Article pubs.acs.org/IECR
Measuring and Predicting Thermodynamic Limitation of an Alcohol Dehydrogenase Reaction Matthias Voges, Florian Fischer, Melanie Neuhaus, Gabriele Sadowski, and Christoph Held* Laboratory of Thermodynamics, Department of Biochemical and Chemical Engineering, Technische Universität Dortmund, Emil-Figge-Straße 70, 44227 Dortmund, Germany S Supporting Information *
ABSTRACT: The knowledge of thermodynamic limitations on enzymatic reactions and of influencing factors thereon is essential for process optimization to increase space−time yields and to reduce the amount of solvent or energy consumption. In this work, the alcohol dehydrogenase (ADH) catalyzed reaction from acetophenone and 2-propanol to 1-phenylethanol and acetone in aqueous solution was investigated in a temperature range of 293.15−303.15 K at pH 7. It serves as a model reaction to demonstrate the use of biothermodynamics in order to investigate and predict limitations of enzymatic reactions. Experimental molalities of the reacting agents at equilibrium were measured yielding the position of reaction equilibrium (Km) at different reaction conditions (temperature, initial reactant molalities). The maximum initial acetophenone molality under investigation was 0.02 mol·kg−1 due to solubility limitations with a 1- to 50-fold excess of 2-propanol. It was shown that Km strongly depends on the initial reactant molalities as well as on reaction temperature. Experimental Km values were in the range of 0.20 to 0.49. Thermodynamic key properties (thermodynamic equilibrium constant, standard Gibbs energy and standard enthalpy of reaction) were determined by measured Km values and activity coefficients of the reacting agents predicted with the thermodynamic model ePC-SAFT. In addition, ePC-SAFT was used to predict Km at different initial molalities. Experimental and predicted results were in quantitative agreement (root-mean-square error of experimental versus predicted Km was 0.053), showing that ePC-SAFT is a promising tool to identify process conditions that might increase/decrease Km values and, thus, shift the position of reactions for industrial applications.
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INTRODUCTION
catalyzed reactions that were done, e.g., for amino transferase reactions, hydrolase reactions or isomerization reactions.5−7 ADH reactions are enzymatic reactions in which alcohols are converted into aldehydes or ketones with the reduction of the cofactor nicotinamide adenine dinucleotide (NAD(P)+ ⇌ NAD(P)H+H+) or in which aldehydes or ketones are converted into alcohol with the oxidation of nicotinamide adenine dinucleotide (NAD(P)H+H+ ⇌ NAD(P)+). As ADHs occur in many metabolic pathways and the use of ADHs in industrial processes is increasing, understanding the thermodynamics and the limitations of ADH reactions for the optimization of biotechnological processes is of great interest.8−10 In this work, the ADH reaction from acetophenone (ACP) to 1-phenylethanol (1-PE) is investigated as an illustrative example (eqs 1, 2).
Generally, the efficiency of enzymatic reactions can be limited by the stability (and thus of the activity) of enzymes in the reaction medium, by the low solubility of reacting agents in mostly aqueous reaction media and by the unfavorable thermodynamic equilibrium. In recent works, the influence of reaction conditions on the yield or kinetics of numerous chemical reactions was investigated experimentally.1−3 This work investigates the limitation of the yield of enzymatic reactions by thermodynamic equilibrium. In general, there are several opportunities to overcome yield limitations by process optimization as summarized by Abu et al.,4 e.g., excess of cosubstrate, in situ product removal, smart substrates or enzyme coupling reactions. However, the characterization of reaction equilibrium from a thermodynamic point of view is indispensable. That is, this work aims at applying thermodynamics to predict equilibria of enzymatic reactions rather than optimizing those by product removal or coupling reactions and plant design. For this purpose a model alcohol dehydrogenase (ADH) reaction is considered to complement investigations on thermodynamics and thermodynamic modeling of enzyme© XXXX American Chemical Society
Received: Revised: Accepted: Published: A
March 24, 2017 April 20, 2017 April 20, 2017 April 20, 2017 DOI: 10.1021/acs.iecr.7b01228 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
Article
Industrial & Engineering Chemistry Research
and the activity-coefficient-based Kγ value, both of which being concentration-dependent. Kth of reaction eq 3 is defined by following equation. The Km value and the Kγ value both depend on pressure, temperature and the composition of the reaction mixture (here mole fraction x of all compounds) and are defined as shown in eqs 5 and 6. a ·a K th(p , T ) = 1 ‐ PE Ac = K m·K γ aACP ·a 2 ‐ P (4) 1-PE is used industrially as a precursor for styrene production.11 The chemical synthesis of 1-PE takes often place at extreme conditions like in the “styrene monomer propylene oxide” process, in which 1-PE is synthesized from ethylbenzene hydroperoxide and propylene at high temperature and pressure.11 In stark contrast, the biochemical synthesis of 1PE takes place at moderate conditions (e.g., 303.15 K, atmospheric pressure) catalyzed by an ADH. The ADH reaction from ACP to 1-PE is an equilibrium reaction that occurs in the presence of NADH+H+ that is converted to its oxidized form NAD+. A second reaction that is also ADHcatalyzed (often by the same ADH that is used in the main ADH reaction) allows recycling NAD+ to NADH+H+. This significantly reduces the amount of added NADH+H+, a strategy that is already well-known and published in the literature since many years.12−14 In this work, the reaction from 2-propanol (2-P) to acetone (Ac) is used to recycle NAD+ back to NADH+H+. Coupling these two single ADH reactions leads to the coupled ADH reaction (eq 3), in which ACP and 2-P are converted to 1-PE and Ac until reaction equilibrium is reached.
K m(p , T , x) =
K γ (p , T , x ) =
EQ m1EQ ‐ PE · mAc EQ mACP ·m2EQ ‐P
m γ1m‐ PE·γAc m γACP ·γ2m‐ P
=
(5)
γ1 ‐ PE·γAc γACP·γ2 ‐ P
(6) −1
Here, is the equilibrium molality (mol·(kg(H2O)) ), γi is the mole-fraction-based activity coefficient and γm i the molalitybased activity coefficient of the reacting agents i (1-PE, Ac, ACP, 2-P). The standard state for γm i and γi is the pure component i. The relation between γm i and γi is given by the activity of reacting agent i: ai = γm i ·mi = γi·xi with xi = molei/ moletotal and mi = molei/kgH2O. Considering the stoichiometry of the coupled ADH reaction eq 3, the total mole and the mass of H2O are eliminated. Thus, Kγ can be calculated straight from the mole-fraction-based activity coefficients as shown in eq 6. In previous works, Km values of several coupled ADH reactions have been reported that also used the cofactor regeneration from 2-P to Ac. Goldberg et al.16 measured and summarized several Km values for ADH reactions from ketones to secondary alcohols in different solvents. Most of these Km values were
