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PubMedDate not supplied

Optimization of protease immobilization by covalent binding using glutaraldehyde.

Immobilization of a protease, Flavourzyme, by covalent binding on various carriers was investigated. Lewatit R258-K, activated with glutaraldehyde, was selected among the tested carriers, because of the highest immobilized enzyme activity. The optimization of activation and immobilization conditions was performed to obtain high recovery yield. The activity recovery decreased with increasing carrier loading over an optimal value, indicating the inactivation of enzymes by their reaction with uncoupled aldehyde groups of carriers. The buffer concentrations for carrier activation and enzyme immobilization were optimally selected as 500 and 50 mM, respectively. With increasing enzyme loading, the immobilized enzyme activity increased, but activity recovery decreased. Immobilization with a highly concentrated enzyme solution was advantageous for both the immobilized enzyme activity and activity recovery. Consequently, the optimum enzyme and carrier loadings for the immobilization of Flavourzyme were determined as 1.8 mg enzyme/mL and 0.6 g resin/mL, respectively.

Buffers↗

PubMedDate not supplied

Immobilization on chitosan of a thermophilic beta-glycosidase expressed in Saccharomyces cerevisiae.

A Sulfolobus solfataricus beta-glycosidase expressed in Saccharomyces cerevisiae (S beta gly) was immobilized on chitosan activated with glutaraldehyde. The yield of immobilization was evaluated as 80%. Compared to the free beta-glycosidase, the immobilized enzyme showed a similar pH optimum (pH = 7.0), the same increasing activity up to 80 degrees C, improved thermostability, and no inhibition by glucose. Functional studies pointed out that the kinetic constant values for both enzymes were comparable. A bioreactor, assembled with the immobilized S beta gly, was used for glucose production. The values of cellobiose conversion increased on increasing residence time in the bioreactor, following a nonlinear trend. However, the highest glucose production/min was obtained at a flow of 0.5 mL/min.

Chitin↗

PubMed1987

Preparation, characterization, and potential application of an immobilized glucose oxidase.

A simple, one-step process, using 0.25 M p-benzoquinone dissolved in 20% dioxane at 50 degrees C for 24 h was applied to the activation of polyacrylamide beads. The activated beads were reacted with glucose oxidase isolated from Aspergillus niger. The coupling reaction was performed in 0.1 M potassium phosphate at pH 8.5 and 0-4 degrees C for 24 h. The protein concentration was 50 mg/mL. In such conditions, the highest activity achieved was about 100 U/g solid. The optimum pH for the catalytic activity was shifted by about 1 pH unit in the acidic direction to pH 5.5. Between 35 and 50 degrees C, the activity of the immobilized form depends on the temperature to a smaller extent than that of the soluble form. Above 50 degrees C, the activity of immobilized glucose oxidase shows a sharper heat dependence. The enzyme-substrate interaction was not profoundly altered by the immobilization of the enzyme. The heat resistance of the immobilized enzyme was enhanced. The immobilized glucose oxidase is most stable at pH 5.5. The practical use of the immobilized glucose oxidase was tested in preliminary experiments for determination of the glucose concentration in blood sera.

Catalysis↗

PubMed1987

Synthesis and properties of vinyl monomer/enzyme conjugates. Conjugation of L-asparaginase with N-succinimidyl acrylate.

Monomer conjugation of an enzyme followed by copolymerization with free monomer is a useful method of enzyme immobilization. L-asparaginase was conjugated with N-succinimidyl acrylate. Analysis of the conjugated enzyme via isoelectric focusing showed that a molar ratio of 9.5 free monomers per enzyme was needed during the conjunction for each vinyl group bound. Only 3% of the enzyme activity was lost per vinyl group added, and conjugation of an average of four monomers per enzyme thermally destabilized the enzyme only at temperatures above 50 degrees C. Activity of the enzyme at physiological temperatures was relatively unaffected.

Acrylates↗

PubMed1987

Enzyme-catalyzed, gas-phase reactions.

