An Alcohol Dehydrogenase from the Short‐Chain Dehydrogenase/Reductase Family of Enzymes for the Lactonization of Hexane‐1,6‐diol

Abstract

Biocatalytic production of lactones, and in particular ϵ‐caprolactone (CL), have gained increasing interest as a greener route to polymer building blocks, especially through the use of Baeyer–Villiger monooxygenases (BVMOs). Despite several advances in the field, BVMOs, however, still suffer several practical limitations. Alcohol dehydrogenase (ADH)‐mediated lactonization of diols in turn has received far less attention and very few enzymes have been identified for the conversion of diols to lactones, with horse‐liver ADH (HLADH) remaining the catalyst of choice. Screening of a diverse panel of ADHs, AaSDR‐1, a member of the short‐chain dehydrogenase/reductase family, was found to produce ϵ‐caprolactone from hexane‐1,6‐diol. Moreover, cofactor regeneration by an NADH oxidase eliminated the requirement of co‐substrates, yielding water as the sole by‐product. Despite lower turnover frequencies as compared to HLADH, higher selectivity was found for the production of CL, with HLADH forming significant amounts of 6‐hydroxyhexanoic acid and adipic acid through aldehyde dehydrogenation/oxidation of the gem‐diol intermediates. Also, CL yield were shown to be dependent on buffer choice, as structural elucidation of a Tris adduct confirmed the buffer amine to react with aliphatic aldehydes forming a Schiff‐base intermediate which through further ADH oxidation, forms a tricyclic acetal product.

Production of Hydroxy Acids: Selective Double Oxidation of Diols by Flavoprotein Alcohol Oxidase

“…Enzymatic synthesis of lactones as polyester building blocks gained great interest as an environmentally sustainable alternative to current chemical methods. The most common biocatalysts to produce lactones are Baeyer–Villiger monooxygenases (BVMOs) and alcohol dehydrogenases (ADHs) . These two classes of enzymes require cofactor NAD(P)H regeneration, making these strategies less suitable for commercial applications.…”

CYP505E3: A Novel Self‐Sufficient ω‐7 In‐Chain Hydroxylase

“…Both these routes require extensive optimization, most likely involving both protein and process engineering. The latter could include using whole cells of E. coli or perhaps a yeast, screening for other alcohol dehydrogenases or oxidases for oxidation of the 1,5‐diol, as well as in situ product removal to avoid over hydroxylation. Hydroxylation of tetradecanoic acid followed by one round of β‐oxidation and lactonisation could in principle also yield δ‐dodecalactone, but would require the expression of CYP505E3 in an organism, probably a yeast, with partially disrupted β‐oxidation …”

Production of Hydroxy Acids: Selective Double Oxidation of Diols by Flavoprotein Alcohol Oxidase

“…We selected just a few to test AOX* (substrates 11, 12, and 13; Table 1). Cyclohexanol (11) was oxidized to cyclohexanone with a low yield. Initially, we tested racemic 1phenylethanol and its conversion was rather low, similar to cyclohexanol.…”

“…The most common biocatalysts to produce lactones are Baeyer-Villiger monooxygenases (BVMOs) and alcohol dehydrogenases (ADHs). [10][11][12][13] These two classes of enzymes require cofactor NAD(P)H regeneration, making these strategies less suitable for commercial applications. Oxidases are an attractive class of enzymes for the production of bulk chemicals since they use oxygen either as an oxidant without the need to regenerate cofactors.…”