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Mathematical modelling and optimization of enzymatic cascades for the synthesis of industrially valuable products

Biocatalysis is in focus due to its use for the synthesis of pharmaceuticals making manufacturing processes more sustainable. In recent years, much interest has been shown in the use of multi- enzyme cascades as a tool in organic synthesis. Reaction engineering methodology was used in this thesis for describing and optimizing three separate biocatalytic cascade reaction systems. The link between all cascades was the use of carbon‒carbon bond forming enzyme in one reaction step per cascade. Cascade reaction that synthesises 3- hydroxyisobutyric acid, a methacrylic acid precursor used as important intermediate for the preparation of polymers, is a novel approach that consists of three enzymes. Proposed biocatalytic synthesis of 3-hydroxyisobutyric acid can be performed by aldolase-catalysed aldol addition of propanal to formaldehyde followed by an enzymatic oxidation catalysed by aldehyde dehydrogenase of the resulting 3-hydroxy-2-methylpropanal to 3- hydroxyisobutyric acid with coenzyme regeneration by NADH oxidase. Developed mathematical model for aldol addition was used for the reaction optimization. At the optimal process conditions, the aldol addition product concentration after 5.5 hours was 814 mM (72 g L–1), product yield was 88.5% and volume productivity was 313.7 g L–1 d–1. Since aldehyde dehydrogenase accepts propanal and formaldehyde as substrates, this cascade could not be performed in a one-pot synthesis, but consecutive, by starting oxidation after aldol addition was completed. Panel of 25 aldehyde dehydrogenases was tested as oxidation step catalyst. Mathematical model of oxidation with coenzyme regeneration by NADH oxidase was developed and validated. It was confirmed by NMR analysis that proposed enzymatic cascade scheme produced desired product, 3-hydroxyisobutyric acid. The yield on oxidation product of 66.5% was achieved with the final product concentration of 26.32 mM (2.7 g L-1). The second optimized cascade reaction in this work is the biocatalytic synthesis of amino acid L-homoserine in a cascade containing Class II pyruvate-dependent aldolase and transaminase as biocatalysts starting from formaldehyde and pyruvate, and L-alanine used in transamination reaction step. Reactions catalysed by separate enzymes (cell free extracts) and E. coli cells containing the same co-expressed enzymes were optimized based on developed mathematical model. Detailed kinetic parameters comparison is made between those two types of biocatalysts. Optimized reaction performed in the fed-batch reactor produced 672 mM (80.1 g L-1) of L- homoserine after 25 hours of reaction with volume productivity of 76.23 g L–1 d–1 with cell free extract enzymes as catalysts, and 640.74 mM (76.3 g L-1) of L-homoserine with volume productivity of 62 g L–1 d–1 when whole cells containing both enzymes were used as catalysts. The third cascade reaction was the synthesis of iminosugar precursor. Two strategies for biocatalytic production of iminosugar precursor were proposed and examined within this doctoral thesis. One approach suggested Cbz-N-3-amino-1, 2- propanediol and second approach involved 3- chloro-1, 2-propanediol as oxidation substrate. Only cascade system from the first approach produced the desired aldol adduct, an iminosugar precursor whose molecular mass was confirmed by LC-MS analysis. Research drawback was caused by incomplete kinetic measurements due to the fact that intermediates and final product molecules are not commercially available chemicals and therefore no comprehensive mathematical model could be developed. Nevertheless, with available kinetic measurements and parameters determined and experience gained during process investigation, some important conclusions regarding this biocatalytic synthesis were withdrawn and substrate conversion of 64% corresponding to product concentration of 13.32 mM (4.2 g L-1) was achieved.

biocatalysis ; enzyme ; iminosugar ; L-homoserine ; methacrylic acid ; process optimization ; reaction engineering

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