Dehydrated preparations of alcohol oxidase adsorbed on DEAE-cellulose vigorously catalyze a gas-phase oxidation of ethanol vapors with molecular oxygen. The gas-phase reaction is strongly dependent on the water activity of the system. The enzymatic activity is severely inhibited by the product hydrogen peroxide. This inhibition can be alleviated, however, by an addition of catalase or peroxidase to the dry preparation. Such dehydrated, bienzymic catalysts afford a complete and selective conversion of the substrate to acetaldehyde. Dry alcohol oxidase is much more thermostable than in aqueous solution. The results of this work suggest that dehydrated enzymes have potential applications in the analysis of gaseous compounds and in the development of novel gas-solid bioreactors.

Alcohol Oxidoreductases↗

PubMed1992

Immobilized beta-glucosidase from Curvularia lunata.

beta-Glucosidase from Curvularia lunata was immobilized in pellets of polyacrylamide, sodium alginate and agar. The activity of the enzyme was estimated at different times by measuring the absorbance of a solution into which 2-nitrophenol was released by the enzyme. The effect of pH and temperature was studied to select the optimum conditions. Thermostability of the beta-glucosidase in each of the carriers was assessed over a period of 12-26 d. The immobilized enzyme on all the three carriers retained its activity longer than free enzyme did. Polyacrylamide was the best carrier both in terms of thermostability and of reusability of the immobilized enzyme preparations. The Michaelis constant (Km) for each of the immobilized enzyme preparation was calculated.

Enzyme Stability↗

PubMed1997

In vivo versus in vitro screening or selection for catalytic activity in enzymes and abzymes.

The recent development of catalytic antibodies and the introduction of new techniques to generate huge libraries of random mutants of existing enzymes have created the need for powerful tools for finding in large populations of cells those producing the catalytically most active proteins. Several approaches have been developed and used to reach this goal. The screening techniques aim at easily detecting the clones producing active enzymes or abzymes; the selection techniques are designed to extract these clones from mixtures. These techniques have been applied both in vivo and in vitro. This review describes the advantages and limitations of the various methods in terms of ease of use, sensitivity, and convenience for handling large libraries. Examples are analyzed and tentative rules proposed. These techniques prove to be quite powerful to study the relationship between structure and function and to alter the properties of enzymes.

Antibodies, Catalytic↗

PubMed1993

Preparation of immobilized proteins covalently coupled through silane coupling agents to inorganic supports.

Enzymes were first immobilized on inorganic supports through silane coupling agents over 25 yr ago. Since that initial report, literally hundreds of laboratories have utilized this methodology for the immobilization of enzymes, antigens, antibodies, receptors, and other high and low mol wt compounds. Today silane coupling is one of the commonly used techniques in the arsenal of the biochemist for the binding of material of all sorts to inorganic surfaces. Inorganic materials come in a variety of shapes, sizes, and characteristics. Today silane coupling is one of the most used coupling methods for the preparation of biosensing devices. Sol-gel entrapped enzymes are also produced by the application of silane technology by the polymerization of the silane to form glass-like materials with entrapped protein. This review will discuss the general preparation and characterization of silane coupled proteins with special emphasis on enzymes and describe in detail the actual methods for the silanization and specific chemical coupling of proteins to the silanized carrier.

Antibodies↗

PubMed1988

Studies on the immobilization of glucuronidase (Part 2). Cleavage of hardly soluble substrates in organic solvents.

Naturally occurring glucuronides and glucosides dissolved in organic solvents can be split with the help of beta-glucuronidase (EC 3.2.1.31) immobilized on controlled pore glass. To protect the enzyme against denaturation by the organic solvents and to promote hydrolytic cleavage of substrates, two methods were used: (a) Immobilization via crosslinking with aged glutaraldehyde in presence of bovine serum albumin; and (b) Adsorption of wet enzyme to the carrier in the presence of organic solvents.

Cross-Linking Reagents↗

PubMed1990

Conversion of ammonia or urea into essential amino acids, L-leucine, L-valine, and L-isoleucine using artificial cells containing an immobilized multienzyme system and dextran-NAD. L-lactic dehydrogenase for coenzyme recycling.

