Characterization of the interaction between hydroxypropyl guar galactomannan and galectin-3

Multivalent galactose ligands have been proposed for selective targeting of carbohydrate-binding proteins on epithelial cell surfaces, both in normal and pathological conditions. One cellular partner is galectin-3, a β-galactoside-binding protein present on many epithelial linings, such as those of the ocular surface. In this study, we investigated the ability of hydroxypropyl guar galactomannan (HPGG) to bind recombinant galectin-3 and to target the apical surface of differentiated human corneal keratinocytes. Pull-down and slot-blot assays demonstrated that fluorescence-labeled HPGG bound recombinant galectin-3 through a galactose-dependent mechanism. In contrast, no binding of HPGG could be detected towards recombinant galectin-8 or -9. In a cell culture system, HPGG bound weakly to biotinylated cell surface corneal isolates containing endogenous galectin-3, and incubation of HPGG with corneal keratinocytes in culture resulted in discrete, galactose-independent, binding to the cell surface. Moreover, HPGG failed to elute the biological counter-receptor MUC16 from galectin-3 affinity columns. We conclude that HPGG binds galectin-3 through the conventional carbohydrate-recognition domain in vitro, but not in a biological system, suggesting that endogenous carbohydrate ligands on epithelial cell surface glycocalyces impair HPGG biorecognition.     

Production of oligoglucuronans by enzymatic depolymerization of nascent glucuronan

An original bioreactor process for production of oligoglucuronans was developed using the Sinorhizobium meliloti M5N1CS strain that produces glucuronan. This anionic homopolysaccharide was composed of beta-D-(1,4)-glucopyranosyluronic residues variably O-acetylated at C-3 and/or C-2 positions according to culture conditions. It was depolymerized during its biosynthesis by addition of a fungal glucuronan lyase activity in broths. After purification by tangential ultrafiltration and low-pressure liquid chromatography, (1)H NMR and ESI-Q/TOF-MS characterized the poly- and oligoglucuronic acid fractions. This enzymatic bioreactor strategy authorized the production in gram quantity of an unsaturated and no acetylated oligoglucuronan with a degree of polymerization of 3.     

Interaction properties of d-galactose-depleted guar galactomannan samples

The interaction of galactose-depleted guar galactomannan with agarose has been studied by optical rotation, and with xanthan using an Instron Materials Tester and a Rheometrics Mechanical Spectrometer. Samples were prepared by treatment of guar galactomannan with highly purified α-d-galactosidase. Modified guar galactomannans with a d-galactose content of 19–25%, in admixture with agarose, showed similar optical rotation changes on heating and cooling as did mixtures of agarose and carob galactomannan (23% d-galactose content). Modified samples with 13–16% d-galactose, in the presence of agarose, showed more pronounced optical rotation changes on heating and cooling, but samples with less than 13% d-galactose were only sparingly soluble even on autoclaving. The degree of interaction of galactose-depleted guar galactomannan samples with xanthan, as measured rheologically, increased as the d-galactose content decreased, paralleling the optical rotation changes with galactomannan/agarose mixtures. In the presence of xanthan, samples with a d-galactose content of 25% or less formed firm rubbery gels.     

Rheological properties of guar galactomannan solutions during hydrolysis with galactomannanase and alpha-galactosidase enzyme mixtures

