Bela Szajani

Ethanol and acetic acid tolerance in free and immobilized cells of Saccharomyces cerevisiae and Acetobacter aceti

Saccharomyces cerevisiae and Acetobacter aceti cells were immobilized by entrapment in Ca-alginate or by adsorption on to preformed cellulose beads and were treated with 0-20% (v/v) ethanol and 0-10% (v/v) acetic acid. At 20% (v/v) ethanol, lethal for free yeast cells, 62-72% of the immobilized cells survived. In 10% (v/v) acetic acid, free and adsorbed Acetobacter aceti cells ceased to grow but 69% of entrapped cells survived. Cells released from the carrier showed an intermediate survival (20-60%).

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

A simple, one-step process, using 0.25Mp-benzoquinone dissolved in 20% dioxane at 50°C for 24 h was applied to the activation of polyacrylamide beads. The activated beads were reacted with glucose oxidase isolated fromAspergillus niger. The coupling reaction was performed in 0.1M potassium phosphate at pH 8.5 and 0–4°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°C, the activity of the immobilized form depends on the temperature to a smaller extent than that of the soluble form. Above 50°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.

Preparation and characterization of immobilized glucose-phosphate isomerase

Abstract Glucose-phosphate isomerase ( d -glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) isolated from rabbit muscle was covalently attached to Sepharose 4B, Akrilex C-100, Akrilex P-100 and Silochrome aldehyde, respectively. The highest immobilized activity (110 units mg −1 solid) was achieved when the enzyme was bound to Akrilex C-100, a polyacrylamide support possessing carboxylic functional groups. The catalytic properties and the stability of this Akrilex-bound enzyme were studied in detail relative to those of the soluble enzyme. For both soluble and immobilized glucose-phosphate isomerase the pH optimum was about pH 8.0. The apparent optimum temperature of the immobilized enzyme was about 50°C, while that for the soluble enzyme lay between 50 and 55°C. The apparent K m for glucose 6-phosphate as substrate was higher (2 × 10 −1 mM) in the case of the immobilized enzyme. Little difference was found between the thermal stabilities of the soluble enzyme (at 45° C t 1 2 = 42 min ; at 50° C t 1 2 = 12 min ) and the immobilized enzyme (at 45° C t 1 2 = 82 min ; at 50° C t 1 2 = 10 min ). As a result of immobilization, the stability against urea was increased.

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 apparentKm 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°C. At 1.5 and 2.5M urea and at higher temperature, there were no marked differences between the soluble and the immobilized enzyme.

A novel polyacrylamide-type support prepared by p-benzoquinone activation.

A new and simple method for the activation of polyacrylamide gels usingp-benzoquinone is described. The optimal conditions of activation have been elaborated. The activated support could be successfully applied to the immobilization of ligands having nucleophilic groups active over a broad pH range.

Diffusional characteristics of a swelling gel and its consequences for bioreactor performance

Abstract A diaphragm diffusion cell was used to determine the effective diffusion coefficient of L -methionine in a modified polyacrylamide gel at different degrees of swelling. As could be expected from theory, the value of the effective diffusion coefficient increased with increasing swelling. The combined effect of increased radius and increased effective diffusion coefficient is analysed in terms of the Thiele modulus and the effectiveness factor. The results can explain the reduced production rate obtained in bioreactors with immobilized enzymes using gel carriers with a shrinking-swelling behaviour during processing.

Effects of immobilization on biomass production and acetic acid fermentation of Acetobacter aceti as a function of temperature and pH

SummaryAcetobacter aceti cells were immobilized using entrapment in Ca-alginate gel and adsorption on preformed cellulose beads. The cell number within the supports showed no significant alterations on changing temperature or pH, whereas the acetic acid production was slightly increased by immobilization.

Stirred fluidised-bed reactor developed for low-density biocatalyst supports

A new type of multistage fluidised-bed reactor was constructed to avoid the fluidisation irregularities by slow stirring of the bed. Aminoacylase immobilized on a polyacrylamide type bead polymer was used as biocatalyst for resolution of racemic amino acids. The efficiency was considerably higher than that of a traditional packed-bed reactor.

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.

Immobilization of pig muscle 3-phosphoglycerate kinase

3-Phosphoglycerate kinase (ATP:3-phospho-d-glycerate 1-phosphotransferase, EC 2.7.2.3) has been covalently immobilized on a polyacrylamide-type support containing carboxylic groups activated by water-soluble carbodiimide. The activity was 88 units g−1 xerogel. The activity versus pH profile showed a sharper maximum at pH 6.5 in the case of the immobilized enzyme. The immobilized enzyme had a broad apparent optimum temperature range between 40 and 50°C. The apparent Km values of the immobilized 3-phosphoglycerate kinase were lower for both 3-phosphoglycerate and ATP than those of the soluble enzyme. In the case of the immobilized enzyme stabilities were enhanced.