Zvjezdana Findrik

Modelling as a tool of enzyme reaction engineering for enzyme reactor development

Strategy of the development of model for enzyme reactor at laboratory scale with respect to the modelling of kinetics is presented. The recent literature on the mathematic modelling on enzyme reaction rate is emphasized.

Enzymatic activity of L-amino acid oxidase from snake venom Crotalus adamanteus in supercritical CO2

L-amino acid oxidase (L-AAO) from snake venom Crotalus adamanteus was successfully tested as a catalyst in supercritical CO2 (SC-CO2). The enzyme activity was measured before and after exposure to supercritical conditions (40°C, 110 bar). It was found that L-AAO activity slightly increased after SC-CO2 exposure by up to 15%. L-AAO was more stable in supercritical CO2 than in phosphate buffer under atmospheric pressure, as well as in the enzyme membrane reactor (EMR) experiment. 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) oxidation was performed in a batch reactor made of stainless steel that could withstand the pressures of SC-CO2, in which L-amino acid oxidase from C. adamanteus was able to catalyze the reaction of oxidative deamination of L-DOPA in SC-CO2. For the comparison L-DOPA oxidation was performed in the EMR at 40°C and pressure of 2.5 bar. Productivity expressed as mmol-s of converted L-DOPA after 3 h per change of enzyme activity after 3 h was the highest in SC-CO2 (1.474 mmol U−1), where catalase was present, and the lowest in the EMR (0.457 mmol U−1).

Aldol addition of dihydroxyacetone to N-Cbz-3- aminopropanal catalyzed by two aldolases variants in microreactors

Aldol addition of dihydroxyacetone to N-Cbz-3-aminopropanal catalyzed by two d-fructose-6-phosphate aldolase variants, FSA A129S and FSA A129S/A165G, overexpressed in Escherichia coli was studied in microreactors. The presence of organic solvent was necessary due to poor solubility of N-Cbz-3-aminopropanal in water. Hence, three co-solvents were evaluated: ethyl acetate, acetonitrile and dimethylformamide (DMF). The influence of these solvents and their concentration on the enzyme activity was independently tested and it was found that all solvents significantly reduce the activity of FSA depending on their concentration. The reaction was carried out in three different microreactors; two without and one with micromixers. By increasing enzyme concentration, it was possible to achieve higher substrate conversion at lower residence time. Enzyme activity measured at the outlet flow of the microreactor at different residence time revealed that enzymes are more stable at lower residence times due to shorter time of exposure to organic solvent. The reaction in the batch reactor was compared with the results in microreactor with micromixers. Volume productivity was more than three fold higher in microreactor with micromixers than in the batch reactor for both aldolases. It was found to be 0.88Md(-1) and 0.80Md(-1) for FSA A129S and FSA A129S/A165G, respectively.

A new concept for production of (3S,4R)-6-[(benzyloxycarbonyl)amino]-5,6-dideoxyhex-2-ulose, a precursor of D-fagomine

A novel cascade reaction for the production of aldol adduct (3S,4R)-6-[(benzyloxycarbonyl)amino]-5,6-dideoxyhex-2-ulose was studied in this work. The strategy combines three enzymes in one pot: (i) horse liver alcohol dehydrogenase for the oxidation of N-Cbz-3-aminopropanol to the corresponding aldehyde, (ii) NADH oxidase for the regeneration of coenzyme NAD+ and (iii) D-fructose-6-phosphate aldolase from E. coli A129S variant for the aldol addition of dihydroxyacetone to N-Cbz-3-aminopropanal. On the basis of preliminary experiments, optimization of the initial reaction conditions was done using statistical methods, i.e. factorial design of experiments. 79% yield of aldol adduct was achieved in the batch reactor after optimization.

