Overcoming Biochemical Limitations of Galactose Oxidase through the Design of a Solid‐Supported Self‐Sufficient Biocatalyst

• Published in: Chembiochem : a European journal of chemical biology 2023-12, Vol.24 (24), p.e202300421-n/a
• Main Authors: Lorente‐Arevalo, Alvaro, Orellana, Guillermo, Ladero, Miguel, Bolivar, Juan M.
• Format: Article
• Language: English
• Subjects: Aeration; Biocatalysis; Biocatalysts; biooxidation; Catalysis; Catalysts; catalytic effectiveness; Dextran; Dextran sulfate; Dextrans; enzyme immobilization; Enzymes; Enzymes, Immobilized - metabolism; Galactose; Galactose oxidase; Galactose Oxidase - metabolism; Hemeproteins - metabolism; Oxidase; Oxidation; Oxygen; oxygen monitoring; Proteins; Sepharose; Stability
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.202300421

Galactose Oxidase (GalOx) has gained significant interest in biocatalysis due to its ability for selective oxidation beyond the natural oxidation of galactose, enabling the production of valuable derivatives. However, the practical application of GalOx has been hindered by the limited availability of active and stable biocatalysts, as well as the inherent biochemical limitations such as oxygen (O2) dependency and the need for activation. In this study, we addressed these challenges by immobilizing GalOx into agarose‐based and Purolite supports to enhance its activity and stability. Additionally, we identified and quantified the oxygen supply limitation into solid catalysts by intraparticle oxygen sensing showing a trade‐off between the amount of protein loaded onto the solid support and the catalytic effectiveness of the immobilized enzyme. Furthermore, we coimmobilized a heme‐containing protein along with the enzyme to function as an activator. To evaluate the practical application of the immobilized GalOx, we conducted the oxidation of galactose in an instrumented aerated reactor. The results showcased the efficient performance of the immobilized enzyme in the 8 h reaction cycle. Notably, the GalOx immobilized into dextran sulfate‐activated agarose exhibited improved stability, overcoming the need for a soluble activator supply, and demonstrated exceptional performance in galactose oxidation. These findings offer promising prospects for the utilization of GalOx in technical biocatalytic applications. This paper presents the novel design of an active and stable self‐sufficient galactose oxidase (GalOx) biocatalyst by co‐immobilization of a hemoprotein as activator. The study of its diffusional constraints by co‐immobilization of a luminescent compound enables the quantification of the oxygen concentration inside the particle. Finally, its application in the oxidation of galactose in an aerated bioreactor is presented.