High-performance liquid chromatography with concanavalin A immobilized by metal interactions on the stationary phase

Concanavalin A (Con A) was immobilized via metal interactions on macroporous, microparticulate silica support having covalently bound iminodiacetic acid functions (IDA-silica) chelated with Cu(II) at the surface. The amount of copper and of Con A in the column could readily be controlled by the conditions used for chelating the metal by IDA-silica and for immobilization of the lectin. The retention behavior of columns packed with the stationary phase did not change under a wide range of elution conditions, indicating no loss of immobilized lectin. However, the Con A proper could readily be removed from the column at pH 3.0 or together with Cu(II) by perfusion with EDTA at neutral pH. Columns containing Con A immobilized by this technique exhibited dual retention behavior for proteins, glycoproteins, and carbohydrates according to the pertinent glycan-lectin or protein-metal interactions. The glycoproteins, peroxidase and alpha 1-acid glycoprotein, were retained by the Con A moiety and eluted with eluents containing competing sugars, whereas the proteins, beta-lactoglobulin, alpha-chymotrypsinogen A, and ribonuclease A and B were retained by the chelated copper and were eluted and separated with eluents containing sodium chloride or borate. Binding constants of glycosides on the immobilized Con A were evaluated chromatographically and found to be one-third to two-thirds those reported in the literature on the basis of experiments in free solution.     

Immobilization of invertase onto poly(3-methylthienyl methacrylate)/poly(3-thiopheneacetic acid) matrix

A novel immobilization matrix, poly(3-methylthienyl methacrylate)–poly(3-thiopheneacetic acid) (PMTM–PTAA), was synthesized and used for the covalent immobilization of Saccharomyces cerevisiae invertase to produce invert sugar. The immobilization resulted in 87% immobilization efficiency. Optimum conditions for activity were not affected by immobilization and the optimum pH and temperature for both free and immobilized enzyme were found to be 4.5 and 55 °C, respectively. However, immobilized invertase was more stable at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined using the Lineweaver–Burk plot. The K_m values were 35 and 38 mM for free and immobilized enzyme, respectively. The V_max values were 29 and 24 mg glucose/mg enzyme min for free and immobilized enzyme, respectively. Immobilized enzyme could be used for the production of glucose and fructose from sucrose since it retained almost all the initial activity for a month in storage and retained the whole activity in repeated 50 batch reactions.     

Studies on the Water-Insoluble Enzyme

Water-insoluble yeast invertase was prepared by binding native invertase to DEAE-cellulose. Some characteristics of the bound invertase and the continuous hydrolysis of sucrose by use of it are described. The activity of bound invertase corresponded to about 1/2 at pH 3.4 when compared with the maximum activity of free form and it could hydrolyze sucrose into invert sugar perfectly. The apparent optimum pH of bound invertase was shifted toward acid pH by about 2 pH units in comparison with free invertase. Stability of bound invertase to temperature was slightly less in comparison with free invertase at pH 5.2. The continuous sucrose hydrolysis was carried out using bound invertase at pH 3.6 and it could be used about ten times until the hydrolysis ratio decreased to the half of the initial.     

Stabilization of yeast invertase by insoluble immunocomplex formation using unfractionated glycosyl-specific and peptide specific polyclonal antiinvertase antibodies

Summary A strategy for the production of non-inhibitory polyclonal antisera for the immobilization of glycoenzymes is described. Insoluble complexes of invertase prepared using antiinvertase glycosyl antisera were more resistant to heat and urea induced denaturation than those prepared using antiinvertase antisera from which the glycosyl specific antibodies were removed. Unfractionated antiinvertase antibodies however gave most stable immunocomplexes with invertase.     

Immobilization of enzyme onto poly(ethylene-vinyl alcohol) membrane

Invertase was ionically bound to the poly(ethylene-vinyl alcohol) membrane surface modified with two aminoacetals with different molecular length, 2-dimethyl-aminoacetoaldehyde dimethylacetal (AAA) and 3-(N, N-dimethylamino-n-propanediamine) propionaldehyde dimethylacetal (APA). Immobilization conditions were determined with respect to enzyme concentration in solution, pH value, ionic strength in immobilization solution, and immobilization time. Various properties of immobilized invertase were evaluated, and thermal stability was found especially to be improved by immobilization. The apparent Michaelis constant, K(m), was smaller for invertase bound by APA with longer molecular lengths than for invertase bound by AAA. We attempted to bind glucoamylase of Rhizopus delemar origin in the same way. The amount and activity of immobilized glucoamylase were much less than of invertase.     

