TADH, the thermostable alcohol dehydrogenase from Thermus sp. ATN1: a versatile new biocatalyst for organic synthesis

Abstract

The alcohol dehydrogenase from Thermus sp. ATN1 (TADH) was characterized biochemically with respect to its potential as a biocatalyst for organic synthesis. TADH is a NAD(H)-dependent enzyme and shows a very broad substrate spectrum producing exclusively the (S)-enantiomer in high enantiomeric excess (>99%) during asymmetric reduction of ketones. TADH is active in the presence of 10% (v/v) water-miscible solvents like 2-propanol or acetone, which permits the use of these solvents as sacrificial substrates in substrate-coupled cofactor regeneration approaches. Furthermore, the presence of a second phase of a water-insoluble solvent like hexane or octane had only minor effects on the enzyme, which retained 80% of its activity, allowing the use of these solvents in aqueous/organic mixtures to increase the availability of low-water soluble substrates. A further activity of TADH, the production of carboxylic acids by dismutation of aldehydes, was investigated. This reaction usually proceeds without net change of the NAD(+)/NADH concentration, leading to equimolar amounts of alcohol and carboxylic acid. When applying cofactor regeneration at high pH, however, the ratio of acid to alcohol could be changed, and full conversion to the carboxylic acid was achieved.

Characterization of an Allylic/Benzyl Alcohol Dehydrogenase from Yokenella sp. Strain WZY002, an Organism Potentially Useful for the Synthesis of α,β-Unsaturated Alcohols from Allylic Aldehydes and Ketones

“…The enzyme was active within a broad temperature range, from 25 to 75°C, in the reduction reaction, while its apparent V max value for crotonaldehyde reduction was as high as 629.4 Ϯ 25.2 U mg Ϫ1 at 65°C, which was remarkably higher than those of other allylic/benzyl alcohol dehydrogenases characterized (13)(14)(15)(16). The enzyme retained 54.5% of the initial activity after 6 h of heat treatment at 55°C, and this moderate thermostability makes it commercially more attractive than its mesophilic counterparts (36).…”

“…Some water-soluble solvents, such as ethanol and 1-propanol, served as the substrate for YsADH, which makes substrate-coupled cofactor regeneration feasible. In addition, YsADH also showed high activity for all the immiscible solvents examined, which can be used as the substrate reservoir and product sink to avoid substrate or product inhibition (36). Given the importance of activity and stability, YsADH could serve as a robust biocatalyst in the synthesis of ␣,␤-unsaturated alcohols from allylic aldehydes.…”

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application

“…For example, Thermus sp. ATN1 alcohol dehydrogenase (TADH) exhibits activity towards a very large range of different alcohols with multiple different functional groups (including both aliphatic and aromatic alcohols) [59], while Zymomonas mobilis alcohol dehydrogenase 2 is only able to oxidize ethanol, 1-propanol and allyl alcohol [60].…”

Structures of Alcohol Dehydrogenases from Ralstonia and Sphingobium spp. Reveal the Molecular Basis for Their Recognition of ‘Bulky–Bulky’ Ketones

“…The natural diversity of ADHs has ensured that enzymes encompassing a broad range of catalytic characteristics have been made available for applications. Hence there are ADHs that offer (R)- [3] or (S)- [4] selectivity, thermostability [5,6] and an impressive tolerance to organic solvents that allows the enzymes to be used in the presence of high substrate concentration [4] or even neat substrate itself [7]. One such niche application of ADHs is served by a subgroup of these enzymes that transforms prochiral ketones that feature large hydrophobic groups on either side of the carbonyl group; 'bulky-bulky' ketones.…”