Comparison of three distinct ELLA protocols for determination of apparent affinity constants between Con A and glycoproteins.

A procedure for determination of apparent affinity constants K(D)(app) between Concanavalin A (Con A) and naturally d-mannose containing glycoproteins using enzyme-linked lectin assay (ELLA) is reported. Three distinct ELLA protocols are compared to each other with 3 different fitting models used (Liliom, Hill with and without a cooperativity factor). The glycoproteins were physisorbed on a highly charged polystyrene solid surface of immunoassay plates and the amount of lectin bound to the glycoproteins was determined by photometry. The interactions of Con A with five mannose-containing glycoproteins, invertase (INV), glucoamylase (GA), glucose oxidase (GOx), ovalbumin (OVA), and transferrin (TRF) were quantified with apparent affinity constant being in the range 2×10(-7) to 9×10(-6)M. The strength of interaction between Con A and glycoproteins is discussed on the basis of glycan structure/exposure on the protein backbone for each glycoprotein.

Modulation of biorecognition of glucoamylases with Concanavalin A by glycosylation via recombinant expression.

Various types of glucoamylases were prepared to modulate their biospecific interaction with Concanavalin A. Glucoamylase Glm was isolated from the native yeast strain Saccharomycopsis fibuligera IFO 0111. Two glycosylated recombinant glucoamylases Glu's of S. fibuligera HUT 7212 were expressed and isolated from the strains Saccharomyces cerevisiae and one, nonglycosylated, from Escherichia coli. The biospecific affinity of those preparations to Concanavalin A was investigated and compared with the commercially available fungal glucoamylase GA from Aspergillus niger. All glycosylated enzymes showed affinity to Concanavalin A characterized by their precipitation courses and by the equilibration dissociation constants within the range from 1.43 to 4.17 x 10(-6) M (determined by SPR method). The results suggested some differences in the interaction of Con A with the individual glucoamylases. The highest affinity to Con A showed GA. The recombinant glucoamylase Glu with the higher content of the saccharides was comprised by two binding sites with the different affinity. The glucoamylases with the lowest affinity (Glm and Glu with a lower content of saccharides) also demonstrated a nonspecific interaction with Con A in the precipitation experiments. The minimal differences between the individual glucoamylases were determined by the inhibition experiments with methyl-alpha-d-mannopyranoside.

Influence of mannan epitopes in glycoproteins--Concanavalin A interaction. Comparison of natural and synthetic glycosylated proteins.

Two natural glycoproteins/glycoenzymes, invertase and glucoamylase, and two neoglycoconjugates, synthetized from Saccharomyces cerevisiae mannan, bovine serum albumin and penicillin G acylase were tested for interaction with lectin Concanavalin A (Con A). The interaction of natural and synthetic glycoproteins with Con A was studied using three different experimental methods: (i). quantitative precipitation in solution (ii). sorption to Con A immobilized on bead cellulose; and (iii). kinetic measurement of the interaction by surface plasmon resonance. Prepared neoglycoproteins were further characterized: saccharide content, molecular weight, polydispersion, kinetic and equilibrium association constants with Con A were determined. It can be concluded that the used conjugation method proved to be able to produce neoglycoproteins with similar properties like natural glycoproteins, i.e. enzymatic activity (protein part) and lectin binding activity (mannan part) were preserved and the neoglycoconjugates interact with Con A similarly as natural mannan-type glycoproteins.

Neoglycoconjugates of mannan with bovine serum albumin and their interaction with lectin concanavalin A.

Neoglycoconjugates were prepared from mannan isolated from yeast Saccharomyces cerevisiae and activated by periodate oxidation to create aldehyde groups. Various degrees of oxidation introduced 11-28 aldehyde groups per mannan molecule and simultaneously resulted in a molar mass decrease from 46 to 44.5-31 kDa. The activated mannans were subsequently conjugated with bovine serum albumin forming neoglycoconjugates. Some parameters of these mannan-bovine serum albumin conjugates were characterized: saccharide content 25-30% w/w, molar mass within the range 169-246 kDa, and polydispersion (M(w)/M(n)) from 2.8 to 3.6. The interaction of these conjugates with lectin concanavalin A was studied using three different methods: (i) quantitative precipitation in solution; (ii) sorption to concanavalin A immobilized on bead cellulose; and (iii) kinetic measurement of the interaction by surface plasmon resonance. Quantitative precipitation assay showed only negligible differences in the precipitation course of original mannan and the corresponding mannan-bovine serum albumin conjugates. Both the sorption method (equilibrium method) and the surface plasmon resonance measurement (kinetic method) demonstrates that the values of dissociation constant K(D) of all synthetic neoglycoconjugates were within the range 10(-7) - 10(-8) mol x L(-1) (close to K(D) = 10(-8) mol x L(-1) determined by the sorption method for the original mannan). In conclusion, characterization of synthetic neoglycoconjugates confirmed that the method used for their preparation retained the ability of mannan moiety to interact with concanavalin A.