Guar galactomannan, a naturally occurring polysaccharide, is susceptible to hydrolysis by three enzymes: beta-mannosidase, beta-mannanase, and alpha-galactosidase. The beta-mannosidase cleaves a single mannose unit from the nonreducing end of the guar molecule, the beta-mannanase cleaves interior glycosidic bonds between adjacent mannose units, and the alpha-galactosidase cleaves the galactose side branches off the guar. In this study, hydrolysis of guar solutions using hyperthermopilic versions of these enzymes together in different proportions and combinations are examined. The enzymatic reactions are carried out in situ in a rheometer, and the progress of the reaction is monitored through measuring the variation in zero shear viscosity. We find the presence of alpha-galactosidase to affect the action of both beta-mannanase and beta-mannosidase with respect to solution rheology. However, this effect is more pronounced when the alpha-galactosidase and beta-mannanase or beta-mannosidase enzymes were added sequentially rather than simultaneously. This likely is the result of debranching of the guar, which facilitates attack on beta-1,4-linkages by both the beta-mannanase and the beta-mannosidase enzymes and increases hydrolytic rates by the individual enzymes. A rheology-based kinetic model is developed to estimate the reaction rate constants and interpret synergistic effects of multiple enzyme contributions. The model fits the experimental data well and reveals that both the native and the debranched guar have the same activation energy for beta-mannanase action, although debranching considerably increases the frequency of enzyme-guar interactions.     

Viscosity-molecular weight relationships,intrinsic chain flexibility,and dynamic solution properties of guar galactomannan

Molecular theories of the viscosity of dilute and more concentrated solutions of random-coil polymers have been applied to five molecular-weight fractions of guar galactomannan. The variation in intrinsic viscosity ([η]; dL.g^?1) with molecular weight (M_r) followed the relationship [η] = 3.8 × 10^?4) M_r^0.723, consistent with random-coil behaviour. The intrinsic stiffness of the galactomannan backbone was estimated by evaluating the “characteristic ratio” C_∝, which is ～ 12.6 and agrees well with results for carboxymethylcellulose, which has a closely similar, β-(1→4)-linked polymer backbone. At intermediate concentrations (c), up to specific viscosities of ～ 10, η_sp was proportional to c^1.3, while at higher concentrations, η_sp was ～c^5.1. This exponent is higher than is usually observed for polymers interacting purely by physical entanglement, and is evidence for more specific polymer-polymer interactions (“hyperentanglements”). The concentration dependence and the time-scale of entanglement coupling have been monitored by both steady-shear and oscillatory measurements, and a generality of response has been demonstrated.     

Assessment of biochemical methods to detect enzymatic depolymerization of polysaccharides

Biochemical methods for detection of depolymerisation were compared. Currently used methods such as reducing sugars assays, double bond monitoring or molecular weight determination were tested to follow the kinetic of depolymerization with different enzyme/polysaccharide combinations. The range of concentrations of different enzymes allowed us to identify the most sensitive and appropriate method to detect polysaccharide degradation. Reducing sugars assays are quantitative, sensitive and usable with all kind of polysaccharide but some compounds may interfere with them. When the polysaccharide is charged, agarose gel electrophoresis, although being a qualitative assay is as sensitive as high performance size exclusion chromatography analysis, easy to handle, high-throughput and this is preferred.     

Analysis of proteins associated with growth of Bacteroides ovatus on the branched galactomannan guar gum.

Bacteroides ovatus, a gram-negative obligate anaerobe from the human colon, can ferment the branched galactomannan guar gum. Previously, three enzymes involved in guar gum breakdown were characterized. The expression of these enzymes appeared to be regulated; i.e., specific activities were higher in extracts from bacteria grown on guar gum than in extracts from bacteria grown on the monosaccharide constituents of guar gum, mannose and galactose. In the present study, we used two-dimensional gel analysis to determine the total number of B. ovatus proteins enhanced during growth on guar gum. Twelve soluble proteins and 20 membrane proteins were expressed at higher levels in guar gum-grown cells than in galactose-grown cells. An unexpected finding was that the expression of the two galactomannanases was induced by glucose as well as guar gum. Three other proteins, one membrane protein and two soluble proteins, had this same expression pattern. The remainder of the guar gum-associated proteins seen on two-dimensional gels and the guar gum-associated alpha-galactosidase were induced in cells grown on guar gum but not in cells grown on glucose. Two transposon-generated mutants (M-5 and M-7) that could not grow on guar gum were isolated. Both mutants still expressed the galactomannanases and the alpha-galactosidase. They also still expressed all of the guar gum-associated proteins that could be detected in two-dimensional gels of glucose-grown or galactose-grown cells. A second transposon insertion that suppressed the guar gum-negative phenotype of M-5 was isolated and characterized. The characteristics of this suppressor mutant indicated that the original transposon insertion was probably in a regulatory locus.     

