Kinetics and metabolism of Bifidobacterium adolescentis MB 239 growing on glucose, galactose, lactose, and galactooligosaccharides

The kinetics and the metabolism of Bifidobacterium adolescentis MB 239 growing on galactooligosaccharides (GOS), lactose, galactose, and glucose were investigated. An unstructured unsegregated model for growth in batch cultures was developed, and kinetic parameters were calculated with a recursive algorithm. The growth rate and cellular yield were highest on galactose, followed by lactose and GOS, and were lowest on glucose. Lactate, acetate, and ethanol yields allowed the calculation of carbon fluxes toward fermentation products. Distributions between two- and three-carbon products were similar on all the carbohydrates (55 and 45%, respectively), but ethanol yields were different on glucose, GOS, lactose, and galactose, in decreasing order of production. Based on the stoichiometry of the fructose-6-phosphate shunt and on the carbon distribution among the products, the ATP yield was calculated. The highest yield was obtained on galactose, while the yields were 5, 8, and 25% lower on lactose, GOS, and glucose, respectively. Therefore, a correspondence among ethanol production, low ATP yields, and low biomass production was established, demonstrating that carbohydrate preferences may result from different distributions of carbon fluxes through the fermentative pathway. During the fermentation of a GOS mixture, substrate selectivity based on the degree of polymerization was exhibited, since lactose and the trisaccharide were the first to be consumed, while a delay was observed until longer oligosaccharides were utilized. Throughout the growth on both lactose and GOS, galactose accumulated in the cultural broth, suggesting that beta(1-4) galactosides can be hydrolyzed before they are taken up.     

Synthesis of potato starch sulfate and optimization of the reaction conditions

Potato starch sulfate was obtained by the reaction between potato starch and chlorosulfonic acid in pyridine. It was characterized by FT-IR and SEM. The reaction conditions were studied systematically, which included the volume ratio of pyridine to chlorosulfonic acid, reaction temperature and time in preparing sulfating agent process, and the ratio of starch mass to chlorosulfonic acid volume, reaction temperature and time in sulfation process. Meantime, the degree of substitution (DS) of each sample was determined via barium sulfate–glutin nephelometery method. By investigating the relationship between these conditions and DS, the optimal conditions were obtained with the maximum DS.     

Glycoengineering of therapeutic glycoproteins: in vitro galactosylation and sialylation of glycoproteins with terminal N-acetylglucosamine and galactose residues

Therapeutic glycoproteins produced in different host cells by recombinant DNA technology often contain terminal GlcNAc and Gal residues. Such glycoproteins clear rapidly from the serum as a consequence of binding to the mannose receptor and/or the asialoglycoprotein receptor in the liver. To increase the serum half-life of these glycoproteins, we carried out in vitro glycosylation experiments using TNFR-IgG, an immunoadhesin molecule, as a model therapeutic glycoprotein. TNFR-IgG is a disulfide-linked dimer of a polypeptide composed of the extracellular portion of the human type 1 (p55) tumor necrosis factor receptor (TNFR) fused to the hinge and Fc regions of the human IgG(1) heavy chain. This bivalent antibody-like molecule contains four N-glycosylation sites per polypeptide, three in the receptor portion and one in the Fc. The heterogeneous N-linked oligosaccharides of TNFR-IgG contain sialic acid (Sia), Gal, and GlcNAc as terminal sugar residues. To increase the level of terminal sialylation, we regalactosylated and/or resialylated TNFR-IgG using beta-1,4-galactosyltransferase (beta1,4GT) and/or alpha-2,3-sialyltransferase (alpha2,3ST). Treatment of TNFR-IgG with beta1,4GT and UDP-Gal, in the presence of MnCl(2), followed by MALDI-TOF-MS analysis of PNGase F-released N-glycans showed that the number of oligosaccharides with terminal GlcNAc residues was significantly decreased with a concomitant increase in the number of terminal Gal residues. Similar treatment of TNFR-IgG with alpha2,3ST and CMP-sialic acid (CMP-Sia), in the presence of MnCl(2), produced a molecule with an approximately 11% increase in the level of terminal sialylation but still contained oligosaccharides with terminal GlcNAc residues. When TNFR-IgG was treated with a combination of beta1,4GT and alpha2,3ST (either in a single step or in a stepwise fashion), the level of terminal sialylation was increased by approximately 20-23%. These results suggest that in vitro galactosylation and sialylation of therapeutic glycoproteins with terminal GlcNAc and Gal residues can be achieved in a single step, and the results are similar to those for the stepwise reaction. This type of in vitro glycosylation is applicable to other glycoproteins containing terminal GlcNAc and Gal residues and could prove to be useful in increasing the serum half-life of therapeutic glycoproteins.     