Stability enhancement of Escherichia coli penicillin G acylase by glycosylation with yeast mannan.

Penicillin G acylase (PGA) from Escherichia coli was cross-linked with mannan dialdehydes. Conjugates were prepared with molecular masses varying from 140 to 580 kDa and containing from 18 to 50% (w/w) saccharides, the values depending on the reaction conditions (mannan/enzyme ratio), and by using mannans with different degrees of oxidation and weight-average molecular mass (M macro(w)). The pH- and thermo-stability of all preparations of glycosylated enzyme were improved remarkably, whereby the influence of the character of the linked mannan dialdehyde, its content, as well as the molecular mass of prepared glycoconjugates, on the stability of PGA, was evaluated. PGA glycosylated with the most oxidized mannan up to an M(w) of 490 kDa, containing 41% (w/w) saccharides, and retaining 90% of its original catalytic activity, showed the highest stability. The half-life of this PGA preparation increased significantly: 13-fold at pH 3, 7-fold at pH 10, and 3.5-fold at pH 8 (all at 37 degrees C), compared with the native enzyme. At higher temperatures (50 degrees C) even more significant stabilization was evident, a 16-fold increase in half-life, from 18 min to 289 min, at pH 8, being measured.

Study of interaction of mannan-BSA neoglycoconjugates with Concanavalin A.

Neoglycoconjugates prepared by synthesis of oxidized mannans from Saccharomyces cerevisiae with bovine serum albumin were studied for interaction with Concanavalin A. The mannan-bovine serum albumin neoglycoproteins, different in degree of mannan oxidation used for synthesis and its content in conjugates were prepared. The interaction of these glycoconjugates with Concanavalin A by using precipitation method was investigated. The conjugates prepared at high weight ratio mannan: protein (4:1) involved 47-65% of saccharides and formed by precipitation with Concanavalin A aggregates with low content of protein. The obtained results showed that conjugates with lower content of mannan (up to 30%) are more efficacious for their aggregation with Concanavalin A than the conjugates with high content of mannan.

Examination of bioaffinity immobilization by precipitation of mannan and mannan-containing enzymes with legume lectins.

The interaction of four lectins from crops of the legume family with Saccharomyces cerevisiae alpha-mannan, and also with two glycoenzymes containing mainly alpha-mannan moieties, has been studied. The interaction was characterized by a quantitative precipitation assay. The results of precipitation differ with respect to both quality (the point of maximum precipitation) and of the quantity (the amount of aggregated lectin and saccharide). The lectin concanavalin A [Con A, from jack bean (Canavalia ensiformis)] was observed to form more extensive precipitates with Saccharomyces cerevisiae mannan and glycoenzymes than did lectins from Lens culinaris (lentil) and Pisum sativum (garden pea), while in the case of Vicia faba (broad or fava bean) no interaction was found with either the examined mannans or with glycosylated enzymes. The complete precipitation of invertase and glucoamylase with Con A (enzymes and also Con A; up to 100%) was achieved at a Con A glycoenzyme molar ratio of 20.2 and 2.3 respectively, whereby about 85% of precipitated and also of initial activities of glycoenzymes were determined in the aggregates. More valuable results were achieved by the technique of enzyme immobilization called 'multiple bioaffinity layering' which is based on the stepwise biospecific adsorption of the glycosylated enzymes and Con A on a matrix precoupled with Con A. A 3-fold repetition of the layering procedure afforded up to a 10-fold increase in catalytic activity of the immobilized invertase, in contrast with a 2.1-fold increase in catalytic activity of the immobilized glucoamylase.

Sonication of chitin-glucan, preparation of water-soluble fractions and characterization by HPLC.

A water-insoluble chitin-glucan complex, isolated from the mycelium of Aspergillus niger, was swollen in various aqueous media and treated subsequently by high-energy sonication. The concentration of the resulting water-soluble polysaccharide fractions was dependent on the swelling medium, the amount of the chitin-glucan complex in the suspension, and on the time of sonication. The yields of water-soluble chitin-glucan were within the range 13.6 to 24.4% relative to the mass of the original chitin-glucan. The nitrogen content obtained for the samples of water-soluble chitin-glucan indicated a higher content of chitin (3.45% of nitrogen in high-molecular fraction) than in the original water-insoluble chitin-glucan sample (1.8%). The distribution of the molecular weights of the water-soluble fractions prepared was determined by high-performance liquid chromatography.

Cellulose as a (bio)affinity carrier: properties, design and applications.

