2‐Deoxyribose‐5‐phosphate aldolase from Thermotoga maritima in the synthesis of a statin side‐chain precursor: characterization, modeling and optimization

Abstract

BACKGROUND Producing statin side‐chains by sequential aldol condensation catalyzed by 2‐deoxyribose‐5‐phosphate aldolase (DERA) (EC 4.1.2.4) is the best‐known and most promising synthetic route. The advantage of this process is the formation of two stereocenters in a single step starting from inexpensive achiral components, acetaldehyde and chloroacetaldehyde. The limitation for the industrial‐scale application is enzyme inactivation caused by substrates as well by the intermediate produced by single aldol addition. RESULTS The DERA enzyme from Thermotoga maritima (DERATm) was investigated extensively in this work. The influence of aldehydes on DERATm stability was studied in detail. Mathematical models in different reactor configurations were developed based on experimentally determined kinetic parameters of all reactions included in the synthesis of statin side‐chain. Models also included enzyme inactivation by all compounds with negative impact on its stability. Mathematical model‐based optimization enabled optimization of process conditions and reactor configuration. The fed‐batch reactor proved to be the most suitable choice achieving product concentration of 78 g L−1, productivity of 56 g L day−1 and yield of 95% under an optimal condition. CONCLUSION The validated mathematical model and applied methodology can be exploited for further process improvement and for process design of similar systems. © 2019 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Synthetic Activity of Recombinant Whole Cell Biocatalysts Containing 2‐Deoxy‐D‐ribose‐5‐phosphate Aldolase from Pectobacterium atrosepticum

“…DERA activity assay : The hydrolysis activity of Pa DERA N‐His and Pa DERA C‐His AA C49M was determined measuring the oxidation of NADH as described previously [37] . The assay mixture contained 100 mM KPi buffer pH 7, 0.2 mM NADH, 0.4 mM DR5P, 4 μL of α‐glycerophosphate dehydrogenase/triosephosphate isomerase mixture (α‐GDH‐TPI) and 5 μL of freshly purified DERA, in a final volume of 0.5 mL.…”

“…The hydrolysis activity of PaDERA N-His and PaDERA C-His AA C49M was determined measuring the oxidation of NADH as described previously. [37] The assay mixture contained 100 mM KPi buffer pH 7, 0.2 mM NADH, 0.4 mM DR5P, 4 μL of αglycerophosphate dehydrogenase/triosephosphate isomerase mixture (α-GDH-TPI) and 5 μL of freshly purified DERA, in a final volume of 0.5 mL. The reaction was initiated by the addition of DERA and the subsequent decrease of NADH concentration was monitored at 340 nm for 1 min at 28 °C with continuous shaking.…”

Robust Model Selection: Flatness-Based Optimal Experimental Design for a Biocatalytic Reaction

“…Please note that we omitted to write out the equations for the second element of the output vector u 2 for model M 1 and model M 2 due to readability reasons. However, keep in mind that the functions F 1 and F 2 in Equations (22) and (23) differ. The inverse model (Equation (22)) with its flat output (Equation (21)) satisfies the set containing all three conditions for differential flatness (Equations (4)-(6)).…”

“…However, biocatalytic production of the related bulk and fine chemicals and active pharmaceutical ingredients (APIs) is insufficiently explored today. Therefore, the application of enzymes in organic synthesis is currently an important research topic to exploit the significant potential of improving manufacturing processes in the chemical and pharmaceutical industry under the agenda of green chemistry [23,24]. An essential problem with all mathematical models and model-based design concepts, including MBDoE, is the uncertainty in model parameters.…”

Biocatalysis for the Production of Pharmaceutical Intermediates

“…The data concerning aldolase-catalysed reactions is scarce 53,84,85 , and for now, only two research papers report mathematical modelling of the reaction towards statin side-chain production. 53,86 Švarc et al 86 have reported a mathematical model (consisting of a formal kinetic model that includes the data-driven or the empirical model that has less kinetic constants) validated in multiple reactor configurations. Using the developed model, process simulations were done and optimal process design was pro-posed.…”