Abstract
Alcohol dehydrogenase from Bacillus (Geobacillus) stearothermophilus (BsADH) is a NADH-dependent enzyme catalyzing the oxidation of alcohols, however its thermal and operational stabilities are too low for its long-term use under non-physiological conditions. Enzyme immobilizations emerges as an attractive tool to enhance the stability of this enzyme. In this work, we have screened a battery of porous carriers and immobilization chemistries to enhance the robustness of a His-tagged variant of BsADH. The selected carriers recovered close to 50% of the immobilized activity and increased enzyme stability from 3 to 9 times compared to the free enzyme. We found a trade-off between the half-life time and the specific activity as a function of the relative anisotropy values of the immobilized enzymes, suggesting that both properties are oppositely related to the enzyme mobility (rotational tumbling). The most thermally stable heterogeneous biocatalysts were coupled with a NADH oxidase/catalase pair co-immobilized on porous agarose beads to perform the batch oxidation of five different 1,ω-diols with in situ recycling of NAD+. Only when His-tagged BsADH was immobilized on porous glass functionalized with Fe3+, the heterogeneous biocatalyst oxidized 1, 5-pentanediol with a conversion higher than 50% after five batch cycles. This immobilized multi-enzyme system presented promising enzymatic productivities towards the oxidation of three different diols. Hence, this strategical study accompanied by a functional and structural characterization of the resulting immobilized enzymes, allowed us selecting an optimal heterogeneous biocatalyst and their integration into a fully heterogeneous multi-enzyme system.
Highlights
Oxidation reactions have been employed as one of the most useful reactions in chemical manufacturing to produce aldehydes as building blocks for the synthesis of more complex molecules such as carboxylic acids and aminoalcohols (Velasco-Lozano et al, 2020; Wu et al, 2021)
Neither was the enzyme released from EziG1 it was immobilized through reversible interactions between the His-tag and the Fe3+-catechol complexes (Supplementary Figure 2)
Once the immobilization was characterized, we investigated the spatial organization of His-BsADH across the surface of the different porous carriers through confocal laser scanning microscopy (CLSM) imaging and through 3D image reconstruction (Figure 1)
Summary
Oxidation reactions have been employed as one of the most useful reactions in chemical manufacturing to produce aldehydes as building blocks for the synthesis of more complex molecules such as carboxylic acids and aminoalcohols (Velasco-Lozano et al, 2020; Wu et al, 2021). Alcohol dehydrogenases (ADHs; EC 1.1.1.1) are widely used for the oxidation of alcohols in combination with redox nicotinamide cofactors (NAD(P)+) as hydride acceptors (Kara et al, 2013) Within this enzyme family, ADH from Bacillus (Geobacillus) stearothermophilus (BsADH) have been successfully exploited for the selective and versatile oxidation of 1,ω-diols (Kirmair et al, 2015). BsADH catalyzes the hydride transfer from one hydroxyl group of the substrate to NAD+ through a compulsory ordered mechanism similar to other alcohol dehydrogenases (Dickinson and Monger, 1973). For this reason, BsADH requires an in situ recycling of NAD+ when exploited in applied biocatalysis. Several enzymatic and chemoenzymatic recycling systems have been proposed for this type of biotransformations using laccases (Pham et al, 2015) and NADH oxidases (Nowak et al, 2015) among others
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