Applications of galactose oxidase in biocatalysis

Abstract

Enzymatic oxidations are reaching maturity for use in organic syntheses however there are still major limitations that prevent universal application. Oxidase enzymes are biocatalysts that mediate selective oxidation with molecular oxygen as the co-substrate. Galactose oxidase (GOase) is a copper radical alcohol oxidase that has been heavily engineered towards a diverse substrate scope including glucosides, sialosides and API precursors. At analytical scale these bio-oxidations are extremely efficient but scaling represents a major bottleneck mainly owing to the poor solubility of molecular oxygen and lack of enzyme stability at process conditions. To overcome such issues, novel continuous flow systems and enzyme immobilization strategies are being developed to greatly improve productivity with immobilization permitting enhanced stability and reusability. In this project, GOase variants were initially tested against bioactive carbohydrates, in particular sialic acids, which are found decorating various components in biological systems. A particular challenge is differentiating the potentially immunogenic N-glycolyl neuraminic acid (Neu5Gc) from its more common precursor N-acetyl neuraminic acid (Neu5Ac). Here an engineered GOase variant was used to selectively oxidise Neu5Gc with no activity towards Neu5Ac, this oxidation combined with a sialidase mediated cleavage enables selective detection of Neu5Gc in complex biological samples such as the biopharmaceutical alpha acid glycoprotein. After the successful demonstration of GOase as an analytical tool, the bio-oxidations were scaled under continuous conditions. Here a multipoint injection reactor (MPIR) was used in the bio-oxidation of lactose with major improvements in space time yield (STY) and productivity. Immobilized oxidases were then applied as heterogeneous catalysts with immobilization enabling the re-use of enzymes under processing conditions. Finally, immobilized and free enzymes were combined in newly developed continuous flow systems to enable biocatalytic cascades towards high value amine intermediates that would be unattainable under conventional batch conditions.

Date of Award	31 Dec 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Sabine Flitsch (Supervisor) & Nicholas Turner (Supervisor)