Analysis of proteins associated with growth of Bacteroides ovatus on the branched galactomannan guar gum.

Bacteroides ovatus, a gram-negative obligate anaerobe from the human colon, can ferment the branched galactomannan guar gum. Previously, three enzymes involved in guar gum breakdown were characterized. The expression of these enzymes appeared to be regulated; i.e., specific activities were higher in extracts from bacteria grown on guar gum than in extracts from bacteria grown on the monosaccharide constituents of guar gum, mannose and galactose. In the present study, we used two-dimensional gel analysis to determine the total number of B. ovatus proteins enhanced during growth on guar gum. Twelve soluble proteins and 20 membrane proteins were expressed at higher levels in guar gum-grown cells than in galactose-grown cells. An unexpected finding was that the expression of the two galactomannanases was induced by glucose as well as guar gum. Three other proteins, one membrane protein and two soluble proteins, had this same expression pattern. The remainder of the guar gum-associated proteins seen on two-dimensional gels and the guar gum-associated alpha-galactosidase were induced in cells grown on guar gum but not in cells grown on glucose. Two transposon-generated mutants (M-5 and M-7) that could not grow on guar gum were isolated. Both mutants still expressed the galactomannanases and the alpha-galactosidase. They also still expressed all of the guar gum-associated proteins that could be detected in two-dimensional gels of glucose-grown or galactose-grown cells. A second transposon insertion that suppressed the guar gum-negative phenotype of M-5 was isolated and characterized. The characteristics of this suppressor mutant indicated that the original transposon insertion was probably in a regulatory locus.     

Rapid isolation of human plasma anti-alpha-galactoside antibody using sugar-specific binding to guar galactomannan or agarose.

A method of purifying the naturally occurring antibody to alpha-galactoside moiety (anti-alpha-Gal) in human plasma by a single-step affinity chromatography on cross-linked guar galactomannan (CLGG) or agarose (Sepharose 4B) is described. IgG nature of the two preparations, as revealed by agar gel diffusion, as well as their preference for alpha-anomer of galactose, as revealed in inhibition of their agglutination of trypsinized rabbit erythrocytes by sugars, identified them with anti-alpha-Gal. The antibody binding capacity of Sepharose 4B was only a third of that of CLGG. Both gels showed similar dependence on ionic strength for binding. The pH optimum for binding of anti-alpha-Gal to CLGG was 8.0. Significantly anti-alpha-Gal binding to Sepharose was unaffected by CNBr activation and ligand coupling to the gel, thus warning that contaminating plasma could introduce artifacts in agarose-based chromatography of human tissue biomolecules.     

Kinetics of actin elongation and depolymerization at the pointed end

We measured the rate of elongation at the pointed filament end with increasing concentrations of G-actin [J(c) function] using villin-capped actin filaments of very small (actin/villin = 3, VA3) and relatively large size (actin/villin = 18, VA18) as nuclei for elongation. The measurements were made under physiological conditions in the presence of both Mg2+ and K+. In both cases the J(c) function was nonlinear. In contrast to the barbed filament end, however, the slope of the J(c) function sharply decreased rather than increased when the monomer concentration was lowered to concentrations near and below the critical concentration c infinity. At zero monomer concentration, depolymerization at the pointed end was very slow with a rate constant of 0.02 s-1 for VA18. When VA3 was used, the nonlinearity of the J(c) function was greatly exaggerated, and the nuclei elongated at actin concentrations below the independently measured critical concentration for the pointed end. This is consistent with and confirms our previous finding [Weber, A., Northrop, J., Bishop, M. F., Ferrone, F. A., & Mooseker, M. S. (1987) Biochemistry (preceding paper in the issue)] that at an actin-villin ratio of 3 a significant fraction of the villin is free and that a series of steady states exist between villin-actin complexes of increasing size and G-actin. The rate constant of elongation seems to increase with increasing G-actin concentrations because of increasing conversion of free villin into villin-actin oligomers during the period of the measurement of the initial elongation rate. The villin-actin oligomers have a much higher rate constant of actin binding than does free villin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interconversion and translocation of free sugars during galactomannan utilization in germinating guar (Cyamopsis tetragonoloba) seed