A mathematical model of oxidative deamination of amino acid catalyzed by two D-amino acid oxidases and influence of aeration on enzyme stability

Two d-amino acid oxidases (DAAO) from different sources (Arthrobacter protophormiae and porcine kidney) were used to oxidatively deaminate d-methionine in the batch reactor. A mathematical model of the process was developed and validated by the experiments carried out without and with oxygen supply by aeration. Kinetic parameters of the model were estimated from the initial reaction rate experiments. Aeration increased the reaction rate in the initial part of the reaction and reduced the time necessary to achieve the final substrate conversion. However, it had a negative influence on the operational stability of enzymes. Operational stability decay rate constants estimated from the experimental data increased with the airflow rate, which indicated lower operational stability of enzymes. It was found that oxygen concentration significantly influenced the stability of DAAO from porcine kidney. Enzyme from microbial source had better operational stability and one order of magnitude lower values of decay rate constants.

Mathematical model for aldol addition catalyzed by two d-fructose-6-phosphate aldolases variants overexpressed in E. coli

Two D-fructose-6-phosphate aldolase variants namely, single variant FSA A129S and double variant FSA A129S/A165G, were used as catalysts in the aldol addition of dihydroxyacetone (DHA) to N-Cbz-3-aminopropanal. Mathematical model for reaction catalyzed by both enzymes, consisting of kinetic and mass balance equations, was developed. Kinetic parameters were estimated from the experimental data gathered by using the initial reaction rate method. The model was validated in the batch and continuously operated ultrafiltration membrane reactor (UFMR). The same type of kinetic model could be applied for both enzymes. The operational stability of the aldolases was assessed by measuring enzyme activity during the experiments. FSA A129S/A165G had better operational stability in the batch reactor (half-life time 26.7 h) in comparison to FSA A129S (half-life time 5.78 h). Both variants were unstable in the continuously operated UFMR in which half-life times were 1.99 and 3.64 h for FSA A129S and FSA A129S/A165G, respectively.

Stabilization of D-amino acid oxidase via covalent immobilization and mathematical model of D-methionine oxidative deamination catalyzed by immobilized enzyme

Porcine kidney D-amino acid oxidase was stabilized by covalent immobilization on spherical particles of Eupergit C because of its low stability in soluble form. The main focus of this work was put on evaluation of operational stability of the immobilized enzyme. To evaluate D-amino acid oxidase’s operational stability during process conditions, repetitive batch reactor experiments of D-methionine oxidation reaction were carried out with continuous aeration for oxygen supply at air-flow rates: of 5 and 10 dm3 h-1. Kinetic analysis of the immobilized enzyme was done as well. The mathematical model of D-methionine oxidative deamination catalyzed by the immobilized D-amino acid oxidase was developed and it described the data well. It enabled the estimation of operational stability decay rate constant. It was possible to achieve 100 % substrate conversion in all batch experiments.

Operational stability of glucoamylase in continuously operated ultrafiltration membrane reactor – experimental methods and mathematical model

Operational stability of glucoamylase was studied at 45, 60 and 70 °C in the reaction of maltose hydrolysis. Experiments were carried out in the continuously operated ultrafiltration membrane reactor (UFMR) at constant residence time of 176.5 minutes. The rate of enzyme operational stability decay increased with temperature. This could be quantitatively observed from the measurements of volume activity during the experiments which were used to estimate enzyme operational stability decay rate constants. Results have shown that stationary conditions in UFMR can be maintained if sufficiently high enzyme concentration is used in the reactor, regardless of the enzyme operational stability decay that occurs. This was shown by the experiment carried out at 45 °C where it was proved that the enzyme operational decay occurs even though maltose conversion was at maximum during the entire experiment. Thus, the operational stability decay can be masked.

The Role of Ionic Liquids in Enzyme-Membrane Integrated Systems

Integration of bioconversion and product separation process, as well as process intensification, have been a focus of the scientific community for some time now because of the necessity of developing processes that are environmentally friendly, spend fewer chemicals and less energy, have higher yields, and generate more profit. In this chapter the role of ionic liquid in such systems integrated with membrane separation processes is presented and discussed. In addition to traditional membrane systems and reactors, newer and modern concepts also are discussed.