| Abstract | 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. |
| Abstract (croatian) | Biokataliza je u središtu pozornosti zbog njezine upotrebe za sintezu lijekova čineći proizvodne procese održivijima. Posljednjih godina pokazalo se veliko zanimanje za korištenje višeenzimskih kaskada kao alata u organskoj sintezi. U ovom je radu korištena metodologija reakcijskog inženjerstva kako bi se opisala i optimizirala tri odvojena biokatalitička kaskadna reakcijska sustava. Veza između istraživanih sustava bila je upotreba enzima koji stvaraju vezu između ugljikovih atoma u jednom od reakcijskih koraka. Kaskadna reakcija kojom se sintetizira 3-hidroksiizomaslačna kiselina, prekursor metakrilne kiseline koja se koristi kao važan međuprodukt za pripravu polimera, novi je pristup koji se sastoji od upotrebe triju enzima. Predložena biokatalitička sinteza 3-hidroksiizomaslačne kiseline može se provesti aldolnom adicijom propanala na formaldehid kataliziranom aldolazom, nakon čega slijedi enzimska oksidacija dobivenog 3-hidroksi-2-metilpropanala katalizirana aldehid dehidrogenazom u 3-hidroksiizomaslačnu kiselinu uz regeneraciju koenzima kataliziranu NADH-oksidazom. Razvijeni matematički model aldolne adicije korištenje za optimizaciju reakcijskih uvjeta. Pri optimalnim procesnim uvjetima, koncentracija produkta aldolne adicije nakon 5,5 sati iznosila je 814 mM (72 g L^-1), iskorištenje na produktu 88,5%, a volumna produktivnost 313,7 g L^-1 d^-1. Budući da aldehid dehidrogenaza prihvaća propanal i formaldehid kao supstrate, ova se kaskada ne može provesti u reaktoru odjednom, već uzastopno pokretanjem oksidacije nakon što reakcija aldolne adicije završi. U svrhu pronalaska katalizatora za reakciju oksidacije ispitano je 25 različitih aldehid dehidrogenaza. Razvijen je i validiran matematički model oksidacije uz regeneraciju koenzima kataliziranu NADH-oksidazom. NMR analizom potvrđeno je da predložena enzimska kaskadna reakcija proizvodi željeni produkt, 3-hidroksiizomaslačnu kiselinu. Postignuto je iskorištenje na oksidacijskom produktu od 66,5% uz koncentraciju produkta od 26,32 mM (2,7 g L^-1). Druga optimizirana kaskadna reakcija u ovom radu je biokatalitička sinteza aminokiseline L-homoserina u kaskadi kataliziranoj piruvat-ovisnom aldolazom klase II i transaminazom, gdje su početni supstrati formaldehid i piruvat te L-alanin koji se koristi u reakciji transaminacije. Na temelju razvijenog matematičkog modela optimizirane su reakcije katalizirane zasebnim enzimima (ekstrakt stanica bez stanične stijenke) i stanicama E. coli koje sadrže iste enzime dobivene ko-ekspresijom. Napravljena je detaljna usporedba kinetičkih parametara za navedena dva tipa biokatalizatora. Provođenjem optimizirane reakcije u šaržnom reaktoru proizvedeno je 672 mM (80,1 g L^-1) L-homoserina nakon
25 sati reakcije s volumnom produktivnosti od 76,23 g L^–1 d^–1 koristeći ekstrakte enzima kao katalizatore te 640,74 mM (76,3 g L^-1) L-homoserina uz volumnu produktivnost od
62 g L^–1 d^–1 kada su kao katalizator korištene cijele stanice koje sadrže oba enzima.
Kao treća kaskadna reakcija, u ovoj su doktorskoj disertaciji predložene i ispitane dvije strategije za biokatalitičku proizvodnju prekursora iminošećera. Jedan pristup temelji se na
Cbz-N-3-amino-1,2-propandiolu, a drugi na 3-kloro-1,2-propandiolu kao početnim supstratima. Kaskadni sustav prvog pristupa jedini je proizveo željeni aldolni produkt, prekursor iminošećera, čija je molekularna masa potvrđena LC-MS analizom. Nedostatak ovog istraživanja proizlazi iz nepotpunih kinetičkih mjerenja zbog činjenice da međuprodukti i produkti ovog reakcijskog sustava nisu komercijalno dostupne kemikalije. Iz tog razloga nije bilo moguće razviti sveobuhvatan matematički model. Ipak, s raspoloživim kinetičkim mjerenjima i procijenjenim parametrima te iskustvom stečenim tijekom istraživanja ove biokatalitičke sinteze, izvedeni su važni zaključci te je postignuta konverzija supstrata od 64% koja odgovara koncentraciji proizvoda od 13,32 mM (4,2 g L^-1). |
