Immobilization Screening and Characterization of an Alcohol Dehydrogenase and its Application to the Multi-Enzymatic Selective Oxidation of 1,-Omega-Diols

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.

Cell‐Free Biosynthesis of ω‐Hydroxy Acids Boosted by a Synergistic Combination of Alcohol Dehydrogenases

“…This insight entails BsADH as the specialist enzyme for the first oxidation within this enzyme cascade. A similar product distribution was found using the immobilized version of this enzyme towards the same substrate and under similar reaction conditions [21] . Likewise, when the system was performed by the HLADH as the unique ADH (even at different amounts, 40 (ratio 0 : 1) or 120 mU(ratio 0 : 3)), the transformation only reached 60 % of ω‐HA yield, similar to the yield achieved with the combination of the two ADHs using an activity ratio 4 : 1.…”

“…A similar product distribution was found using the immobilized version of this enzyme towards the same substrate and under similar reaction conditions. [21] Likewise, when the system was performed by the HLADH as the unique ADH (even at different amounts, 40 (ratio 0 : 1) or 120 mU(ratio 0 : 3)), the transformation only reached 60 % of ω-HA yield, similar to the yield achieved with the combination of the two ADHs using an activity ratio 4 : 1. Pleasantly, the activity ratio 4 : 3 (BsADH/HLADH) achieved the highest ω-HA yield (97 %) with a quantitative conversion of the diol (2 a), attaining a total turnover number for NAD + (TTN NAD þ ) of 38.8 (close to the theoretical maximum of 40).…”

“…S2), together with an excess of the widely used NAD + recycling system formed by a pure thermostable NOX from Thermus thermophilus (TtNOX) and the catalase from bovine liver (BlCAT) (commercial crude extract) [20] . We did not select the water‐forming NOX, since our previous studies show that the TtNOX outperforms the former one exhibiting higher operational stability [20b,21] . Furthermore, the tandem TtNOX BlCAT stoichiometrically demands half of the oxygen for the NAD + recycling than the water‐forming oxidases, a fact that positively impacts on the atom economy of the process.…”

Intraparticle Kinetics Unveil Crowding and Enzyme Distribution Effects on the Performance of Cofactor-Dependent Heterogeneous Biocatalysts

“…As a result, the enzymes are oriented through their His-tag at the N-terminus and irreversibly attached to the surface of the carriers via covalent bonds formed between the epoxy groups and the Lys residues neighboring the His-tag ( Figure 1 ). 31 Irreversible attachment of the enzymes to the AG-Co 2+ /E surface was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis ( Figure S1 ). For the co-immobilization of the phosphorylated cofactors, the microbeads that harbor the immobilized enzymes were coated with the following cationic polymers ( Figure 1 ): polyethyleneimine (PEI), polyallylamine (PAH), and polydiallyldimethylammonium chloride (PDADMAC), which contain different types of amines whose chemical structures are illustrated in Table 1 .…”

Intraparticle Macromolecular Migration Alters the Structure and Function of Proteins Reversibly Immobilized on Porous Microbeads

“…Irreversible immobilization was achieved through covalent bonds formed between the nucleophile and solvent exposed residues (Lys, His, Cys, or Tyr) at the protein surface and the epoxy groups at the surface of the agarose microbeads functionalized with both cobalt-chelates and epoxy groups (AG-Co 2+ /E, Figure 6a). [33,34] Irreversible immobilization of the enzymes at the AG-Co 2+ /E surface was demonstrated by SDS-PAGE analysis (Figure S5, Supporting Information) as bound proteins were not eluted from the support upon their incubation under denaturing conditions (100 °C in Laemmli buffer). Reversibly and irreversibly immobilized proteins were incubated for 15 days at 4 °C, (typical storage conditions).…”