An Engineered Alcohol Oxidase for the Oxidation of Primary Alcohols

Abstract

Structure‐guided directed evolution of choline oxidase has been carried out by using the oxidation of hexan‐1‐ol to hexanal as the target reaction. A six‐amino‐acid variant was identified with a 20‐fold increased kcat compared to that of the wild‐type enzyme. This variant enabled the oxidation of 10 mm hexanol to hexanal in less than 24 h with 100 % conversion. Furthermore, this variant showed a marked increase in thermostability with a corresponding increase in Tm of 20 °C. Improved solvent tolerance was demonstrated with organic solvents including ethyl acetate, heptane and cyclohexane, thereby enabling improved conversions to the aldehyde by up to 30 % above conversion for the solvent‐free system. Despite the evolution of choline oxidase towards hexan‐1‐ol, this new variant also showed increased specific activities (by up to 100‐fold) for around 50 primary aliphatic, unsaturated, branched, cyclic, benzylic and halogenated alcohols.

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

“…Another recent demonstration of the biocatalytic potential of a flavoprotein alcohol oxidase was recently reported by Turner and co‐workers. They showed that choline oxidase can be engineered towards a wider substrate acceptance, for the selective oxidation of primary alcohols to aldehydes …”

Natural Heterogeneous Catalysis with Immobilised Oxidase Biocatalysts

“…16,17,19 Increased use of alcohol oxidases is advantageous as the selective generation of aldehyde intermediates from alcohols has wide applications in the fine chemical industry and as part of biosynthetic cascades. 19,28 MAOs have been broadly exploited for the resolution and functionalization of amines but can be limited by substrate and product inhibition. 29,30 Immobilisation of these enzymes could overcome these limitations.…”

“…Particularly attractive oxidase biocatalysts for immobilisation are galactose oxidase (GOase), choline oxidase and monoamine oxidase from Aspergillus niger (MAO-N), as all have demonstrated broad applications in biocatalysis. [13][14][15] Previous engineering efforts for GOase and choline oxidase have developed a number of variants capable of oxidising a myriad of alcohols and have been demonstrated in both batch and flow reactor configurations, 1,[16][17][18][19][20] including the implementation of GOase in the gram scale biocatalytic manufacture of the investigational anti-HIV drug Islatravir. 21 Additionally, MAO-N versatility has been extensively shown in amine resolution and functionalization.…”

“…To further demonstrate this general immobilisation strategy, an engineered choline oxidase (AcCO6), 19 GOase M3-5 and MAO-N D9 were immobilized using the same procedure. Similarly to GOase M 1 , AcCO6, MAO-N D9 and GOase M 3-5 retained activity for over a week (supporting information).…”

Natural heterogeneous catalysis with immobilised oxidase biocatalysts

“…Particularly attractive oxidase biocatalysts for immobilisation are galactose oxidase (GOase), choline oxidase and monoamine oxidase from Aspergillus niger (MAO-N), as all have demonstrated broad applications in biocatalysis. [13][14][15] Previous engineering efforts for GOase and choline oxidase have developed a number of variants capable of oxidising a myriad of alcohols and have been demonstrated in both batch and ow reactor congurations, 1,[16][17][18][19][20] including the implementation of GOase in the gram scale biocatalytic manufacture of the investigational anti-HIV drug Islatravir. 21 Additionally, MAO-N versatility has been extensively shown in amine resolution and functionalization.…”