Inactivation and reactivation of horseradish peroxidase immobilized by various procedures.

Horseradish peroxidase (HRP) immobilized by coupling the amino acid side chain amino groups or carbohydrate spikes to the matrix has been studied for its resistance to heat, urea-induced inactivation and ability to regain activity after denaturation in order to understand the influence of the nature of immobilization procedure on these processes. The various immobilized preparations were obtained and their properties studied: Sp-HRP was obtained by direct coupling of HRP to cyanogen bromide-activated Sepharose, Sp-NHHRP by coupling periodate oxidized and diamine-treated enzyme to the cyanogen bromide activated Sepharose, SpNH-COHRP by coupling periodate-treated enzyme to amino-Sepharose and SpCon A-HRP by binding of the enzyme on Con A-Sepharose. All the immobilized preparations exhibited higher stability against heat-induced inactivation as compared to the native HRP. Sp-NHHRP was most stable followed by Sp-HRP, SpNH-COHRP and SpCon A-HRP. Sp-NHHRP was also superior in its ability to regain enzyme activity after thermal denaturation, although Sp-HRP regained maximum activity after urea denaturation. Inclusion of Ca2+ was essential for the reactivation of all preparations subsequent to denaturation by urea.     

Immobilization of catalase on cotton fabric oxidized by sodium periodate

Cotton fabric was first oxidized with sodium periodate, and then employed to immobilize catalase. Optimization studies for oxidation of the fabric and immobilization of the enzyme were performed. The properties of the immobilized catalase were examined and compared with those of the free enzyme. A high activity of the immobilized enzyme was obtained when the fabric was oxidized at 40°C and pH 6.0 for 8h in a bath containing 0.20 mol L^?1 sodium periodate and the enzyme was immobilized at 4°C for 24h with a catalase dosage of 120.0 U mL^?1. The immobilized enzyme exhibited optimum activity at 40°C, while the free enzyme had optimal temperature of 30°C, suggesting that the immobilized catalase could be used in a broader temperature range. Both the immobilized and free enzyme had pH optima of 7.0. The staining test and reusability showed that the catalase was fixed covalently on the oxidized cotton fabric.     

Purification of anti-chymotrypsin antibodies for the preparation of a bioaffinity matrix with oriented chymotrypsin as immobilized ligand

Polyclonal antibodies suitable for the oriented immobilization of chymotrypsin we prepared by chromatography on a bioaffinity matrix which had the enzyme immobilized through its active site to antilysin, covalently linked to bead cellulose. After periodate oxidation of their carbohydrate moieties, the isolated antibodies were coupled to a hydrazide derivative of bead cellulose. The periodate oxidation step, which led to greater efficiency and stability of the immunosorbent, had no deleterious effect on antibody activity as assessed by ELISA. Addition of chymotrypsin to the immunosorbent yielded an enzymically active bioaffinity matrix with the optimum molar enzyme/antibody ratio of 2.     

Immobilization of glycoenzymes through carbohydrate side chains.

Glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y were covalently bound to water-insoluble supports through their carbohydrate side chains. Two approaches were used. First, the carbohydrate portions of the enzymes were oxidized with periodate to generate aldehyde groups. Treatment with amines (ethylenediamine or glycyltyrosine) and borohydride provided groups through which the protein could be immobilized. Ethylenediamine was attached to glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y to the extent of 24, 20, 30, and 15 mol/mol of enzyme, respectively. These derivatives were coupled to an aminocaproate adduct of CL-Sepharose via an N-hydroxysuccinimide ester or to CNBr-activated Sepharose. Coupling yields were in the range of 37–50%. Retained activities of the bound aminoalkyl-enzymes were 41% (glucoamylase), 79% (peroxidase), 71% (glucose oxidase), 83% (carboxypeptidase Y). A glycyltyrosine derivative of carboxypeptidase Y was bound to diazotized arylamine-glass. Coupling yield was 42% and retained esterase activity was 84%. In the second approach, the enzyme was adsorbed to immobilized concanavalin A and the complex was crosslinked. Adsorption of carboxypeptidase Y on immobilized concanavalin A followed by crosslinking with glutaraldehyde was also effective. The bound enzyme retained 96% of the native esterase activity and showed very good operational stability.     

