D-Methionine was converted to L-methionine in a reaction system where four enzymes were used. D-amino acid oxidase (D-AAO) from Arthrobacter protophormiae was used for the complete conversion of D-methionine to 2-oxo-4-methylthiobutyric acid. Catalase was added to prevent 2-oxo-4-methylthiobutyric acid decarboxylation. In the second reaction step, L-phenylalanine dehydrogenase (L-PheDH) from Rhodococcus sp. was used to convert 2- oxo-4-methylthiobutyric acid to L-methionine, and formate dehydrogenase (FDH) from Candida boidinii was added for NADH regeneration. Enzyme kinetics of all enzymes was analyzed in detail. Mathematical models for separate reactions steps, as well as for the complete system were developed and validated in the batch reactor experiments. Complete conversion of D-methionine to L-methionine was achieved. Considering that both enzymes act on different substrates, such a system could be easily employed for the synthesis of other amino acids from D-isomer, as well as from the racemate of a certain amino acid (DL-amino acid).
NADPH-dependent alcohol dehydrogenase (ADH) from Thermoanaerobacter sp. was kinetically characterized using reduction of acetophenone as a model. To achieve 98% conversion of acetophenone, cofactor regeneration by oxidation of 2-propanol with the same enzyme was used. The enzyme was stable in the batch reactor. It was enantioselective towards (S)-1-phenylethanol (ee>99.5%). Due to its high deactivation in continuously operated stirred tank reactor (kd=0.0141 min-1) there was no way to keep high conversion of acetophenone at 98%. The deactivation occurred in the repetitive batch as well. A mathematical model for the acetophenone reduction with cofactor regeneration describing the behaviour in a batch, repetitive-batch and continuously stirred tank reactor was developed.
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.
A commercial enzyme Dextrozyme was tested as catalyst for maltose hydrolysis at two different temperatures: 40 and 65°C at pH 5.5. Its operational stability was studied in different reactor types: batch, repetitive batch, fed-batch and continuously operated enzyme membrane reactor. Dextrozyme was more active at 65°C, but operational stability decay was observed during the prolonged use in the reactor at this temperature. The reactor efficiencies were compared according to the volumetric productivity, biocatalyst productivity and enzyme consumption. The best reactor type according to the volumetric productivity for maltose hydrolysis is batch and the best reactor type according to the biocatalyst productivity and enzyme consumption is continuously operated enzyme membrane reactor. The mathematical model developed for the maltose hydrolysis in the different reactors was validated by the experiments at both temperatures. The Michaelis-Menten kinetics describing maltose hydrolysis was used.List of symbols EC Enzyme consumption (kU g -1 ) K m Michaelis constant (g dm -3 ) k d Enzyme operational stability decay rate constant (min -1 ) q v Flow rate of the reaction solution (cm 3 min -1 ) Q BP Biocatalyst productivity (g kU -1 ) Q p Volumetric productivity (kg dm -3 d -1 )Maximal reaction rate (g dm -3 min -1 )Greek symbols c Concentration (g dm -3 ) u Enzyme concentration expressed as enzyme volume per volume of reaction mixture (cm 3 /cm 3 )
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