Studies on kinetic parameters and stability of aminoacylase in non-conventional media.

Catalytic properties and conformational stability of aminoacylase (N-acylamino acid amidohydrolase, EC 3.5.1.14) were studied in water-N,N-dimethylformamide (DMF) and water-dioxane solvent mixtures. Beside the prompt inhibition the solvents caused further inactivation during incubations. In the presence of 5% DMF content the inactivation proceeds with a well-measurable rate (t1/2 39 min), while in the case of 20% DMF the enzyme practically lost its starting activity during 50 min incubation (t1/2 13 min). The K(m) value of the enzyme increased about three times with increasing DMF concentrations up to about 2.6 M DMF, while the Vmax value decreased practically to zero in the same concentration range.

Continuous production of ethanol using yeast cells immobilized in preformed cellulose beads.

Saccharomyces cerevisiae cells were immobilized on preformed cellulose beads by adsorption. The fermentation capacity of the immobilized yeast cells was found to be practically independent of the hydrogen ion concentration between pH 3.1 and 6.25. The fermentation capacity was maximal at 30 degrees C. The immobilized yeast cells were used for continuous production of ethanol in a fluidized-bead reactor. The average values characteristic for the process were an ethanol concentration of 41.9 +/- 0.1 g1(-1), a fermentation efficiency of 82.9 +/- 2.1% and a volumetric productivity of 3.94 +/- 0.52 g1(-1) h-1.

Immobilization, characterization, and laboratory-scale application of bovine liver arginase.

Arginase isolated from beef liver was covalently attached to a polyacrylamide bead support bearing carboxylic groups activated by a water-soluble carbodiimide. The most favorable carbodiimide was N-cyclohexyl-N'-(methyl-2-p-nitrophenyl-2-oxoethyl) aminopropyl carbodiimide methyl bromide, but for practical purposes, N-cyclohexyl-N'-morpholinoethyl carbodiimide methyl tosylate was used. The optimal conditions for the coupling procedure were determined. The catalytic activity of the immobilized arginase was 290-340 U/g solid or 2.9-3.4 U/mL wet gel. The pH optimum for the catalytic activity was pH 9.5, the apparent temperature maximum was at 60 degrees C and Kmapp was calculated to be 0.37M L-arginine. Immobilization markedly improved the conformational stability of arginase. At 60 degrees C, the pH for maximal stability was found to be 8.0. The immobilized arginase was used for the production of L-ornithine and D-arginine.

Coenzyme production using immobilized enzymes. III. Immobilization of glucose-6-phosphate dehydrogenase from bakers' yeast.

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) from Bakers' yeast was immobilized with the highest activity on polyacrylamide beads possessing carboxylic functional groups activated by a water-soluble carbodiimide. The optimal pH values for the catalytic activity of the soluble and the immobilized glucose-6-phosphate dehydrogenase were practically identical, lying between pH 9.0 and 9.2. The optimal temperature for both the soluble and the immobilized enzyme was about 50 degrees C. The apparent Km values of the immobilized enzyme were slightly higher than those of the soluble enzyme. The immobilization improved the stability of the enzyme in the pH range 6.0-9.0 at 45 degrees C. The operational stability of the immobilized glucose-6-phosphate dehydrogenase proved favorable in a column experiment during 37 days of operation.

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.

A re-evaluation of the lipid-bound sialic acid determination.

The origin of serum sialic acid measured in the lipid-bound sialic acid determination reported by Katopodis et al. (1980) was investigated in detail. By varying the experimental conditions of sample preparation the protein, lipid and sialic acid contents of the methanol-water extract obtained from human sera were analyzed and compared in healthy controls and cancer patients. Using polyacrylamide gel electrophoretic and gel chromatographic methods it has been shown that most of the lipid-bound sialic acid was attributed to the acid alpha 1-glycoprotein (orosomucoid) fraction of human sera. Based on these observations a re-evaluation of the molecular background of the LBSA determination seems to be necessary.

Effects of carbodiimide structure on the immobilization of enzymes.

A series of water-soluble disubstituted carbodiimides of different structure was tested for enzyme immobilization. In the experiments, a polyacrylamide-type bead polymer possessing carboxylic functional groups was used as support. The enzymes immobilized were aminoacylase (N-acylamino acid amidohydrolase; EC 3.5.1.14), arginase (L-arginine amidinohydrolase; EC 3.5.3.1), cyclodextrin glycosyltransferase (alpha-1,4-glucan 4-glycosyltransferase, cyclizing; EC 3.2.1.19), glucoamylase (1,4-alpha-D-glucan glycohydrolase, EC 3.2.1.3), and carboxypeptidase B (peptidyl-L-lysine [L-arginine] hydrolase; EC 3.4.17.2). It was found that the degree of immobilization strongly depended on the structure of carbodiimide used.

