Viera Illeová

Chromatographic separation and kinetic properties of fructosyltransferase from Aureobasidium pullulans

Efficient chromatographic separation of fructosyltransferase from Aureobasidium pullulans was achieved on a preparative scale using a weak anion-exchanger Sepabeads FP-DA. The recovery yield was about 70% and the purification factor reached a value of 28. The molecular weight of the enzyme determined by size-exclusion chromatography was 570,000. The enzyme exhibited both hydrolytic and transferase activity when the latter was higher in the whole concentration range and completely dominating at higher sucrose concentrations. It was found that sucrose was the only donor of fructosyl moiety used in the transfer reaction. The initial rate method was used for the investigation of the kinetics of the action of fructosyltransferase on sucrose in the concentration range 30-2,430 mM. The initial transfructosylation rate was well fitted with a linear function of the sucrose activity where the activity coefficient was an exponentially decreasing function of the sucrose concentration.

Kinetics of acetophenone reduction to (R)-1-phenylethanol by a whole-cell Pichia capsulata biocatalyst

Abstract (R)-1-phenylethanol is an important substance in fragrance and flavor industry. In this work, the reduction of acetophenone to (R)-1-phenylethanol in an aqueous medium was examined using Pichia capsulata as a whole-cell biocatalyst. Progress curve and initial rate measurements were used to obtain kinetic data. The experiments were carried out at pH 5, temperature of 25 °C, and in the presence of glucose to maintain in vivo regeneration of NADH. A model of the reversible reaction kinetics considering the substrate inhibition of the forward reaction was developed. Five kinetic parameters of this model were determined by a simultaneous fit of a reaction rate dependence on substrate concentration and 18 substrate and product concentration progress curves with very good accuracy. Equilibrium constant of the reaction and equilibrium conversion of acetophenone to (R)-1-phenylethanol were 13.7 and 93%, respectively.

Production of fructosyltransferase in mechanically stirred and air-lift bioreactors

Aureobasidium pullulans cells were cultivated in a 3 dm3 stirred bioreactor to optimize the production of fructosyltransferase (FTase). Batch experiments were focused on the influence of the initial sucrose concentration (150–350 g dm−3), sodium nitrate concentration (10–25 g dm−3), and stirring rate (180–540 min−1) on the process. It was found that the FTase specific activity per cell mass was positively influenced by the conditions that impeded the cell growth such as higher sucrose concentrations or lower oxygen transfer rates. Higher content of inorganic nitrogen substrate slightly increased the overall FTase production. The presence of fructose moiety-containing saccharides in the cultivation medium was observed to be indispensable for FTase production. An addition of a concentrated sucrose solution in the 40th cultivation hour therefore essentially resumed the FTase production. Finally, scale-up experiments were performed in 12 dm3 and 100 dm3 mechanically stirred bioreactors and in 20 dm3 and 60 dm3 air-lift bioreactors.

Spray drying of the mixtures of mono-, di-, and oligosaccharides

Abstract Optimal conditions of spray drying of a fructooligosaccharide-rich mixture prepared by enzymatic conversion of sucrose were investigated. It was found that efficient drying of the mixture containing about 40 % of monosaccharides and sucrose required addition of a compound with a high glass transition temperature. The addition of maltodextrin helped to achieve satisfactory solids yield and moisture. Optimized process parameters were the feed flow rate and solids concentration, drying air flow rate and inlet temperature.

Biotransformation of acetophenone to R-1-phenylethanol with immobilized Pichia capsulata in batch reactor

Abstract Biotransformation of acetophenone to R-1-phenylethanol with Pichia capsulata immobilized in Caalginate gel beads was studied. Experiments were carried out in a batch reactor stirred on an orbital shaker (300 rpm) at 25 ℃. Based on experimental data, uncoupled parameters of reaction kinetics (measurements with free cells) and diffusion coefficients (sorption method) were estimated. The model expressing the conversion of acetophenone with immobilized cells in the batch reactor consisted of mass balance equations of acetophenone and R-1-phenylethanol in the liquid and solid phases. A satisfactory agreement between the experimental and predicted values of acetophenone and R-1-phenylethanol concentrations was achieved

Investigation of plant sources of hydroperoxide lyase for 2(E)-hexenal production

Abstract 2(E)-hexenal is a green note flavour molecule that is widely used in various compositions of aromas, flavours and perfumery. As there is considerable demand for naturally produced aromas this article deals with some aspects of this C6-volatile production with regard to the selection of plant source material and reaction conditions. The following plants were tested for this purpose: runner bean (Phaseolus coccineus), common bean (Phaseolus vulgaris), three (Capsicum annuum) bell pepper varieties, garden cress (Lepidium sativum), green slicing cucumber (Cucumis sativa), mung beans (Vigna mungo) and brown lentils (Lens culinaris). Selection of source material was considered on the basis of 2(E)-hexenal yield and productivity. The common bean leafs were able to produce up to 35 mg of 2(E)-hexenal/kg fresh leaves.