Preparation and important functional properties of water-soluble chitosan produced through Maillard reaction

The objective of this research was to improve the solubility of chitosan at neutral or basic pH using the Maillard-type reaction method. To prepare the water-soluble chitosans, various chitosans and saccharides were used under various operating conditions. Biological and physicochemical properties of the chitosan-saccharide derivatives were investigated as well. Results indicated that the solubility of modified chitosan is significantly greater than that of native chitosan, and the chitosan-maltose derivative remained soluble when the pH approached 10. Among chitosan-saccharide derivatives, the solubility of chitosan-fructose derivative was highest at 17.1 g/l. Considering yield, solubility and pH stability, the chitosan-glucosamine derivative was deemed the optimal water-soluble derivative. Compared with the acid-soluble chitosan, the chitosan-glucosamine derivative exhibited high chelating capacity for Zn(2+), Fe(2+) and Cu(2+) ions. Relatively high antibacterial activity against Escherichia coli and Staphylococcus aureus was noted for the chitosan-glucosamine derivative as compared with native chitosan. Results suggest that the water-soluble chitosan produced using the Maillard reaction may be a promising commercial substitute for acid-soluble chitosan.     

Cell surface labeling of erythrocyte glycoproteins by galactose oxidase and Mn++-catalyzed coupling reaction with methionine sulfone hydrazide.

Methionine sulfone hydrazide (MSH) was coupled to 6-aldehydosugars and the reaction was found to be catalytically enhanced by Mn⁺⁺ ion under physiological condition. The reaction was applied to label surface glycoproteins of erythrocytes with [³⁵S]-MSH after treating cells with galactose oxidase. The slab gel electrophoretic pattern of surface glycoproteins in sodium dodecylsulfate-polyacrylamide can be printed on autoradiogram. At least ten glycoproteins of normal human erythrocytes were printed; five (c, d, e, g, and k) were major bands, and of these four (c, d, e, and g) corresponded to “PAS I, II′, II, and III”. Others are hitherto unrecognized. Two intense bands each corresponds to c, and g, and two new bands, d′ and e′, were printed in desialylated fetal erythrocytes; intact fetal erythrocytes did not show significant label.     

The estimation of galactose, mannose and fucose in glycoproteins by radioisotope dilution

1. The principle of radioisotope dilution, as used previously for the estimation of mannose in egg albumin, was applied on a semi-micro scale to the estimation of fucose, mannose and galactose in some glycoproteins. The sugars were separated by partition chromatography on columns of Celite 545. 2. The release of mannose from egg albumin in 2n-hydrochloric acid at 100 degrees after various times was determined by the radioisotope-dilution method and found to have a half-time of 7min. 3. The destruction of mannose in 2n-hydrochloric acid after 3hr. at 100 degrees was found to be small if air was excluded. The destruction was slightly increased by the presence of lysozyme containing tryptophan in an amount equimolar with the mannose. The same amount of free tryptophan caused considerable loss of mannose. 4. Analytical values are reported for the non-amino sugar contents of egg albumin, rabbit gamma-globulin and some samples of blood-group-specific substances. The values found were similar to the most reliable estimates published previously.     