A multienzyme system consisting of leucine dehydrogenase (EC 1.4.1.9), L-lactic dehydrogenase (EC 1.1.1.27), urease (EC 3.5.1.5), and dextran-NAD+ was microencapsulated within artificial cells. This system could convert ammonia and urea into essential amino acids, L-leucine, L-valine, and L-isoleucine. L-lactate acted as a cosubstrate for the regeneration of dextran-NADH. Greater concentrations of L-lactate favored the higher conversion ratios. The effects of ammonium salts and urea on reaction rate were also studied. The relative reaction rates in ammonium salts solutions were 44.6-78.8% of those in urea solutions. More than 90% of the original activity was retained when artificial cells were kept at 4 degrees C for 6 wk.

Amino Acid Oxidoreductases↗

PubMed1991

Entrapment of proteins by aggregation within sephadex beads.

When a protein is aggregated by chemical crosslinking inside Sephadex beads of appropriate pore size, it gets trapped inside the beads. The above approach was used for immobilization of beta-galactosidase, acid phosphatase, trypsin, and concanavalin A. It was found to be a simple, convenient, and fast method for immobilization of proteins.

Acid Phosphatase↗

PubMed1991

Immobilization of penicillin acylase in porous beads of polyacrylamide gel.

A procedure is described for the immobilization of benzylpenicillin acylase from Escherichia coli within uniformly spherical, porous polyacrylamide gel beads. Aqueous solutions of the enzyme and sodium alginate and of acrylamide monomer, N,N'-methylene-bis-acrylamide, N,N,N,N'-tetramethylethylenediamine (TEMED) and sodium alginate are cooled separately, mixed, and dropped immediately into ice-cold, buffered calcium formate solution, pH 8.5, to give calcium alginate-coated beads. The beads are left for 30-60 min in the cold calcium formate solution for polyacrylamide gel formation. The beads are then treated with a solution of glutaraldehyde and the calcium alginate subsequently leached out with a solution of potassium phosphate. Modification of the native enzyme with glutaraldehyde results in a slight enhancement in the rate of hydrolysis of benzylpenicillin at pH 7.8 and 0.05M substrate concentration. The enzyme entrapped in porous polyacrylamide gel beads shows no measurable diffusional limitation in stirred reactors, catalyzing the hydrolysis of the substrate at a rate comparable to that of the glutaraldehyde-modified native enzyme. The immobilized enzyme preparation has been used in batch mode over 90 cycles without any apparent loss in hydrolytic activity.

Acrylamide↗

PubMed1991

Hydrolysis of nucleic acids in single-cell protein concentrates using immobilized benzonase.

Hydrolysis of nucleic acids for single-cell protein concentrates has been carried out in one step using immobilized benzonase on corn cob. The immobilization is carried out by tosylation of primary alcohols of cellulose of corn cob. The immobilized benzonase is more stable vs pH changes than native benzonase, but the same optimum values of [Mg(II)] and temperature are obtained. The DNase activity is greater than the RNase activity. The percentage of DNA is reduced to 3-6% and that of RNA to 50%. The protein loss is negligible (1%). The enzymatic activity per weight unit of enzyme is greater in the case of benzonase that in reported data for other nucleases insolubilized on corn cob by the same procedure.

DNA, Fungal↗

PubMed1991

Detoxification of organophosphate pesticides using a nylon based immobilized phosphotriesterase from Pseudomonas diminuta.

A partially purified phophotriesterase was successfully immobilized onto nylon 6 and 66 membranes, nylon 11 powder, and nylon tubing. Up to 9000 U of enzyme activity was immobilized onto 2000 cm2 of a nylon 6 membrane where 1 U is the amount of enzyme necessary to catalyze the hydrolysis of 1.0 mumol of paraoxon/min at 25 degrees C. The nylon 66 membrane-bound phosphotriesterase was characterized kinetically where the apparent Km value for the immobilized enzyme was 0.35 mM. This is 5-6 times higher than that observed for the soluble enzyme. However, nylon immobilization limited the maximum rate of paraoxon hydrolysis to less than 10% of the value measured for the soluble enzyme. The addition of the cosolvent, methanol, resulted in an increase in the apparent Km value for paraoxon hydrolysis but concentrations up to 40% had no negative effect on the catalytic effectiveness with the soluble or immobilized phosphotriesterase. Based on the kinetic analysis, methanol appears to be a competitive inhibitor for both forms of enzyme. The nylon powder immobilized enzyme was shown to be stable for at least 20 mo. The immobilization of the phosphotriesterase onto nylon provides a practical method for the detoxification of organophosphate pesticides.