Enzyme immobilization on a low-cost magnetic support: kinetic studies on immobilized and coimmobilized glucose oxidase and glucoamylase.

Glucose oxidase (GOx) and glucoamylase (GA) were immobilized and coimmobilized through their carbohydrate moieties onto polyethyleneimine-coated magnetite crosslinked with glutaraldehyde and derivatized with adipic dihydrazide. The carbohydrates were oxidized with sodium periodate, and at optimal concentration, their Vm increased up to 18% for GOx and up to 16% for GA. After immobilization, a remaining activity as high as 88% and 70% for GA with maltose and maltodextrin respectively as substrates was obtained, independently of the particle loading. On the contrary, the remaining activity of GOx strongly decreased at high particle loading. Nevertheless, half of its initial activity was recovered at low loading and was not significantly affected when GA was coimmobilized by saturating the reactive groups left on the particle. The Vm of both immobilized enzymes was improved by crosslinking their carbohydrates with adipic dihydrazide, a treatment which allows further coimmobilization of the other enzyme on a second layer.     

Oriented immobilization of galactose oxidase to bead and magnetic bead cellulose and poly(HEMA-co-EDMA) and magnetic poly(HEMA-co-EDMA) microspheres

In order to obtain an active and stable oxidation reactor for daily use in biochemical laboratory we decided to immobilize galactose oxidase orientedly through a carbohydrate chain to the magnetic carriers. We used hydrazide derivatives of non-magnetic and magnetic bead cellulose and of magnetic and non-magnetic poly(HEMA-co-EDMA) microspheres. Activation of the enzyme molecules was done by sodium periodate in the presence of supplements (fucose, CuSO4, catalase). Orientedly immobilized galactose oxidase presents high storage stability and lower susceptibility to inappropriate microenvironmental conditions. Reactor reactivated by three pulses of D-galactose retained practically 100% of its native activity after 6 months. The positive properties of both magnetic carriers were entirely confirmed.     

Effect of periodate oxidation on properties of antibodies

The subject of the present research is the investigation of the influence of sodium periodate on the properties of immunoglobulin G molecules. It is shown that 100 and 300 M of periodate cause a slight enhancement of the sedimentation coefficient which is a result of the higher protein density. However high concentrations (2000 M) of periodate decrease sedimentation coefficient considerably and disturb the protein structure homogeneity. Studies of the immunologic activity in the periodate-treated antibodies by the reaction of passive hemagglutination showed that in low concentrations it did not decrease significantly the activity but with an increase in the concentration up to 2000 M the activity lowered. The conjugation of antibodies with the enzyme markers was fulfilled due to periodate oxidation. The conjugates obtained were successfully used for improving sensitivity of the precipitation line in immunologic tests.     

固定化克鲁维酵母Y-179外切菊粉酶的研究

鹰嘴豆孢克鲁维酵母(Kluveromyces cicerisporus Y-179)分泌的糖基化菊粉外切酶经高碘酸钠氧化其分子表面的糖链产生醛基,再共价结合于氨基型固定化载体ZH-HA上,固定化酶活力达到4 000 U/g湿载体。所制备的固定化酶在pH 3.5和70℃温度下表现出最大反应活性,该固定化酶pH稳定性和热稳定性较游离酶明显提高。固定化酶在分批式反应器中重复水解菊粉50批次,活力没有明显损失,表现出良好的工作稳定性。     

Immobilization of lipase from Candida rugosa on Eupergit C supports by covalent attachment