Acid and Neutral Invertases in the Mesocarp of Developing Muskmelon (Cucumis melo L. cv Prince) Fruit

Acid and neutral invertases were found in the mesocarp of developing muskmelon (Cucumis melo L. cv Prince) fruit and the activities of these enzymes declined with maturation of the fruit, concomitantly with the accumulation of sucrose. Neutral invertase was only present in the soluble fraction and acid invertase was present in both the soluble and cell-wall fractions. The cell-wall fraction contained three types of acid invertase: a NaCl-released invertase; an EDTA-released invertase, and a tightly bound invertase that still remained on the cell wall after treatment with NaCl and EDTA. The soluble acid and neutral invertases could be separated from one another by chromatography on DEAE-cellulose and they exhibited clear differences in their properties, namely, in their pH optima, substrate specificity, K_m values for sucrose, and inhibition by metal ions. The EDTA-released invertase and the soluble acid invertase were similar with regard to their chromatographic behavior on DEAE-cellulose, but the NaCl-released invertase was different because it was adsorbed to a column of CM-cellulose. The soluble acid invertase and two cell-wall bound invertases had very similar characteristics with regard to optimal pH and temperature, K_m value for sucrose, and substrate specificity.     

Characterization of wall-bound invertase isoforms of Picea abies cells and regulation by ectomycorrhizal fungi

In culture, the ectomycorrhiza-forming fungi Amanita muscaria (Pers. ex Fries) Hock. and Hebeloma crustuliniforme (Bull. ex Fries) Quel. only grow on media with glucose or fructose but not with sucrose as sole carbohydrate source. This is due to their lack of wall-bound invertase activity. Therefore, utilization of sucrose by the fungi within a mycorrhizal association is believed to depend on the wall-bound invertase activity of the host. This enzyme activity was studied in the apoplast of suspension cultured cells of Picea abies (L.) Karst. An ionically and a tightly wall-bound isoform of acid invertase were found that function as β-d -fructofuranoside-fructohydrolases (EC 3.2.1.26). The ionically bound enzyme could be easily released from walls of intact cells with buffer of high ionic strength. In its native form, the ionically bound invertase isoform is a monomeric protein with a molecular mass of 61 kDa, as determined by gel filtration and SDS-PAGE. Glycoprotein nature of the enzyme was demonstrated with antibodies directed against the digoxigenin-labeled protein. The K_m values of both enzymes for sucrose, their natural substrate, are relatively high (ionically bound invertase K_m= 16 mM, tightly bound invertase K_m= 8.6 mM). Activity of both wall-bound invertase isoforms strongly depends on the apoplastic pH. They have a narrow pH-optimum and exhibit highest activity at pH 4.5. with elevated activity between pH 4.5 and 6.0. Furthermore, fructose acts as competitive inhibitor of both isoforms, whereas glucose is not inhibitory. Unloading of sucrose from host cells to the apoplastic interface of the Hartig net in ectomycorrhizae appears to depend on the rate of hydrolysis by the wall-bound invertase of the host. Since the activity of the plant invertase depends on the actual pH value and the fructose concentration in the mycorrhizal interface, we suggest that the fungus can actively influence the activity of the plant invertase by acidification of the cell wall and by fructose uptake. Thus, the fungus itself can regulate its own supply of glucose and fructose.     

Invertase immobilization via its carbohydrate moiety

After periodate oxidation of its glycosidic component, invertase was covalently bound onto three types of modified solid supports: glycidyl methacrylate, styrene-divinylbenzene copolymers, and bead cellulose. Direct reaction of the invertase aldehyde groups that were formed with amino groups of the support and use of the modified Ugi reaction have been employed as immobilization procedures. Apart from binding methods, the important effects of the buffer, support, conditions of periodate oxidation, and the length of the spacer on the activity of the enzyme conjugate have been investigated. Superior conjugate activity was obtained, via modified Ugi reaction, by the immobilization of a suitably oxidized invertase to a styrene-divinylbenzene copolymer having free amino groups.     

Utilization of powdered pig bone as a support for immobilization of lipase

Pig bone was examined for its suitability as a support material for lipase immobilization. It was observed that pig bone (PB) particles dispersed readily in both polar and nonpolar solvents, and lipase was easily adsorbed. In particular lipase adsorbed on olive oil-soaked pig bone (OPB) particles exhibited a higher hydrolytic activity than that in lipase adsorbed on a selection of other representative supports, regardless of removing the presoaked olive oil from the particles after immobilization of lipase. The optimum pH and temperature for hydrolytic activity of OPB-adsorbed lipase were the same as those for free lipase, although thermal resistance was increased by immobilization. When OPB-adsorbed lipase was used for repeated batch reactions of olive oil hydrolysis, an activity of more than 80% of the initial activity of each run could he retained after 46 h reaction. The results suggest that PB is an excellent support material.     