With the onset of the degradation of galactomannan, the galactose and mannose levels increased in the endosperm. The hydrolysis of galactomannan was more or less complete within the first 3 days of germination. In the cotyledons, sucrose was the predominant free sugar during the period of rapid galactomannan hydrolysis and reducing sugars (glucose + fructose) were present in only 10–20% proportion. The level of soluble acid invertase activity was in the order of embryonic axis > endosperm > cotyledons. On the basis of (a) absence of galactose and mannose, (b) high proportion of sucrose, (c) very fast conversion of [¹⁴C]glucose and [¹⁴C]mannose to [¹⁴C]sucrose and (d) very low levels of both soluble and bound invertases in cotyledons, we conclude that there is an active synthesis of sucrose in this tissue where disaccharide seems to be least hydrolysed during the period of galactomannan mobilization. A rapid hydrolysis of galactomannan in endosperm during early germination resulted in the synthesis of some starch, as a temporary reserve, in cotyledons. When the cotyledons entered the phase of first leaf formation, cotyledonary sucrose was hydrolysed giving rise to invert sugars. In the embryonic axis, the increase in the ratio of reducing sugars to sucrose coupled with a higher level of invertase, compared with sucrose-UDP glucosyl transferase, indicated that free sugars from the cotyledons are translocated to the embryonic axis as sucrose.     

Kinetics of enzymatic degradation of cyanide

CYANIDASE(@) is a new enzyme preparation capable of degrading cyanide in industrial wastewaters to ammonia and formate in an apparently one-step reaction, down to very low concentrations. This enzyme has both a high selectivity and affinity toward cyanide. A granular form of the biocatalyst was used in a recirculation fixed bed reactor in order to characterize the new biocatalyst with respect to pH, ionic strength, common ions normally present in wastewaters, mass transfer effects, and temperature. Long term stability was investigated. The kinetics of the enzymatic degradation of cyanide were studied in a batch reactor using the powdered immobilized enzyme preparation and modeled using a simple Michaelis-Menten equation.     

Microbial system for polysaccharide depolymerization: enzymatic route for xanthan depolymerization by Bacillus sp. strain GL1.

An enzymatic route for the depolymerization of a heteropolysaccharide (xanthan) in Bacillus sp. strain GL1, which was closely related to Brevibacillus thermoruber, was determined by analyzing the structures of xanthan depolymerization products. The bacterium produces extracellular xanthan lyase catalyzing the cleavage of the glycosidic bond between pyruvylated mannosyl and glucuronyl residues in xanthan side chains (W. Hashimoto et al., Appl. Environ. Microbiol. 64:3765-3768, 1998). The modified xanthan after the lyase reaction was then depolymerized by extracellular beta-D-glucanase to a tetrasaccharide, without the terminal mannosyl residue of the side chain in a pentasaccharide, a repeating unit of xanthan. The tetrasaccharide was taken into cells and converted to a trisaccharide (unsaturated glucuronyl-acetylated mannosyl-glucose) by beta-D-glucosidase. The trisaccharide was then converted to the unsaturated glucuronic acid and a disaccharide (mannosyl-glucose) by unsaturated glucuronyl hydrolase. Finally, the disaccharide was hydrolyzed to mannose and glucose by alpha-D-mannosidase. This is the first complete report on xanthan depolymerization by bacteria. Novel beta-D-glucanase, one of the five enzymes involved in the depolymerization route, was purified from the culture fluid. This enzyme was a homodimer with a subunit molecular mass of 173 kDa and was most active at pH 6.0 and 45 degrees C. The enzyme specifically acted on xanthan after treatment with xanthan lyase and released the tetrasaccharide.     