The present study compares the results of three different covalent immobilization methods employed for immobilization of lipase from Candida rugosa on Eupergit^® C supports with respect to enzyme loadings, activities and coupling yields. It seems that method yielding the highest activity retention of 43.3% is based on coupling lipase via its carbohydrate moiety previously modified by periodate oxidation. Study of thermal deactivation kinetics at three temperatures (37, 50 and 75 °C) revealed that the immobilization method also produces an appreciable stabilization of the biocatalyst, changing its thermal deactivation profile. By comparison of the t_1/2 values obtained at 75 °C, it can be concluded that the lipase immobilized via carbohydrate moiety was almost 2-fold more stable than conventionally immobilized one and 18-fold than free lipase. The immobilization procedure developed is quite simple, and easily reproduced, and provides a promising solution for application of lipase in aqueous and microaqueous reaction system.     

Hydrolysis of milk lactose by immobilized beta-galactosidase-hen egg white powder

Immobilized beta-galactosidase was obtained by crosslinking the enzyme with hen egg white using 2% glutaraldehyde. The gel obtained could be lyophilized to give a dry enzyme powder. The pH optimum of both the soluble and immobilized enzyme was found to be 6.8. The immobilized enzyme showed a higher K(m) for the substrates. The extent of enzyme inhibition by galactose was reduced upon immobilization. The stability towards inactivation by heat, urea, gamma irradiation, and protease treatment were enhanced. The bound enzyme as tested in a batch reactor could be used repeatedly for the hydrolysis of milk lactose. The possible application of this system for small-scale domestic use has been suggested.     

Immobilized luciferase of Luciola mingrelica fireflies. The kinetic properties and thermostability of luciferase immobilized on cellulose films

Luciferase of fireflies Luciola mingrelica was immobilized on cellulose films activated by cyanuric chloride or sodium periodate. Kinetic properties and the contribution of diffusional obstacles to the kinetics of the immobilized enzyme were examined. External and internal diffusion were found to influence the kinetic parameters. The stability of the enzyme was investigated at 25 degrees C and pH 7.8. Thermoactivation of the immobilized enzyme was shown to proceed in two stages: fast and slow. Dithiotreitol and cystein stabilized the enzyme at the fast stage while salt supplements at both stages. The fast thermoinactivation stage was apparently associated with the oxidation of luciferase SH-groups. It is demonstrated that the immobilized enzyme of Luciola mingrelica can be employed to measure ATP traces with the detection limit 0.1 mM. The enzyme immobilized on cellulose films can be used repeatedly.     

Immobilization of lipase from Candida rugosa on Sepabeads(®): the effect of lipase oxidation by periodates

The objective of this paper was the investigation of a suitable Sepabeads^? support and method for immobilization of lipase from Candida rugosa. Three different supports were used, two with amino groups, (Sepabeads^? EC-EA and Sepabeads^? EC-HA), differing in spacer length (two and six carbons, respectively) and one with epoxy group (Sepabeads^? EC-EP). Lipase immobilization was carried out by two conventional methods (via epoxy groups and via glutaraldehyde), and with periodate method for modification of lipase. The results of activity assays showed that lipase retained 94.8% or 87.6% of activity after immobilization via epoxy groups or with periodate method, respectively, while glutaraldehyde method was inferior with only 12.7% of retention. The immobilization of lipase, previously modified by periodate oxidation, via amino groups has proven to be more efficient than direct immobilization of lipase via epoxy groups. In such a way immobilized enzyme exhibited higher activity at high reaction temperatures and higher thermal stability.     

Various properties of galactose oxidase from Fusarium graminearum IMV-F-1060 immobilized on aminoorganosilochromes

Galactose oxidase preparations are obtained from Fusarium graminearum IMV-F-N 1060 immobilized on aminoorganosilochromes activated by cyanuron chloride and 2.4-toluylene diizocyanate. The immobilized preparations were studied for their selective action on different carbohydrate substrates and for the pH-medium dependence of the obtained preparation activity. Potassium ferricyanide is established to have an activating effect on the immobilized enzyme. It is shown that the immobilized galactose oxidase preparations may be used for the analysis of galactose and lactose.     