A new lectin from the tuberous rhizome of Kaempferia rotunda: isolation, characterization, antibacterial and antiproliferative activities

A lectin (designated as KRL) was purified from the extracts of Kaempferia rotunda Linn. tuberous rhizome by glucose-sepharose affinity chromatography. KRL was determined to be a 29.0 ± 1.0 kDa polypeptide by SDS-PAGE under both reducing and non-reducing conditions. KRL was a divalent ion dependent glycoprotein with 4% neutral sugar which agglutinated different groups of human blood cells. Methyl-α-D-mannopyranoside, D-mannose and methyl-α-D-glucopyranoside were the most potent inhibitors. N-terminal sequence of KRL showed similarity to some mannose/ glucose specific lectins but the main differences with their molecular masses and sugar content. KRL lost its activity markedly in the presence of denaturants and exhibited high agglutination activity from pH 6.0 to 8.2 and temperature 30 to 60° C. The lectin showed toxicity against brine shrimp nauplii with the LC50 value of 18 ± 6 μg/ml and strong agglutination activity against seven pathogenic bacteria. KRL inhibited the growth of six bacteria partially and did not show antifungal activity. In addition, antiproliferative activity against Ehrlich ascites carcinoma (EAC) cells showed 51% and 67% inhibition in vivo in mice administered 1.25 mg/kg/day and 2.5 mg/kg/day of KRL respectively by injection for five days.     

Concanavalin A: a useful ligand for glycoenzyme immobilization--a review.

Concanavalin A is finding increasing applications as a useful ligand in glycoenzyme immobilization. An attempt therefore, has been made to summarize the work available in the area. Glycoenzymes that are recalcitrant to immobilization procedures involving covalent coupling to solid supports can be immobilized in high yields by binding to matrices precoupled with concanavalin A. In addition, glycoenzymes associated with concanavalin A matrices usually exhibit high retention of activity and enhanced stability against various forms of inactivation. Binding of the glycoenzymes on the concanavalin A supports, being noncovalent, can be reversed by incubating the preparation with a high concentration of sugars/glycosides or at acidic pH. The association can be, however, rendered covalent by crosslinking the preparations with bifunctional reagents like glutaraldehyde. Crosslinking may be accompanied by further increase in stability, albeit at the expense of the loss of some enzyme activity. Several laboratory-size reactors containing concanavalin A matrix-bound glycoenzyme have been successfully operated for reasonably long durations with only small losses in catalytic activity. Insoluble glycoenzyme preparation can also be obtained by precipitating them from solution as concanavalin A complexes. Such complexes have small particle dimensions but can be successfully used in column reactors after a subsequent immobilization step. Insoluble concanavalin A-flocculates containing various microorganisms and glycoenzymes that successfully carry out multistep transformations have also been obtained by several investigators.     

Preparation and properties of immobilized amyloglucosidase on nonporous PS/PNaSS microspheres

Nonporous polystyrene/poly(sodium styrene sulfonate) (PS/PNaSS) microspheres were used for immobilization of amyloglucosidase and the properties of immobilized enzyme was studied and compared with those of free enzyme. Sulfonated groups on the PS/PNaSS microspheres present a very simple, mild, and time-saving process for enzyme immobilization. Nonporous microspheres provide their surface for immobilization of enzyme and prevent the diffusion limitation problem in the pore. Despite the high concentration of bound enzyme the influence of immobilization on kinematic parameters, K(m) and V(max), is relatively low compare to other porous supports. Simple and time-saving immobilization procedure as well as the effects of pH and temperature on immobilized enzyme also showed that the PS/PNaSS microspheres could be good support.     

Immobilization of porcine pancreatic <Emphasis Type="Italic">α</Emphasis>-amylase on magnetic Fe<Subscript>2</Subscript>O<Subscript>3</Subscript> nanoparticles: Applications to the hydrolysis of starch

