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Alcohol Oxidase

Mechanism + Description

Alcohol oxidases contain either a Cu or flavin adenine dinucleotide (FAD) cofactor and oxidise alcohols to aldehydes in a two-electron process thus producing H2O2 as a by-product.

 

General comments

Alcohols can be oxidised by ADH/KRED enzymes if the NAD/NADP cofactor recycle is pushed in the right direction. Alcohol oxidases use a different mechanism and use a metal or flavin cofactor O2 as the terminal oxidant generating peroxide. Since this by-product is highly deactivating to most enzymes, catalase is often added to cause dismutation to O2 and H2O. Thus, alcohol oxidase represents a selective green oxidation of primary alcohols to aldehydes avoiding stoichiometric heavy metals or hypervalent iodine reagents, etc. Alcohol oxidase have also been evolved that will oxidise secondary alcohols to ketones with O2.

 

Key references

Aalbers, F. S.; Fraaije, M. W. Design of Artificial Alcohol Oxidases: Alcohol Dehydrogenase–NADPH Oxidase Fusions for Continuous Oxidations. ChemBioChem 2018, 19, 51-56.

Van Schie, M. M. C. H.; de Almeida, T. P.; Laudadio, G.; Tieves, F.; Fernández-Fueyo, E.; Noël, T.; Arends, I. W. C. E.; Hollmann, F. Biocatalytic synthesis of the Green Note trans-2-hexenal in a continuous-flow microreactor. Beilstein J. Org. Chem. 2018, 14, 697–703.

Liu, J.; Wu, S.; Li, Z. Recent advances in enzymatic oxidation of alcohols. Curr. Opin. Chem. Biol. 2018, 43, 77–86.

Heath, R. S.; Birmingham, W. R.; Thompson, M. P.; Taglieber, A.; Daviet, L.; Turner, N. J. An Engineered Alcohol Oxidase for the Oxidation of Primary Alcohols. ChemBioChem. 2019, 20, 276–281.

Ewing, T. A.; Kühn, J.; Segarra, S.; Tortajada, M.; Zuhse, R.; van Berkel, W. J. H. Multigram Scale Enzymatic Synthesis of (R)-1-(4′-Hydroxyphenyl)ethanol Using Vanillyl Alcohol Oxidase. Adv. Synth. Catal. 2018, 360, 2370–2376.

 

Relevant scale up examples

Org. Process Res. Dev. 2015, 19,1580−1589
5 g scale

 

Green Review

  1. Atom efficiency (by-products, molecular weight)
    Catalytic technology—but enzyme loading should be optimized and steps using stoichiometric reagents like cofactor recycle should be optimized to avoid unnecessary excess of reagents.
  2. Safety Concerns
    Generally considered a safe technology to scale-up. Issues—enzymes/proteins can be sensitizers by inhalation, and some by mode of action can be skin irritants (proteases). If viable GMO cells are used, local regulations relating to use of GMOs need to be followed.
  3. Toxicity and environmental/aquatic impact
    No real concerns—enzymes are non-toxic and readily biodegradable. If viable GMO cells are used, these need to be passivated before discharge into the environment.
  4. Cost, availability & sustainable feedstocks
    Enzymes are produced from natural sustainable feedstocks.
  5. Sustainable implications
    Enzymes are made via fermentation; cloned and recombinant enzymes would be at no risk from depletion. Enzymes break down in the environment and the constituent amino acids are recycled in nature. The use of over expressed recombinant enzymes is generally much better than natural enzymes on a life cycle impact basis.