have reported that glucosamine has equivocal efficacy. Glucosamine is also used in cell cultures to stimulate hexosamine flux and protein O-glycosylation, but at many-fold greater concentrations than those in human plasma following oral dosing. Lean Zucker rats were dosed orally for 6 weeks with glucosamine hydrochloride at doses (0–600 mg/kg/day) that produced peak serum concentrations of <1–35 μM, spanning the human exposure range. Relative expression of both TGFβ1 and CTGF mRNA were significantly increased up to 2.3-fold in liver, kidney and articular cartilage when evaluated 4 h after final dose. Apparent threshold serum glucosamine ( C max) concentration required to increase TGFβ1 expression in cartilage was 10–20 μM. These increases were associated with significant increases in UDP- N-acetylglucosamine concentrations suggesting increased hexosamine flux. Both TGFβ1 and CTGF are mediators of chondrocyte proliferation and cartilage repair. Study demonstrates that oral glucosamine doses that produce clinically relevant serum glucosamine concentrations can induce tissue TGFβ1 and CTGF expression in vivo and provides a mechanistic rationale for reported beneficial effects of glucosamine therapy. Induction of renal TGFβ1 and CTGF mRNA suggests that potential sclerotic side-effects may occur following consumption of potent glucosamine preparations.

Liquid chromatography–tandem mass spectrometry method for determination of N-acetylglucosamine concentration in human plasma

A simple, reliable and sensitive liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for quantification of N-acetylglucosamine in human plasma. Plasma samples were pretreated with acetonitrile for protein precipitation. The chromatographic separation was performed on Hypersil Silica column (150 mm × 2 mm, 5 μm). The deprotonated analyte ion was detected in negative ionization mode by multiple reaction monitoring mode. The mass transition pairs of m/ z 220.3 → 118.9 and m/ z 226.4 → 123.2 were used to detect N-acetylglucosamine and internal standard 13C 6- N-acetylglucosamine, respectively. The assay exhibited a linear range from 20 to 1280 ng/ml for N-acetylglucosamine in human plasma. Acceptable precision and accuracy were obtained for concentrations of the calibration standard and quality control. The validated method was successfully applied to analyze human plasma samples in a pharmacokinetic study.

The sugar phosphotransferase system of Streptomyces coelicolor is regulated by the GntR‐family regulator DasR and links N‐acetylglucosamine metabolism to the control of development

Members of the soil-dwelling, sporulating prokaryotic genus Streptomyces are indispensable for the recycling of the most abundant polysaccharides on earth (cellulose and chitin), and produce a wide range of antibiotics and industrial enzymes. How do these organisms sense the nutritional state of the environment, and what controls the signal for the switch to antibiotic production and morphological development? Here we show that high extracellular concentrations of N-acetylglucosamine, the monomer of chitin, prevent Streptomyces coelicolor progressing beyond the vegetative state, and that this effect is absent in a mutant defective of N-acetylglucosamine transport. We provide evidence that the signal is transmitted through the GntR-family regulator DasR, which controls the N-acetylglucosamine regulon, including the pts genes ptsH, ptsI and crr needed for uptake of N-acetylglucosamine. Deletion of dasR or the pts genes resulted in a bald phenotype. Binding of DasR to its target genes is abolished by glucosamine 6-phosphate, a central molecule in N-acetylglucosamine metabolism. Extracellular complementation experiments with many bld mutants showed that the dasR mutant is arrested at an early stage of the developmental programme, and does not fit in the previously described bld signalling cascade. Thus, for the first time we are able to directly link carbon (and nitrogen) metabolism to development, highlighting a novel type of metabolic regulator, which senses the nutritional state of the habitat, maintaining vegetative growth until changing circumstances trigger the switch to sporulation. Our work, and the model it suggests, provide new leads towards understanding how microorganisms time developmental commitment.

Glycoconjugate Expression in Normal Human Inferior Turbinate Mucosa: A Lectin Histochemical Study

Further study on the composition and concentration of normal nasal mucosal glycoconjugates is needed. A battery of ten lectins was used to stain six carbohydrates residing in the inferior turbinate mucosa of 21 healthy individuals. The concentrations of glycoconjugates of the glycocalyx exceeded that of epithelial goblet, ciliated, and basal cells, and also of mucous, serous, and mixed submucosal glands. Epithelial goblet cells and submucosal mucous glands contained relatively high concentrations of glycoconjugates, whereas in epithelial ciliated and basal cells, similar to serous and mixed submucosal glands, concentrations were scanty. The concentrations of N-acetylgalactosamine and galactose were higher in goblet cells than in mucous glands (p = 0.006, and p = 0.056, respectively). Differences in the concentrations of N-acetylglucosamine, fucose, mannose, and sialic acid were insignificant. The findings provide a baseline for comparison of the composition and concentration of carbohydrates of the normal and pathologic inferior turbinate mucosa.

Cuticle-induced endo/exoacting chitinase CHIT30 from Metarhizium anisopliae is encoded by an ortholog of the chi3 gene

The characterization of chitinase genes and enzymes is an important step toward global understanding of the chitinolytic system in entomopathogenic fungi. Chitinase CHIT30 from Metarhizium anisopliae var. anisopliae (strain E6) has both endo- and exochitinase activities and is a potential determinant of pathogenicity. Serum anti-CHIT30 specifically detected this chitinase amongst five isoenzymes shown in glycol-chitin activity gels. Chitinase CHIT30 secretion is upregulated by chitin, tick cuticle and low concentrations of N-acetylglucosamine (0.25%) and is downregulated by both high N-acetylglucosamine (1%) and glucose (1%) concentrations. Chitinase CHIT30 was produced at tick cuticle during fungal infection. The chi3 gene was assigned to code chitinase CHIT30 in M. anisopliae var. anisopliae.

