engleski

Biocatalytic synthesis of statin side-chain precursors

Statins, also known as 3-hydroxy-3- methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are the most widely prescribed lipid-lowering drugs in the world. Even though well-established chemical approaches for the production of statins are employed, they are time-consuming and require a large number of steps. The increasing commercial demand for statins and requirements for their high chemical and stereochemical purity have led to immense efforts to find a more efficient and economical production of chiral statin side- chain precursors. Due to the nature of enzymes to perform highly stereoselective attacks under mild conditions, biocatalysis represents the most attractive alternative. The objective of this thesis was the development of two enzymatic cascades for the synthesis of two distinctive statin side-chain precursors. The kinetics of each elementary reaction within the multi-enzyme system and the impact of all present compounds (organic compounds: substrates and products) on the enzyme stability were determined. The developed mathematical models were validated in different reactor types (batch, repetitive batch, and fed-batch reactor). Using the validated models, the reaction conditions were optimized with respect to production of optimum quantity of key molecules and volume productivity. The research was based on the following hypotheses: 1. The kinetic models and enzyme stability properties will enable determination of the most suitable strategy for the implementation of a multi-enzyme process. 2. The mathematical models will enable determination of optimal reaction conditions. 3. The optimized multi-enzyme process for the synthesis of statin side-chain precursors will become an attractive alternative to chemical methods. Two different routes towards the production of statin side-chain precursors were examined, which differed in starting materials and used enzymes. The first statin side-chain precursor was obtained by conducting a reaction that involved a 2-deoxyribose-5-phosphate aldolase (DERA), an NAD(P)-dependent dehydrogenase (DH), and a halohydrin dehalogenase (HHDH), while the second precursor was synthesized by carrying out a tandem reaction consisting of DERA and DH, whereby different genetically modified DERA and DH variants were tested, using inexpensive, easily-accessible aldehydes. To make the DH- catalyzed reactions economically feasible, for each reaction an in situ NAD(P)+ regeneration system was examined using an NAD(P)H-dependent oxidase (NOX). The influence of different pH, buffers and organic compounds (substrates and products) on the activity and/or stability of all applied enzymes was investigated. The kinetic data of each elementary reaction within the multi- enzyme system was obtained applying the initial reaction rate method. The developed kinetic models and the data obtained from the enzyme stability measurements enabled the determination of the most suitable strategy for the implementation of a multi-enzyme process. The mathematical models were developed for different reactor modes and the proposed models were experimentally validated. The possibility of running multi-enzyme one-pot reactions was examined as well. It was experimentally proven that all cascade reactions could be conducted in a sequential multi-step one-pot manner with an integrated coenzyme regeneration system working as simultaneous one-pot in order to obtain industrially relevant metrics. The validated mathematical models were used for further optimization of reaction conditions. Based on the obtained results and calculated metrics, optimal reaction setups and enzymes were found. The validated mathematical models represent a valuable basis for future application in the examined multi-enzyme- catalyzed synthesis of statin side-chain precursors.

aldolase, cascade reaction, dehydrogenase, enzyme kinetics, halohydrin dehalogenase, mathematical modelling, optimization, statins

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