Factors influencing the operation of a vertical bioreactor segmented with perforated plates.

Factors influencing the operation of a vertical bioreactor segmented with perforated plates supporting immobilized yeast cells were studied. It was found that the most important factors are the length-diameter (L/D) ratio of the reactor and the dilution rate. It was supposed that the optimal L/D ratio is about 1. The operation of the reactor was more favourable at a low liquid phase-solid phase ratio. The spatial distribution of the biocatalyst had only a slight, if any effect. The periodically changed direction of feed flow has no improving effect on the fermentation process.

Effects of polyethylene terephthalate on yeast alcohol dehydrogenase.

Yeast alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, EC 1.1.1.1) was adsorbed onto polyethylene terephthalate, a synthetic polymer. The effects of the polymer on the properties of the enzyme were studied. The specific activity of the bound enzyme on protein basis was only 1.2 per cent of the specific activity of the soluble enzyme. The optimum pH for the catalytic activity was strongly shifted toward acidic direction. The apparent temperature optimum of the bound enzyme was identical with that of the soluble form. The apparent Michaelis constants of the bound enzyme were higher for both ethanol and NAD+. The conformational stability of the enzyme against heat treatment and urea was decreased as a consequence of adsorption.

Effects of paraquat treatment on fish transaminase molecular subforms.

The effects of paraquat (PQ; 1,1'-dimethyl-4,4'-bipyridylium dichloride) treatment were investigated in carp, silver carp and wels. The serum glutamic-oxalacetic transaminase (GOT; L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1.) level was enhanced by 50% at 1 ppm exposure and by 100% at 10 ppm exposure in all species, and there was a change in the distribution of the molecular subforms of GOT in the liver and heart. The activities of the individual subforms decreased with increasing PQ concentration or after a longer exposure. In some cases, one of the subforms was no longer present in the liver. An increased serum GOT activity, a decreased enzyme activity in different organs and the disappearance of molecular subform indicate tissue damage.

Immobilization of pig muscle aldolase on a silica-based support.

Pig muscle aldolase was covalently attached to a silica-based support possessing aldehyde functional groups. The activity of the immobilized enzyme was 37 U/g solid, and the specific activity calculated on a bound protein basis was 1.9 U/mg protein. The optimum pH for the catalytic activity was pH 7.5. The apparent optimum temperature was found to be 45 degrees C. The Km app value of the immobilized aldolase with D-fructose 1,6-diphosphate as substrate was 1.25 X 10(-4) M. The conformational stability was improved by the immobilization. The immobilized aldolase was used for the continuous splitting of D-fructose 1,6-diphosphate.

Preparation, characterization, and application of a novel immobilized carboxypeptidase B.

Pig pancreas carboxypeptidase B has been immobilized by covalent attachment to a polyacrylamide-type bead support possessing carboxylic functional groups activated by water-soluble carbodiimide. The optimum conditions of immobilization were determined. The activation of the support and the coupling reaction were performed in 0.1 M sodium citrate/sodium phosphate buffer (pH 4.5) using a support-carbodiimide-enzyme weight ratio 4:8:1 at 0-4 degrees C. Under such conditions, the highest activity achieved was 6700 U/g solid. The catalytic properties and stability of immobilized carboxypeptidase B were studied and compared with the corresponding properties of the soluble enzyme. The specific activity of the immobilized enzyme calculated on bound protein basis was about 70% of that of soluble enzyme. The optimum pH for the catalytic activity of the immobilized carboxypeptidase B was practically identical with that of soluble enzyme (pH 7.6-7.7). The apparent optimum temperature of the immobilized carboxypeptidase B was about 7 degrees C higher than that of the soluble enzyme. With hippuryl-L-arginine as substrate, Kmapp value of the immobilized enzyme was tenfold higher than the Km value of the soluble enzyme. The conformational stability of the enzyme was markedly enhanced by the strongly hydrophylic microenvironment in a wide temperature and pH range. The immobilized carboxypeptidase B was used for stepwise digestion of cytochrome C.