Effect of periodate oxidation on the structure and properties of glucose oxidase.

In order to elucidate the molecular structure of glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) and the roles of its carbohydrate moiety, chemical, physiochemical and immunological experiments were performed with enzyme samples before and after periodate oxidation. Hydrodynamic parameters indicated that the native enzyme was a globular protein with values of 1.21 for the frictional ratio and 43 A for the Stokes radius. The enzyme contained about 12% carbohydrate by weight, of which the main component was mannose. The periodate treatment decreased the carbohydrate content to about 40% of its original value. Slight modifications were detected in the absorbance spectrum and the content of arginyl residue. However, no significant alteration was brought about by this treatment in the catalytic parameters, immunological reactivities of the gross structure, not in the secondary and quaternary structures of the protein moity. Thermal denaturation temperature (about 72.5 degrees C) and the enthalpy of denaturation (about 450 kcal/mol) were common to the native and the periodate-oxodozed enzymes. The native was found to be quite resistant to sodium dodecyl sulfate and fairly stable to urea and heating. The periodate-oxidized enzyme was also stable to heat treatment, but it showed a diminished stability when denaturing agents were present. Kinetic analyses of the thermal inactivation processes showed that the entropy of activation was greatly decreased by the denaturing agents, especially in the case of the periodate-oxidized enzyme. It is concluded that the carbohydrate moiety of the enzyme plays a role in increasing the stability of the protein moiety, but does not directly participate in the catalytic activity, the immunological reactivity, or in maintaining the conformation of the enzyme protein.     

IMMOBILIZATION OF CARBONIC ANHYDRASE ENZYME PURIFIED FROM Bacillus subtilis VSG-4 AND ITS APPLICATION AS CO(2) SEQUESTERER

The purification, immobilization, and characterization of carbonic anhydrase (CA) secreted by Bacillus subtilis VSG-4 isolated from tropical soil have been investigated in this work. Carbonic anhydrase was purified using ammonium sulfate precipitation, Sephadex-G-75 column chromatography, and DEAE-cellulose chromatography, achieving a 24.6-fold purification. The apparent molecular mass of purified CA obtained by SDS-PAGE was found to be 37 kD. The purified CA was entrapped within a chitosan-alginate polyelectrolyte complex (C-A PEC) hydrogel for potential use as an immobilized enzyme. The optimum pH and temperature for both free and immobilized enzymes were 8.2 and 37°C, respectively. The immobilized enzyme had a much higher storage stability than the free enzyme. Certain metal ions, namely, Co(2+), Cu(2+), and Fe(3+), increased the enzyme activity, whereas CA activity was inhibited by Pb(2+), Hg(2+), ethylenediamine tetraacetic acid (EDTA), 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB), and acetazolamide. Free and immobilized CAs were tested further for the targeted application of the carbonation reaction to convert CO(2) to CaCO(3). The maximum CO(2) sequestration potential was achieved with immobilized CA (480?mg CaCO(3)/mg protein). These properties suggest that immobilized VSG-4 carbonic anhydrase has the potential to be used for biomimetic CO(2) sequestration.     

A comparative study of free and immobilized soybean and horseradish peroxidases for 4-chlorophenol removal: protective effects of immobilization

Horseradish peroxidase (HRP) and soybean peroxidase (SBP) were covalently immobilized onto aldehyde glass through their amine groups. The activity yield and the protein content for the immobilized SBP were higher than for the immobilized HRP. When free and immobilized peroxidases were tested for their ability to remove 4-chlorophenol from aqueous solutions, the removal percentages were higher with immobilized HRP than with free HRP, whereas immobilized SBP needs more enzyme to reach the same conversion than free enzyme. In the present paper the two immobilized derivatives are compared. It was found that at an immobilized enzyme concentration in the reactor of 15 mg l(-1), SBP removed 5% more of 4-chlorophenol than HRP, and that a shorter treatment was necessary. Since immobilized SBP was less susceptible to inactivation than HRP and provided higher 4-chlorophenol elimination, this derivative was chosen for further inactivation studies. The protective effect of the immobilization against the enzyme inactivation by hydrogen peroxide was demonstrated.     