Chitin/chitosan transformation by thermo-mechano-chemical treatment including characterization by enzymatic depolymerization

Chitosan, the deacetylated derivative of chitin, was until recently produced by hydrolysis in 50% (w/v) NaOH. Application of thermo-mechano-chemical technology to chitin deacetylation was evaluated as an alternative method of chitosan production. This process consists of a cascade reactor unit operating under reduced alkaline conditions of 10% (w/v) NaOH. Prior mercerization of chitin at 4 degrees C for 24 h was required for high deacetylation yields. Sudden decompression of the aqueous alkaline suspension of mercerized chitin resulted in near complete deacetylation of chitin. Reactor residence time was 90 s at 230 degrees C prior to decompression. The chitosan produced was characterized by elemental analysis, (13)C-NMR and enzymatic depolymerization. Enzymatic determination of the degree of acetylation of chitin/chitosan mixtures was also investigated. Relative chitinase and/or chitosanase digestibilities were shown to be strongly dependent on chitin deacetylation. Based on enzymatic digestibilities, the alkaline aqueous high shear process does not appear to produce significant secondary products. Correlation of chitosanase digestibility with percentage of deacetylation provides a simple biological assay to study chitosan composition.     

Inter-conversion of free sugars and accumulation of galactomannan in response to supplied metabolic mediators during pod filling in guar

Detached inflorescences of guar (Cyamopsis tetragonoloba), each bearing 4 uniformly-developing pods at 42 days post anthesis (DPA), were cultured for 6 days in complete liquid medium manipulated with a fixed concentration of mannose and varying concentration of myo-inositol. Such inflorescences, but with 2 pods, were also maintained in the solutions of (i) glucose(U-14C) containing myo-inositol or phytohormones, and (ii) mannose(U-14C) containing galactose for 36 hr. Effect of such exogenously supplied metabolic mediators on interconversion of free sugars in pod wall, endosperm and cotyledons and galactomannan accumulation in endosperm was studied. Myo-inositol decreased, over control, the relative proportion of invert sugars in pod wall, endosperm and cotyledons and at lower concentration (27.75 mM) it decreased the level of free sugars in pod wall and galactomannan in endosperm. In all pod tissues, 14C from both glucose and mannose got incorporated into myo-inositol as well as various sugars and maximum incorporation occurred in sucrose. High concentration of total free sugars and their 14C activity in pod wall indicated that this pod tissue was a potent accumulator of free sugars. With myoinositol, the relative proportion of 14C from glucose into raffinose sugars of pod wall and endosperm increased with a simultaneous decrease in this incorporation into galactomannan of the latter. Accompanying this, relative proportion of 14C into hexoses and myo-inositol decreased in pod tissues. Galactose increased 14C incorporation from mannose into total free sugars, sucrose and galactomannan with a concomitant decline in the labelling of hexoses. IAA and ABA enhanced 14C incorporation from glucose into total free sugars and this enhancement was much higher with IAA than ABA. The latter inhibited 14C incorporation into galactomannan. Based on these results, it was suggested that myo-inositol at lower concentration was inadequate to mediate the metabolism of sugars and, thereby, galactomannan synthesis. Galactose and mannose exhibited a mutual beneficial effect on their transportation to pods. Phytohormones stimulated the accumulation of sucrose in pod wall for its obligatory unloading into the seed.     