This contribution presents a framework for the rational design of affinity sorbents based on cellulose materials as a support. A three-level evaluation procedure, utilizing the knowledge of physical, chemical and engineering theories, is discussed, which integrates the design of support, affinity sorbent and chromatographic contactor. The principal support properties, such as morphological, diffusional, hydrodynamic, mechanical or ligand-binding properties, are presented and literature data on them are surveyed.

The glycoprotein character of multiple forms of Aspergillus polygalacturonase.

Comparisons of known primary structures of polygalacturonases show that extent and localization of potential N-glycosylation sites differ. Some sites are similar in position and adjacent to strictly conserved residues at the potential active site. The presence of N-acetylglucosamine and mannose in the molecules of two homogeneous, major Aspergillus sp. polygalacturonase forms was confirmed by IR spectroscopy. The purification method, based on interaction of the carbohydrate part with concanavalin A immobilized on chlorotriazine bead cellulose, was optimized. Deglycosylation with N-glycosidase F under denaturating and nondenaturating conditions led to molecular mass decreases followed by complete inactivation of the polygalacturonase enzyme activity. These results show the importance of glycosylation in these protein forms, while the comparative patterns establish both variability and some similarities in overall glycosylation architectures.

The glycosylated enzyme-binding assay for the study of the interaction of free and immobilized lectins with carbohydrates.

The glycosylated enzymes (invertase and glucose oxidase) were used as the competitive markers for a simple and rapid determination of the lectin-saccharide interactions. The method, based on the formation of the conjugate of an appropriate glycoenzyme with the specific carbohydrate-binding lectins and the inhibition of the conjugate formation with a monosaccharide, was described. This method was used to estimate the relative carbohydrate specificity of Concanavalin A for monosaccharides derived from D-mannose. The inhibition effect of the saccharides on the formation of Concanavalin A-glycosylated enzyme precipitate was compared with their influence on the enzyme sorption on conjugate Concanavalin A-bead cellulose support. The amount of the interacting enzyme was estimated either indirectly from its concentration in a supernatant that was determined spectrophotometrically (Con A was in a free or immobilized form) or directly in the immobilized form linked to Con A-sorbent using the flow microcalorimetric method. The results obtained, using different methods, agreed in general.

Insight into the distribution of molecular weights and higher-order structure of hyaluronans and some beta-(1 --> 3)-glucans by size exclusion chromatography.

The effects of high energy ultrasound and slightly raised temperature combined with the denaturing action of dimethylsulphoxide on the molecular weight and higher-order structure of hyaluronans and some beta-(1 --> 3)-glucans were studied by means of size exclusion chromatography (SEC) technique. Some experimental conditions connected with the (bio-)polymer sample preparation prior to its SEC analysis are overviewed in the light of informational relevance of studies where the action of a physical and/or chemical agent changes the hydrodynamic size of the m omolecule.

Production of extracellular beta-mannanases by yeasts and yeast-like microorganisms.

A new screening method for simultaneous detection of endo-beta-1,4-mannanase and endo-beta-1,4-xylanase producing microorganisms is described. Two differently dyed substrates Ostazin Brilliant Red-galactomannan and Remazol Brilliant Blue-xylan were incorporated into the same agar media. Decolorizing of one or both substrates around the cell colonies indicates secretion of the corresponding enzyme(s). The method was used to screen 449 yeasts and yeast-like microorganisms belonging to 68 different genera. The secretion of endo-beta-1,4-mannanases and/or endo-beta-1,4-xylanases was found within 10 genera (42 positive strains out of 261 tested). A low frequency of occurrence of endo-beta-1,4-mannanases was observed within the genera Cryptococcus (1 positive strain out of 15 tested), Geotrichum (1 of 6) and Pichia (1 of 35). The highest frequency of occurrence of endo-beta-1,4-mannanases was found within the genera Stephanoascus (2 of 2) and Aureobasidium (14 of 14). Strains hydrolyzing Ostazin Brilliant Red-galactomannan were cultivated in liquid media containing 1% locust bean gum. The best producers of extracellualr endo-beta-1,4-mannanases were found to be the strains of Aureobasidium pullulans.

Lectin-glycoenzyme column chromatography monitored by enzyme flow microcalorimetry.

A method based on the flow microcalorimetric determination of catalytic activity of immobilized enzyme in a so-called enzyme thermistor was used to monitor the process of lectin affinity chromatography of invertase on Concanavalin A-bead cellulose. The strong biospecific interaction between Concanavalin A and invertase was employed to determine the bound enzyme and this principle was used for the investigation of an alternative direct method for monitoring the lectin affinity chromatography of glycoenzymes. The results obtained by flow microcalorimetry showed that the catalytic activity of invertase immobilized on Concanavalin A-bead cellulose can be compared directly with the thermometric value delta Tmax. The validity of the method was also confirmed by the enzyme thermistor post-column method, which is based on the determination of the product from the immobilized invertase enzymatic reaction. The adsorption and desorption in the chromatography column were examined by flow microcalorimetry in small samples withdrawn from the column. Attention has been given to the operating parameters and the storage stability of the affinity sorbent. The binding ability of the affinity matrix decreased with the number of consecutive chromatographic runs, although its storage stability was satisfactory.