Comparative studies on immobilization of human prostatic acid phosphatase.

Acid phosphatase (othophosphoric monoester phosphohydrolase (acid optimum), EC 3.1.3.2) from the human prostate was immobilized by its protein moiety on cyanogen bromide-activated Sepharose, by carbohydrate moiety on Concanavalin-A-Sepharose, and by Schiff base formation with partially oxidized carbohydrate groups on ethylenediamine-Sepharose. The highest retention of enzyme activity, 80%, was found for the noncovalent immobilization on Concanavalin-A-Sepharose. It was demonstrated that the optimal pH changes for the Concanavalin-A-Sepharose and CNBr-Sepharose-enzyme complexes are electrostratic in character. In all cases of immobilization the enzyme has higher thermostability than that for the native enzyme under the same conditions. The effects of the enzyme stabilization were interpreted in terms of the multipoint interaction between the enzyme molecule and the carrier.     

Highly efficient solubilization of natural lignocellulosic materials by a commercial cellulase immobilized on various solid supports

Summary A commercial preparation of cellulase was immobilized on CNBr-sepharose, ConA-sepharose, and CNBr-glass beads. When filter paper was used as the substrate, the specific activity of the enzyme immobilized on ConA-sepharose was more than twice that of the soluble enzyme, while the activity of the enzymes immobilized on the other two substrates was either very slightly (CNBr-sepharose) or slightly (CNBr-glass beads) reduced. The immobilized enzymes showed alterations both in the Km and V max values: these were generally either slightly increased (Km) or reduced (V max). In addition, the immobilized enzymes were more resistant to inhibition both by glucose and cellobiose, they were all more stable than the soluble enzyme and solubilized three different natural lignocellulosic materials (alfa-alfa, wheat straw, and pine needles) to a much greater or significantly greater extext than the soluble enzyme: the ConA-sepharose cellulase was the most efficient. The possibility of reusing the immobilized enzyme was also tested. It was found that the ConA-sepharose cellulase could be reused five times with a final loss of activity that ranged between 30% and 50%.     

Covalent immobilization of a glucoamylase to bagasse dialdehyde cellulose

Cellulose fibres from bagasse were oxidized by sodium periodate in sulphuric acid media at positions 2 and 3 of the anhydroglucose unit to produce dialdehyde cellulose. The aldehyde groups of the dialdehyde cellulose were able to react with amino groups of a glucoamylase to form covalent bonds and result in a dialdehyde cellulose immobilized enzyme. The optimum pH of this immobilized enzyme and free enzyme were in the range of 3.0–5.0 and 3.5–5.0, respectively. The optimum temperature for both the free and immobilized enzymes was 60–65 °C. The relative remaining activity of the immobilized enzyme was 36% and its stability was very good, since it could be reused for over 30 cycles. Its activity decreased from the first to the seventh reuse cycles, due to the slow detachment of non-covalently bound enzyme. However, activity tended to stabilize after the seventh cycle of reuse, indicating very stable covalent binding between the enzyme and dialdehyde cellulose.     

The role of carbohydrate in the glycoenzyme invertase of Neurospora crassa.

Data obtained concerning the carbohydrate moieties of the glycoenzyme invertase (EC 3.2.1.26, beta-D-fructofuranoside fructohydrolase) from Neurospora crassa were consistent with a linkage of some carbohydrate chains by O-glycosidic bonds to serine and threonine residues; the possibility of N-glycosylamine linkage of some of the carbohydrate to the amide group of asparagine is also indicated. The invertase was remarkably stable on storage at low temperatures. Oxidation of the carbohydrate residues in the enzyme by sodium periodate markedly affected the heat-stability of the enzyme. It is suggested that the carbohydrate moieties function as stabilizers of the tertiary structure of the glycoenzyme.