TY - JOUR

T1 - Unraveling Alcohol Dehydrogenase Catalysis in Organic–Aqueous Biphasic Systems Combining Experiments and Molecular Dynamics Simulations

AU - Zhang, Ningning

AU - Bittner, Jan Philipp

AU - Fiedler, Marius

AU - Beretta, Thomas

AU - María, Pablo Domínguez de

AU - Jakobtorweihen, Sven

AU - Kara, Selin

N1 - Funding Information: The authors thank the Deutsche Forschungsgemeinschaft (DFG) (Grant Nos. KA 4399/3-1 and JA 2500/5-1) for financial support. This research used computational resources provided by the North-German Supercomputing Alliance (HLRN). The authors thank Assoc. Prof. Dr. Diederik Johannes Opperman (University of the Free State, South Africa) for the recombinant plasmid containing the HLADH gene. Furthermore, we would like to thank Michelle Leganger Juul Sørensen for the technical assistance and Nico Briewig for the assistance with the MD simulations.

PY - 2022/8/5

Y1 - 2022/8/5

N2 - The use of oxidoreductases in organic-aqueous biphasic systems is advantageous (effective solvation of reactants, minimization of substrate/product-induced inhibition, improved volumetric productivity, and straightforward downstream processing). This paper explores the effects of organic solvents on horse liver alcohol dehydrogenase (HLADH) by combining experimental and computational studies. Various organic solvents displaying a broad range of hydrophobicity and functionalities are used, namely, ethyl acetate, 2-methyltetrahydrofuran, methyl tert-butyl ether, cyclopentyl methyl ether, toluene, cyclohexane, heptane, and dodecane. The catalytic performance of model enzyme horse liver alcohol dehydrogenase concerning its activity, stability, and selectivity is experimentally evaluated. The results are interpreted with molecular dynamics simulations by assessing the (i) protein location in biphasic media, (ii) organic solvent distribution, and (iii) enzyme conformation. Herein, the stability states the robustness of the enzyme while storing it in biphasic media without catalysis taking place. Overall, different toxicities of the solvent to the enzyme can be pinpointed: "molecular toxicity", related to the solvent functional groups, and "interfacial toxicity", related to the position of the enzyme at the interface. Likewise, some solvents are more prone to be located close to the active site of the enzyme, triggering other effects on the enzymatic performance. Thus, methyl tert-butyl ether resulted as an optimal option for the enzyme, whereas other solvents like toluene and 2-methyltetrahydrofuran were detrimental. The combined forces of experiments and simulations have been shown to be useful tools to study the effects of reaction media, thus guiding solvent selection.

AB - The use of oxidoreductases in organic-aqueous biphasic systems is advantageous (effective solvation of reactants, minimization of substrate/product-induced inhibition, improved volumetric productivity, and straightforward downstream processing). This paper explores the effects of organic solvents on horse liver alcohol dehydrogenase (HLADH) by combining experimental and computational studies. Various organic solvents displaying a broad range of hydrophobicity and functionalities are used, namely, ethyl acetate, 2-methyltetrahydrofuran, methyl tert-butyl ether, cyclopentyl methyl ether, toluene, cyclohexane, heptane, and dodecane. The catalytic performance of model enzyme horse liver alcohol dehydrogenase concerning its activity, stability, and selectivity is experimentally evaluated. The results are interpreted with molecular dynamics simulations by assessing the (i) protein location in biphasic media, (ii) organic solvent distribution, and (iii) enzyme conformation. Herein, the stability states the robustness of the enzyme while storing it in biphasic media without catalysis taking place. Overall, different toxicities of the solvent to the enzyme can be pinpointed: "molecular toxicity", related to the solvent functional groups, and "interfacial toxicity", related to the position of the enzyme at the interface. Likewise, some solvents are more prone to be located close to the active site of the enzyme, triggering other effects on the enzymatic performance. Thus, methyl tert-butyl ether resulted as an optimal option for the enzyme, whereas other solvents like toluene and 2-methyltetrahydrofuran were detrimental. The combined forces of experiments and simulations have been shown to be useful tools to study the effects of reaction media, thus guiding solvent selection.

KW - alcohol dehydrogenases

KW - biocatalysis

KW - experimental analysis

KW - molecular dynamics simulations

KW - organic-aqueous biphasic systems

UR - http://www.scopus.com/inward/record.url?scp=85136510947&partnerID=8YFLogxK

U2 - 10.1021/acscatal.2c02881

DO - 10.1021/acscatal.2c02881

M3 - Article

VL - 12

SP - 9171

EP - 9180

JO - ACS catalysis

JF - ACS catalysis

SN - 2155-5435

IS - 15

ER -