Synthesis of prebiotic galactooligosaccharides from lactose using bifidobacterial β-galactosidase (BbgIV) immobilised on DEAE-Cellulose,Q-Sepharose and amino-ethyl agarose

The bifidobacterial β-galactosidase BbgIV was immobilised on DEAE-Cellulose and Q-Sepharose via ionic binding and on amino-ethyl- and glyoxal-agarose via covalent attachment, and was then used to catalyse the synthesis of galactooligosaccharides (GOS). The immobilisation yield exceeded 90% using ionic binding, while it was low using amino-ethyl agarose (25–28%) and very low using glyoxal agarose (<3%). This was due to the mild conditions and absence of chemical reagents in ionic binding, compared to covalent attachment. The maximum GOS yield obtained using DEAE-Cellulose and Q-Sepharose was similar to that obtained using free BbgIV (49–53%), indicating the absence of diffusion limitation and mass transfer issues. For amino-ethyl agarose, however, the GOS yield obtained was lower (42–44%) compared to that obtained using free BbgIV. All the supports tried significantly (P < 0.05) increased the BbgIV operational stability and the GOS synthesis productivity up to 55 °C. Besides, six successive GOS synthesis batches were performed using BbgIV immobilised on Q-Sepharose; all resulted in similar GOS yields, indicating the possibility of developing a robust synthesis process. Overall, the GOS synthesis operation performance using BbgIV was improved by immobilising the enzyme onto solid supports, in particular on Q-Sepharose.     

External labeling of human erythrocyte glycoproteins. Studies with galactose oxidase and fluorography.

Glycoproteins of the human erythrocyte membrane were labeled with tritiated sodium borohydride after oxidation of terminal galactosyl and N-acetylgalactosaminyl residues with galactose oxidase. After separation of the polypeptides on polyacrylamide slab gels, a scintillator was introduced into the gel, and the radioactive proteins were visualed by autoradiography (fluorography). The following results were obtained. (a) The erythrocyte membrane contains at least 20 glycoproteins, many of which are minor components. (b) The carbohydrate of all the labeled glycoproteins is exposed only to the outside, since no additional glycoproteins can be labeled in isolated unsealed ghosts. (c) The membrane contains two major groups of glycoproteins. The first group of proteins contains sialic acids linked to the penultimate galactosyl/N-acetylgalactosaminyl residues, which are efficiently labeled only after pretreatment with neuraminidase. The second group has terminal galactosyl/N-acetylgalactosaminyl residues which can be easily labeled without neuraminidase treatment. The glycoproteins from fetal erythrocytes all belong to the first group, whereas only five glycoproteins of erythrocytes from adults belong. (d) Trypsin cleaves the proteins containing sialic acids, and fragments containing carbohydrate remain tightly bound and exposed in the membrane. (e) Pronase cleaves Band 3 in addition to the sialic acid containing glycoproteins, but most of the glycoproteins still remain unmodified in the membrane. (f) No difference is seen between membrane glycoproteins from cells of different ABH blood groups.     

Induction of GM-CSF production of macrophages by advanced glycation end products of the Maillard reaction

We previously reported that AGEs can induce macrophage growth. In this paper, we examined whether advanced glycation end products (AGE) of protein induced GM-CSF production of macrophages. AGE of bovine serum albumin markedly stimulated not only the expression of GM-CSF mRNA, but also GM-CSF secretion in macrophage supernatant. Thus GM-CSF is suggested to be an endogenous signal for macrophage growth induction by AGEs.     

Pyridinium-carbaldehyde: active Maillard reaction product from the reaction of hexoses with lysine residues

Besides the formation of the aminotriazine N6-[4-(3-amino-1,2,4-triazin-5-yl)-2,3-dihydroxybutyl]-L-lysine, the reaction of [1-13C]D-glucose with lysine and aminoguanidine leads to the generation of 6-[2-([[amino(imino)methyl]hydrazono]methyl)pyridinium-1-yl]-L-norleucine (14-13C1). The dideoxyosone N6-(2,3-dihydroxy-5,6-dioxohexyl)-L-lysine was shown to be a precursor in the formation of 14-13C1, which proceeds via the reactive carbonyl intermediate 6-(2-formylpyridinium-1-yl)-L-norleucine (13-13C1). In order to study the reactivity of 13-13C1, the model compound 1-butyl-2-formylpyridinium (18) was prepared in a two-step procedure starting from 2-pyridinemethanol. The reaction of the pyridinium-carbaldehyde 18 with L-lysine yielded the Strecker analogous degradation product 2-(aminomethyl)-1-butylpyridinium and another compound, which was shown to be as 1-butyl-2-[(2-oxopiperidin-3-ylidene)methyl]pyridinium. Reaction of 18 with the C-H acidic 4-hydroxy-5-methylfuran-3(2H)-one leads to the formation of the condensation product 1-butyl-2-[hydroxy-(4-hydroxy-5-methyl-3-oxofuran-2(3H)-ylidene)methyl]-pyridinium.     