Affinity chromatography of invertase on concanavalin A-bead cellulose matrix: the case of an extraordinary strong binding glycoenzyme.

This work presents confirmation of the biospecific character of the interaction of Concanavalin A (Con A) immobilized on bead cellulose with invertase. In spite of the extraordinary strong binding of invertase to this Con A conjugate (KD = 5 x 10(-9) M), conditions have been found to use Con A-bead cellulose as an affinity chromatography medium. The effective factor in the release of the bound invertase by the counter ligand (alpha-methyl-D-mannopyranoside) is the time of incubation. This phenomenon was demonstrated in both batch and flow-through experiments. A concentration of 1.5 mg Con A per ml of gel was found to be suitable with regard to the maximal invertase/Con A binding ratio and the optimal invertase recovery (94%). As a result of the strong biospecific interaction the purification of invertase was very effective (above ten-fold). Verification by FPLC and PAGE of the product purity revealed only one significant protein band.

Screening of concanavalin A-bead cellulose conjugates using an enzyme thermistor with immobilized invertase as the reporter catalyst.

Screening and design of immobilized biocatalysts (IMBs) is a time-consuming process. An ideal process should be universal, fast, convenient, precise, and reproducible. Many of these requirements are met by enzymic flow microcalorimeters, also known as enzyme thermistors (ETs) or thermal assay probes (TAPs). Adaptation of ETs to real measurements of reaction rates requires coupling of the mathematical description of the reaction-diffusion phenomena in the ET column with heat balance and, subsequently, experimental verification of the mathematical model. This article presents such a process developed as an adaptation of ETs for the characterization of the microkinetic properties of IMBs and their further application for screening of IMBs. The IMBs characterized were the preparations of invertase, biospecificaly adsorbed on concanavalin A conjugated to activated bead cellulose. (c) 1994 John Wiley & Sons, Inc.

Application of adsorption kinetics for estimation of dissociation constants.

Substances such as drugs, as well as special ligands with expressive biospecific properties, all with different affinities, interact with proteins which can be characterized by dissociation constants. The method for estimation of the dissociation constant on the basis of adsorption kinetics was verified for two typical cases: adsorption of lactate dehydrogenase onto bead cellulose derivatized by reactive dyes C.I.2. or C.I.19, and adsorption of different drugs (neuroleptics and local anesthetics) onto calmodulin immobilized on agarose gel. The real equilibrium values obtained by using the complete time-concentration model of adsorption were fitted according to the respective adsorption isotherms by non-linear regression.

A new chromogenic substrate for assay and detection of alpha-amylase.

A new soluble chromogenic substrate for alpha-amylase was prepared by coupling partially hydrolyzed starch with a dye, Ostazin brilliant red H-3B. The substrate is precipitable from buffered solutions with ethanol and is equally suitable for assay of alpha-amylase, detection of separated alpha-amylase isoenzymes in gels, and selection of microbial producers of the enzyme.

Soluble chromogenic substrates for the assay of endo-1,4-beta-xylanases and endo-1,4-beta-glucanases.

New soluble chromogenic substrates were prepared for specific and rapid assays of endo-1,4-beta-xylanases and endo-1,4-beta-glucanases. A soluble beechwood 4-O-methyl-D-glucurono-D-xylan was dyed with Remazol brilliant blue R, and hydroxyethylcellulose was coupled to Ostazin brilliant red H-3B. The assays are based on photometric measurements of the enzyme-released dyed fragments soluble in the presence of organic solvents which precipitate the original substrates and their high-molecular-weight fractions. The assays are advantageous for rapid analyses of large amount of samples and also permit evaluation of the activities of both enzymes in the presence of exo-beta-glycanases and beta-glycosidases, at a high level of reducing compounds and viable cells, on the cell surface and on cell membranes and organelles.

Sensitive detection of endo-1,4-beta-glucanases and endo-1,4-beta-xylanases in gels.

A simple, highly sensitive zymogram technique for detection of endo-1,4-beta-glucanases and endo-1,4-beta-xylanases in polyacrylamide gels after electrophoresis or isoelectric focusing was developed. The detection employs transparent agar replicas containing soluble covalently dyed polysaccharides, hydroxyethylcellulose dyed with Ostazin brilliant red H-3B and beechwood 4-O-methyl-D-glucurono-D-xylan dyed with Remazol brilliant blue R, as the respective substrates. The high sensitivity of the detection is achieved by selective removal of depolymerized dyed substrates from the agar replicas by solvents which neither solubilize nor precipitate the original nondegraded dyed polysaccharides present in the agar gel.