Kinetics and mechanisms of depolymerization of alginate and chitosan in aqueous solution

The kinetics and mechanisms of depolymerization of aqueous chitosan and alginate solutions at elevated temperatures have been investigated. Chitosan salts of different degree of acetylation (F_A), type of counterions (-glutamate, -chloride) and degree of purity were studied. One commercially available highly purified sodium alginate sample with high content of guluronic acid (G) was also studied. Furthermore, the influence of oxygen, H⁺ and OH⁻ ions on the initial depolymerization rates was investigated. Depolymerization kinetics was followed by measuring the time courses of the apparent viscosity and the intrinsic viscosity. The initial rate constants for depolymerization were determined from the intrinsic viscosity data converted to a quantity proportional to the fraction of bonds broken. The activation energies of the chitosan chloride and chitosan glutamate solutions with pH close to 5 and the same degree of acetylation, F_A = 0.14, were determined from the initial rate constants to be 76 ± 13 kJ/mol and 80 ± 11 kJ/mol, respectively.The results reported herein suggest that the stability of aqueous chitosan and alginate solutions at pH values 5–8 will be influenced by oxidative–reductive depolymerization (ORD) as the primary mechanism as long as transition metal ions are presented in the samples. Acid – and alkaline depolymerization will be the primary mechanisms for highly purified samples.     

Kinetics of the enzymatic hydrolysis of cellulose

Enzymatic hydrolysis of cellulose for sugar production offers advantages of higher conversion, minimal by-product formation, low energy requirements, and mild operating conditions over other chemical conversions. The development of a kinetic model, based on observable, macroscopic properties of the overall system, is helpful in design and economic evaluation of processes for sugar conversion and ethanol production. A kinetic model is presented, incorporating enzyme adsorption, product inhibition, and considers a multiple enzyme and substrate system. This model was capable of simulating saccharification of a lignocellulosic material, rice straw, at high substrate (up to 333 g/L) and enzyme concentrations (up to 9.2 FPU/mL) that are common to proposed process designs.     

Kinetics of the enzymatic synthesis of benzylpenicillin

The kinetics of the enzymatic synthesis of benzylpenicillin catalysed by penicillin amidase (EC 3.5.1.11) from Escherichia coli have been studied. Both free phenylacetic acid (PAA) and its activated derivative, phenylacetylglycine (PAG), were used in the synthesis as acylating agents for 6-aminopenicillanic acid (6-APA). The catalytic rate constants for synthesis carried out at pH 6.0 were 11.2 and 25.2 s⁻¹, respectively, i.e. they are close and have high absolute values. The main feature of the enzymatic synthesis of benzylpenicillin from phenylacetylglycine, compared with the synthesis from phenylacetic acid, is the shape of the progress curve of antibiotic accumulation. In the former case, benzylpenicillin gradually accumulates until equilibrium is reached. Thus, if the reaction is carried out at the thermodynamically optimum pH of synthesis (low pH), penicillin can be obtained in high yield. In the case of phenylacetylglycine, the kinetic curves are more complex and are characterized by a clear-cut maximum. The presence of the maximum, its value and position on the time axis depend on reagent concentration and on the pH used. A kinetic scheme is proposed which describes well the experimental dependencies. The possibility of using activated acid derivatives in synthesis and the advantages of using computer calculations for process optimization are discussed.     

Investigating the kinetics of the enzymatic depolymerization of polygalacturonic acid in continuous UF-membrane reactors

A stirred tank membrane reactor is used to study the kinetics of polygalacturonic acid (PGA) enzymatic hydrolysis. The reactor operates in semicontinuous configuration: the native biopolymer is loaded at the initial time and the system is continuously fed with the buffer. The effect of retention time (from 101 to 142 min) and membrane molecular weight cutoff (from 1 to 30 kDa) on the rate of permeable oligomers production is investigated. Reaction products are clustered in two different classes, those sized below the membrane cutoff and those above. The reducing power measured in the permeate is used as an estimate of total product concentration. The characteristic breakdown times range from 40 to 100 min. The overall kinetics obeys a first-order law with a characteristic time estimated to 24 min. New mathematical data handling are developed and illustrated using the experimental data obtained. Finally, the body of the experimental results suggests useful indications (reactor productivity, breakdown induction period) for implementing the bioprocess at the industrial scale.