Development of patatin knockdown potato tubers using RNA interference (RNAi) technology,for the production of human-therapeutic glycoproteins

Background  

Patatins encoded by a multi-gene family are one of the major storage glycoproteins in potato tubers. Potato tubers have recently emerged as bioreactors for the production of human therapeutic glycoproteins (vaccines). Increasing the yield of recombinant proteins, targeting the produced proteins to specific cellular compartments, and diminishing expensive protein purification steps are important research goals in plant biotechnology. In the present study, potato patatins were eliminated almost completely via RNA interference (RNAi) technology to develop potato tubers as a more efficient protein expression system. The gene silencing effect of patatins in the transgenic potato plants was examined at individual isoform levels.     

On the reaction of potato starch with isocyanates

The reactions of potato starch in aqueous suspension with some isocyanates were investigated. In contrast to previously reported results we found that no starch carbamates were formed but only the corresponding urea compounds. These urea compounds were physically bound to the potato starch and could be washed from the starch by several organic solvents.     

Separation of bovine heart galactose lectin from endogenous glycoproteins co-purified with the lectin during affinity chromatography

During affinity chromatographic purification of bovine heart 14 kDa galactose-binding lectin (galectin 1) on lactose-Sepharose, several high molecular weight non-lectin glycoproteins were co-purified with the lectin. Glycoprotein binding to the affinity matrix was neither hydrophobic nor ionic, but galactose-dependent since lactose abolished binding. Purification of galectin from the co-purified glycoproteins by affinity electrophoresis in presence of the specific sugar lactose increased agglutination activity about 65-fold, indicating that a complex containing galectin molecules bound sugar specifically to endogenous glycoproteins with sugar binding sites still available had been retained on lactose-Sepharose.     

Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose

A significant percentage of eukaryotic proteins contain posttranslationalmodifications, including glycosylation, which are required forbiological function. However, the understanding of the structure–functionrelationships of N-glycans has lagged significantly due to themicroheterogeneity of glycosylation in mammalian produced proteins.Recently we reported on the cellular engineering of yeast toreplicate human N-glycosylation for the production of glycoproteins.Here we report the engineering of an artificial glycosylationpathway in Pichia pastoris blocked in dolichol oligosaccharideassembly. The PpALG3 gene encoding Dol-P-Man:Man₅GlcNAc₂-PP-Dolmannosyltransferase was deleted in a strain that was previouslyengineered to produce hybrid GlcNAcMan₅GlcNAc₂ human N-glycans.Employing this approach, combined with the use of combinatorialgenetic libraries, we engineered P. pastoris strains that synthesizecomplex GlcNAc₂Man₃GlcNAc₂ N-glycans with striking homogeneity.Furthermore, through expression of a Golgi-localized fusionprotein comprising UDP-glucose 4-epimerase and ß-1,4-galactosyltransferase activities we demonstrate that this structure isa substrate for highly efficient in vivo galactose addition.Taken together, these data demonstrate that the artificial invivo glycoengineering of yeast represents a major advance inthe production of glycoproteins and will emerge as a practicaltool to systematically elucidate the structure–functionrelationship of N-glycans. ¹ These authors contributed equally to this work. ² To whom correspondence should be addressed; e-mail: swildt{at}glycofi.com     

Thermal luminescence spectra of polyamides and their Maillard reaction with reducing sugars