[Immobilization of glucose oxidase on silica-based supports]. / Immobilizatsiia gliukozooksidazy na nositeliakh s silikatnymi osnovami.

Glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) was covalently coupled to silica-based supports containing aldehyde functional groups. The activity of the immobilized enzyme was about 1000 U/g support. The optimum pH of the catalytic activity was 5.5 for the soluble enzyme and 6.0 for the immobilized enzyme. With glucose as a substrate the Km value of the immobilized enzyme was higher than in case of the soluble enzyme. The immobilized enzyme was found to be more thermostable than the soluble one. The immobilization did not affect the stability of glucose oxidase against the denaturing effect of urea.

Determination of glycolytic intermediates in a flow injection system using immobilized enzymes.

Some glycolytic enzymes (lactate dehydrogenase, pyruvate kinase, enolase and phosphoglyceromutase) were immobilized on a polyacrylamide-type bead polymer containing carboxylic functional groups activated by water-soluble carbodiimide. The immobilized enzymes were used for the determination of pyruvic acid, phosphoenolpyruvic acid, 2-phosphoglyceric acid and 3-phosphoglyceric acid in a flow injection system. The immobilized lactate dehydrogenase column was repeatedly employed for the determination of pyruvic acid in clinical samples. The results of the flow injection method accorded well in accuracy, sensitivity and reproducibility with those of soluble enzyme analysis.

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.

Targeted immobilization of neurotransmitters and neuropeptides on agarose and on Acrylex polymers.

A new strategy was devised for the targeted immobilization of ligands on aminohexyl- and carboxyhexyl-agarose. Selectively protected neurotransmitter amino acids and neuropeptides were coupled to amino or carboxyl group-containing agarose derivatives using activated esters, mixed anhydrides or carbodiimides. After coupling, agarose beads were dehydrated and the protecting groups were cleaved in non-aqueous media with acids (trifluoroacetic acid, formic acid). Agarose beads were rehydrated and applied for affinity chromatography and cell surface recognition. The same compounds were coupled to derivatized polyacrylamide beads containing primary amino (Acrylex A), acyl hydrazide (Acrylex AH-100) or carboxyl (Acrylex C-100) groups. Protecting groups were removed by acidolytic cleavage. Oxytocin, vasopressin, tetra- and pentagastrin, cholecystokinin, leucine-enkephalin and carboxyl-bearing derivatives of the neurotransmitters noradrenaline, dopamine, histamine, serotonin, acetylcholine and gamma-aminobutyric acid were immunobilized on agarose and on derivatized polyacrylamide gels.

Immobilization of lactate dehydrogenase on polyacrylamide beads.

Pig muscle lactate dehydrogenase (L-lactate:NAD oxidoreductase, EC 1.1.1.27) was covalently immobilized on polyacrylamide beads containing carboxylic functional groups activated by water-soluble carbodiimide. The effects of immobilization on the catalytic properties and stability of the lactate dehydrogenase were studied. There was no shift in the pH optimum of the immobilized enzyme compared to that of the soluble one. The apparent optimum temperature of the soluble enzyme was 65 degrees C, while that of the immobilized enzyme was between 50 and 65 degrees C. The apparent Km values of the immobilized enzyme with pyruvate and NADH substrates were higher than those of the soluble enzyme. As a result of immobilization, enhanced stabilities were found against heat treatment, changes in pH, and urea denaturation.

A novel method for the isolation of carboxypeptidase B.

Carboxypeptidase B was isolated from porcine pancreas by selective heat treatment of the autolyzed tissue at 60 degrees C and pH 6.0 the presence of phosphate ions. The heat treatment was followed by ammonium sulfate fractionation and ion-exchange in a batch system. The method could also be used for the isolation of carboxypeptidase B from beef pancreas.

Comparative studies on soluble and immobilized rabbit muscle pyruvate kinase.

Rabbit muscle pyruvate kinase was immobilized by covalent attachment to a polyacrylamide support (Akrilex C) containing carboxylic functional groups. As a result of immobilization, the pH optimum for catalytic activity shifted into a more alkaline direction. The apparent Km value with phosphoenolpyruvate increased, and that with ADP slightly decreased. With respect to the stability against urea and thermal inactivation, the immobilized pyruvate kinase seemed to be the more stable at lower urea concentrations and between 45 and 55 degrees C. At 1.5 and 2.5M urea and at higher temperature, there were no marked differences between the soluble and the immobilized enzyme.