Enzymes play a pivotal role in catalyzing diverse reactions. However, their instability upon repetitive/prolonged use, as well as their inhibition by high substrates and product concentration, remains an area of concern. In this study, porcine pancreatic α-amylase was immobilized on magnetic Fe₂O₃ nanoparticles (Fe₂O₃-NPs) in order to hydrolyze starch. The magnetic nanoparticle bound enzymes retained 94% of their initial enzyme activity. X-ray diffraction and atomic force microscopy analyses showed that the prepared matrix had advantageous microenvironment and a large surface area for binding significant amounts of protein. Functional groups present in enzyme and support were monitored by Fourier transform infrared spectroscopy. Immobilized enzyme exhibited lowered pH optimum (pH 6.0) to a greater degree than its soluble counterpart (pH 7.0). Optimum temperature for the immobilized enzyme shifted towards higher temperatures. The immobilized enzyme was significantly more resistant to inactivation caused by various metal ions and chemical denaturants. Immobilized α-amylase hydrolyzed 92% starch in a batch process, after 8 h at 40°C; while the free enzyme could hydrolyze only 73% starch under similar experimental conditions. A reusability experiment demonstrated that the immobilized enzyme retained 83% of its original activity even after its 8^th repeated use.     

Optimization of invertase immobilization by adsorption in ionic exchange resin for sucrose hydrolysis

This work presents as a main objective to study the immobilization process of yeast invertase by adsorption in the ion exchanging resin Duolite A-568 for invert sugar production. Initially, a kinetic study of the soluble form of the enzyme was carried out. At the sequence was studied the immobilization process of yeast invertase in the weakly exchanging anionic resin Duolite A-568. The influences of the pH, enzyme concentration and temperature in the enzyme immobilization were analyzed through a central composite design (CCD). The results indicated that the retention of the catalytic activity in immobilization was strongly dependent of these variables, being maximum in a pH value of 5.0, with an enzyme concentration of 12.5 g/L (1.875 g of protein per liter) and temperature of 30 °C. The simultaneous influence of pH and temperature on the free and immobilized invertase activity was also studied through a CCD.     

Purification and characterization of a thermostable mycelial lectin from basidiomycete Lentinus squarrosulus

Lectins are non-immune carbohydrate-binding proteins or glycoproteins with specific binding sites for certain glycoconjugates. Fungal lectins have been documented for their antitumour, antiproliferative, immunomodulatory, hypotensive and insecticidal effects. In the present study, a mycelial lectin having molecular mass 55 kDa has been purified and characterized from Lentinus squarrosulus. Biological action spectrum of the lectin revealed agglutination of all human blood types (A, B, O, AB), goat, sheep, rabbit and pig erythrocytes. Neuraminidase treatment of blood type O erythrocytes considerably augmented hemagglutination titre. Carbohydrate inhibition studies showed its high affinity to mucin and asialofetuin. Lectin was purified by a combination of ammonium sulphate precipitation, dialysis, ion exchange chromatography and gel filtration chromatography. Optimum pH for lectin activity was observed to be 6.5–8.0 and optimum temperature was 25–30°C. Lectin showed poor pH stability and was stable within pH 7.0–7.5. It was highly thermostable and could withstand temperature upto 70°C. Lectin activity was sensitive to ethylenediaminetetraacetic acid and denaturants.     

A Study on the Comparative Stability of Insoluble Complexes of Glucose Oxidase Obtained with Concanavalin A and Specific Polyclonal Antibodies

Summary The formation of insoluble complexes of glycoenzymes with lectins and antibodies is one of the simplest methods of enzyme immobilization. Insoluble complexes of glucose oxidase were simply obtained by mixing the enzyme with concanavalin A or a specific polyclonal antibodies solution. The concanavalin A and immunocomplexes of glucose oxidase retained more than 80% of the original enzyme activity. Expression of very high enzyme activity in insoluble complexes suggested that these aggregates were quite porous and easily accessible to substrates. Insoluble complexes of glucose oxidase showed very high stability against denaturation induced by pH, temperature, urea and water-miscible organic solvents. Complexes of glucose oxidase obtained with concanavalin A and glycosyl-specific antiglucose oxidase polyclonal antibodies were quite comparable in stability while complexes prepared using polyclonal antibodies raised against the native glucose oxidase were slightly less stable. The complexes of glucose oxidase obtained with glycosyl-specific antiglucose oxidase polyclonal antibodies showed very high stability against inactivation mediated by exposure to water-miscible organic solvents. Insoluble complexes of glucose oxidase were cross-linked with glutaraldehyde to maintain their integrity in the presence of substrates. The cross-linking of complexes resulted in a slight decrease in enzyme activity but showed a pronounced enhancement in stability against various forms of denaturation.     