Aryldialkylphosphatase↗

PubMedDate not supplied

Immobilization of glucose isomerase onto granular chicken bone.

Glucose isomerase was immobilized onto granular chicken bone (BIOBONE) by adsorption. The amount of activity bound relative to an equal amount of free enzyme was 32 +/- 1%, with the estimated specific activity decreasing from 11.1 +/- 0.7 to 3.9 +/- 0.5 U/mg protein with immobilization. Compared with the free enzyme, immobilized glucose isomerase showed a threefold increase in the Km for fructose and a fivefold decrease in Vmax. High operating temperatures were possible (greater than 55 degrees C), but continuous use and long-term storage studies showed gradual losses of activity. Both the binding and the activity of the bone-immobilized enzyme were highly resistant to treatments with detergent, ethanol, and KCl. Studies to determine mass transfer limitation effects on immobilized glucose isomerase showed that these were insignificant for this system.

Adhesiveness↗

PubMedDate not supplied

Immobilization of invertase through its carbohydrate moiety on Ocimum basilicum seed.

Yeast invertase, a glycoprotein, was covalently coupled to Ocimum basilicum seeds either through its protein or carbohydrate moiety. Of the various methods investigated, binding of the enzyme through its carbohydrate moiety resulted in the retention of considerably higher amounts of enzyme activity. Immobilized invertase showed a shift in the pH optimum toward the alkaline side without appreciable change in temperature optimum. However, the immobilized preparation was more thermostable than the free enzyme. Invertase bound to the seeds could be used repeatedly for the hydrolysis of sucrose syrups in a batch process without appreciable loss in activity. The seeds could serve as an inexpensive, ready-to-use, natural pellicular polysaccharide support for immobilizing enzymes.

Carbohydrates↗

PubMed1992

Immobilized triosephosphate isomerases. A comparative study.

Pig muscle triosephosphate isomerase was covalently attached to polyacrylamide and silica-based supports possessing carboxylic or aldehyde functional groups or activated with p-benzoquinone. A silica-based support activated with p-benzoquinone proved to be the most advantageous. There were no profound alterations in the catalytic properties as a result of the immobilization. The immobilization enhanced the resistance against urea and heat treatment. At the start of the treatments, the enzyme was activated. The extent of activation depended on the pH, and on the buffer and salt concentrations. Increase of the ionic strength decreased or eliminated the activation. The phosphate ion had a specific effect on the thermal inactivation.

Animals↗

PubMed2003

Amperometric biosensor with HRP immobilized on a sandwiched nano-Au / polymerized m-phenylenediamine film and ferrocene mediator.

An amperometric biosensor has been developed for the determination of H(2)O(2) in plant samples. Horseradish peroxidase (HRP) is immobilized on a sandwiched nano-Au particle / m-phenylenediamine polymer film by glutaraldehyde cross-linking. The film is formulated on the carbon paste electrode (CPE) blended with ferrocene as an electron transfer mediator. On the low concentration range, the current response is related to the H(2)O(2) concentration linearly from 0 to 8x10(-6) M with a detection limit of 1.3x10(-7) M. On a wider concentration range of 8x10(-6) to 1.4x10(-4) M, the reciprocal of current response is linearly related to the reciprocal of H(2)O(2) concentration. The apparent Michaelis-Menten constant (K(m)(app)) was calculated to be 0.0334 mM. The sensor has been tested by determining H(2)O(2) concentration in plant leaf samples.

Biosensing Techniques↗