Thermal luminescence (TL) spectra of polyamides were measured with a Fourier‐transform chemiluminescence spectrometer to elucidate the emission mechanism. A TL band of ε‐polylysine with a peak at 542 nm observed at 403 K was assigned to the emission due to the interaction of the –CO–NH– group with oxygen molecules by comparison with nylon‐6, polyglycine, and polyalanine. When the sample was kept at 453 K, the intensity of the TL band decreased and the wavelength of the peak shifted to 602 nm, which was assigned to the emission due to the interaction of the NH₂ group on the side chain with oxygen molecules by comparison with monomeric lysine. A weak emission with a peak at 668 nm was assigned to the advanced glycosylation end products (AGEs) yielded by the Maillard reaction with a catalytic amount of water. To understand this reaction and to examine the TL emission of AGEs, we measured TL spectra of mixtures of polylysine and reducing sugars such as glucose, maltose, lactose, and dextrin. The minimum temperature for TL emission, wavelength of the peak and the relative intensities of the TL emission were found to depend on the size of the sugars. Copyright © 2011 John Wiley & Sons, Ltd.     

Cell surface glycoproteins of rat lymphocytes. I. Correlation of mitogenic stimulation by periodate or neuraminidase and galactose oxidase with the presence of papain-sensitive glycoproteins.

Oxidation of viable rat lymph node lymphocytes with either periodate or a combination of neuraminidase and galactose oxidase (NGO), followed by reduction with tritiated sodium borohydride, labels similar sets of cell-surface molecules as assessed by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Periodate and NGO induce blast transformation of lymph node lymphocytes (oxidative mitogenesis), and borohydride reduction inhibits the proliferative response. Thus, it is inferred that some or all of the glycoproteins that are labeled with tritiated borohydride may be involved in mediating the stimulation caused by the oxidizing agents. Treatment of lymph node lymphocytes with 5 units/ml papain abolishes the response to periodate or NGO but does not significantly affect the response to Con A. At the same time, papain treatment eliminates the labeled bands representing six high m.w. glycoproteins (175,000, 170,000, 160,000, 155,000, 100,000, and 70,000 daltons). No significant effect is seen on the labeling of the other components visualized in the slab gels. The results implicate the subset of six high m.w. papain-sensitive sialoglycoproteins in mediating oxidative mitogenesis of rat lymph node lymphocytes.     

Modulation of antibody galactosylation through feeding of uridine, manganese chloride, and galactose

Through process transfer and optimization for increased antibody production to 3 g/L for a GS-CHO cell line, an undesirable drop in antibody Fc galactosylation was observed. Uridine (U), manganese chloride (M), and galactose (G), constituents involved in the intracellular galactosylation process, were evaluated in 2-L bioreactors for their potential to specifically increase antibody galactosylation. These components were placed in the feed medium at proportionally increasing concentrations from 0 to 20 × UMG, where a 1× concentration of U was 1 mM, a 1× concentration of M was 0.002 mM, and a 1× concentration of G was 5 mM. Antibody galactosylation increased rapidly from 3% at 0× UMG up to 21% at 8× UMG and then more slowly to 23% at 20× UMG. The increase was primarily due to a shift from G0F to G1F, with minimal impact on other glycoforms or product quality attributes. Cell culture performance was largely not impacted by addition of up to 20× UMG except for suppression of glucose consumption and lactate production at 16 and 20× UMG and a slight drop in antibody concentration at 20× UMG. Higher accumulation of free galactose in the medium was observed at 8× UMG and above, coincident with achieving the plateau of maximal galactosylation. A concentration of 4× UMG resulted in achieving the target of 18% galactosylation at 2-L scale, a result that was reproduced in a 1,000-L run. Follow-up studies to evaluate the addition of each component individually up to 12× concentration revealed that the effect was synergistic; the combination of all three components gave a higher level of galactosylation than addition of the each effect independently. The approach was found generally useful since a second cell line responded similarly, with an increase in galactosylation from 5% to 29% from 0 to 8× UMG and no further increase or impact on culture performance up to 12× UMG. These results demonstrate a useful approach to provide exact and specific control of antibody galactosylation through manipulation of the concentrations of uridine, manganese chloride, and galactose in the cell culture medium.