Immobilization of invertase via carbohydrate moiety on chitosan to enhance its thermal stability

A new technique using chitosan as support for covalent coupling of invertase via carbohydrate moiety improved the activity and thermal stability of immobilized invertase. The best preparation of immobilized invertase retained 91% of original specific activity (412 U mg^–1). The half-life at 60°C was increased from 2.3 h (free invertase) to 7.2 h (immobilized invertase). In contrast, the immobilization of invertase via protein moiety on chitosan or using Sepharose as support resulted in less thermostable preparations. Additionally, immobilization of invertase on both supports caused the optimal reaction pH to shift from 4.5 to 2.5 and the substrate (sucrose) concentration for maximum activity to increase from 0.5 M to 1.0 M.     

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.     

Binding of human fibroblast interferon to concanavalin A-agarose. Involvement of carbohydrate recognition and hydrophobic interaction.

Human fibroblast interferon binds to a concanavalin A-agarose (Con A-Sepharose) equilibrated with methyl alpha-D-mannopyranoside, or levan; in contrast, it is only partially retarded on a similar column equilibrated with ethylene glycol. Interferon does not bind, however, to a lectin column equilibrated with both methyl alpha-D-mannopyranoside and ethylene glycol. Thus, a hydrophobic interaction between fibroblast interferon and the immobilized lectin seems to account for a large portion of the binding forces involved. Other hydrophobic solutes, such as dioxane, 1, 2-propanediol, and tetraethylammonium chloride, were found equally or more efficient than ethylene glycol in displacing interferon from the lectin column. The elution pattern of interferon from a concanavalin A-agarose (Con A-Sepharose) column, at a constant ehtylene glycol concentration and with an increasing mannoside concentration, reveals the existence of four distinct interferon components. The selective adsorption to and elution from a concanavalin A-agarose (Con A-Sepharose) column resulted in about a 3000-fold purification of human fibroblast interferon and complete recovery of activity. The specific activity of the partially purified interferon preparation is about 5 X 10(7) units per mg of protein. The chromatographic behavior of human leukocyte interferon is remarkable in that it does not bind to concanavalin A-agarose at all indicating the absence of carbohydrate moieties recognizable by the lectin, or if present, their masked status. When concanavalin A was coupled to an agarose matrix (cyanogen bromide activated) at pH 8.0 and 6.0 human fibroblast interferon bound to both lectin-agarose adsorbents and could be recovered with methyl alpha-D-mannopyranoside. Concanavalin A, immobilized directly on agarose matrix at pH 8.0 and 6.0, thus displays only carbohydrate recognition toward interferon. By contrast, unless a hydrophobic solute was included in the solvent containing methyl mannoside, human fibroblast interferon could not be recovered from concanavalin A-agarose coupled at pH 9.0. When concanavalin A was immobilized via molecular arms, in tetrameric as well as dimeric forms, the binding of interferon again occurred exclusively through carbohydrate recognition. Thus, the hydrophobic interaction can be eliminated by appropriate immobilization of the lectin, and then adsorbed glycoproteins, as exemplified here by interferon, can be recovered readily with methyl mannoside alone.     

Lectin specificity and binding characteristics of human C-reactive protein.

C-reactive protein (CRP) is thought to play an important role in immunomodulation. The exact biologic function of this pentraxin protein is, however, still unclear. Here we report experiments designed to further characterize the binding properties of CRP. Using purified human CRP it could be shown that CRP immobilized onto polystyrene surfaces or onto latex beads binds distinct plasma glycoproteins including IgG, asialofetuin, asialo-beta 2-glycoprotein I and, likewise, synthetic glycoproteins as a lectin, exhibiting binding specificity for terminal galactosyl residues of the glycoprotein glycans. Binding of CRP to IgA, IgM, IgG, asialofetuin, asialo-beta 2-glycoprotein I and to synthetic glycoproteins requires immobilization onto surfaces of both CRP and the ligand. Fibronectin and fibrinogen are bound by surface-immobilized CRP also in soluble phase. Comparing various mono-, di-, and trisaccharides as competitive inhibitors of the lectin binding activity of CRP, only beta-D-Gal-(1-3)-D-GalNAc, beta-D-Gal-(1-4)-D-GalNAc, and beta-D-Gal-(1-4)-beta-D-Gal-(1-4)-D-GlcNAc had significant inhibitory power at a concentration of 8 mmol/liter. Binding activity of CRP was pH-dependent with an optimum at pH 5 to 6 and was reduced by 90% when pH was shifted from 6 to the physiologic pH value of 7.4. CRP exhibited lectin-like properties with binding specificity for galactosyl residues also when bound to K-562 erythroleukemia cells. It is therefore suggested that CRP immobilized onto surfaces exhibits lectin activity toward galactosyl groups preferentially in a mildly acidic environment as present at sites of inflammation.