Lec3 Chinese Hamster Ovary Mutants Lack UDP-N-acetylglucosamine 2-Epimerase Activity Because of Mutations in the Epimerase Domain of the Gne Gene

Lec3 Chinese hamster ovary (CHO) cell glycosylation mutants have a defect in sialic acid biosynthesis that is shown here to be reflected most sensitively in reduced polysialic acid (PSA) on neural cell adhesion molecules. To identify the genetic origin of the phenotype, genes encoding different factors required for sialic acid biosynthesis were transfected into Lec3 cells. Only a Gne cDNA encoding UDP-GlcNAc 2-epimerase:ManNAc kinase rescued PSA synthesis. In an in vitro UDP-GlcNAc 2-epimerase assay, Lec3 cells had no detectable UDP-GlcNAc 2-epimerase activity, and Lec3 cells grown in serum-free medium were essentially devoid of sialic acid on glycoproteins. The Lec3 phenotype was rescued by exogenously added N-acetylmannosamine or mannosamine but not by the same concentrations of N-acetylglucosamine, glucosamine, glucose, or mannose. Sequencing of CHO Gne cDNAs identified a nonsense (E35stop) and a missense (G135E) mutation, respectively, in two independent Lec3 mutants. The G135E Lec3 mutant transfected with a rat Gne cDNA had restored in vitro UDP-GlcNAc 2-epimerase activity and cell surface PSA expression. Both Lec3 mutants were similarly rescued with a CHO Gne cDNA and with CHO Gne encoding the known kinase-deficient D413K mutation. However, cDNAs encoding the known epimerase-deficient mutation H132A or the new Lec3 G135E Gne mutation did not rescue the Lec3 phenotype. The G135E Gne missense mutation is a novel mechanism for inactivating UDP-GlcNAc 2-epimerase activity. Lec3 mutants with no UDP-GlcNAc 2-epimerase activity represent sensitive hosts for characterizing disease-causing mutations in the human GNE gene that give rise to sialuria, hereditary inclusion body myopathy, and Nonaka myopathy.

Tn551-mediated insertional inactivation of the fmtB gene encoding a cell wall-associated protein abolishes methicillin resistance in Staphylococcus aureus.

A Tn551 insert in a gene termed fmtB was shown to reduce oxacillin as well as Triton X-100 resistance in highly methicillin-resistant Staphylococcus aureus (MRSA) COL. Backcrosses of fmtB::Tn551 into S. aureus COL and into two genetically distinct MRSA strains, KSA8 and NCTC10443, confirmed the linkage of fmtB::Tn551 with loss of oxacillin resistance. The fmtB gene codes for a protein of a deduced molecular mass of 263 kDa that contains 17 tandem repeats of 75 amino acids and a C-terminal LPXTG cell wall-sorting motif. Immunoblots with anti-FmtB antibodies confirmed its localization in the cell wall fraction. The fmtB gene was mapped downstream of the phosphoglucosamine mutase operon glmM which catalyses formation of glucosamine-1-phosphate. Oxacillin resistance was not restored in fmtB mutants by trans-complementation with fmtB. However, although GlmM production was not affected by fmtB inactivation, oxacillin resistance was increased in fmtB mutants by introducing a plasmid-borne glmM gene, presumably by GlmM overexpression. Interestingly, a similar phenotypic complementation was obtained in fmtB mutants by including substrate level concentrations of N-acetylglucosamine or glucosamine in the growth medium. Inactivation of the fmtB gene seems therefore to have an indirect effect on methicillin resistance which can be relieved by increasing the production of the cell wall precursor glucosamine-1-phosphate.

Isolation and partial characterization of giraffine, a lectin from the Mexican endemic alga Codium giraffa Silva

A lectin from the alga Codium giraffa, reported as an endemic species of Mexico, was purified by a combination of ammonium sulfate fractionation, affinity and anion exchange chromatographies. Giraffine, as we called this lectin, shares some characteristics with other lectins isolated from the same genus. It is a monomeric protein with a relative molecular weight of 17,800, as shown by native and SDS-PAGE and confirmed by mass spectrometry; its isoelectric point was 6.4 and it did not show multiple molecular forms. The agglutinating activity of this lectin was inhibited with low concentrations of N-acetylglucosamine and N-acetylgalactosamine and was independent of divalent cations.

Differences in N-acetyllactosamine synthesis between beta-1,4-galactosyltransferases I and V.

Unlike classical beta-1,4-galactosyltransferase (beta-1,4-GalT I), beta-1,4-GalT V (formerly IV**) has little activity towards 1 mM N-acetylglucosamine [Sato et al. (1998) Proc Natl Acad Sci USA 95:472-477]. The human beta-1,4-GalTs I and V were expressed individually in Sf-9 cells by transfection of the full coding sequences, and their N-acetyllactosamine synthetase activities were determined towards different N-acetylglucosamine concentrations. Kinetic studies using the cell homogenates as an enzyme source revealed that beta-1,4-GalTs I and V possess Km values of 0.6 mM and 33 mM towards N-acetylglucosamine, and of 48 microM and 41 microM towards UDP-Gal, respectively. No significant inhibition of N-acetyllactosamine synthesis with alpha-lactalbumin was observed for beta-1,4-GalT V but the significant inhibition with alpha-lactalbumin was observed for beta-1,4-GalT I.