Influence of water‐miscible organic solvents on kinetics and enantioselectivity of the (R,‐specific alcohol dehydrogenase from Lactobacillus brevis

Abstract

Using the organic solvents acetonitrile and 1,4-dioxane as water-miscible additives for the alcohol dehydrogenase (ADH)-catalyzed reduction of butan-2-one, we investigated the influence of the solvents on enzyme reaction behavior and enantioselectivity. The NADP(+)-dependent (R)-selective ADH from Lactobacillus brevis (ADH-LB) was chosen as biocatalyst. For cofactor regeneration, the substrate-coupled approach using propan-2-ol as co-substrate was applied. Acetonitrile and 1,4-dioxane were tested from mole fraction 0.015 up to 0.1. Initial rate experiments revealed a complex kinetic behavior with enzyme activation caused by the substrate butan-2-one, and increasing K(M) values with increasing solvent concentration. Furthermore, these experiments showed an enhancement of the enantioselectivity for (R)-butan-2-ol from 37% enantiomeric excess (ee) in pure phosphate buffer up to 43% ee in the presence of 0.1 mol fraction acetonitrile. Finally, the influence of the co-solvents on water activity of the reaction mixture and on enzyme stability was investigated.

How carbonyl reductases control stereoselectivity: Approaching the goal of rational design

“…A few additional examples of tuning enantioselectivity of ketone reduction by changing the reaction medium have been reported for the alcohol dehydrogenases from Thermoanaerobacterium sp. KET4B1 [12], Lactobacillus brevis [13], and W110A mutant from Thermoanaerobacter ethanolicus [14,15].…”

Immobilized redox mediators for electrochemical NAD(P)+ regeneration

“…No reagents or by-products of the regeneration/substitution step should interfere with product isolation, and they should be compatible with the rest of the reaction system. In the literature, several enzyme-coupled (Geueke et al 2003;Riebel et al 2002Riebel et al , 2003Seelbach et al 1996) and substrate-coupled (Kroutil et al 2004b;Mertens et al 2003;Schumacher et al 2006;Stampfer et al 2002) cofactor regeneration systems are described. There are some disadvantages using an enzyme-coupled regeneration system: costs for a second enzyme and a co-substrate, interferences during product purification caused by secondary products, and reaction conditions have to be optimized for both enzyme reactions.…”

Reduction and cyclization in biotransformation of carbonyl compounds by cultured cells ofTaxusspecies

“…The use of organic solvents in enzymatic reactions is well documented (Klibanov 2001;Schumacher et al 2006). The solubility of rac-2-benzoylcyclohexanone in citrate-phosphate buffer is as low as 5 mg/ 20 mL, but if a mixture of dioxane-acetonitrile is added, the solubility increases.…”