Galactaric acid production by engineering substrate specificity in glucose oxidase from Aspergillus niger

Sugar oxidation and its products, such as organic acids derived from monosaccharides, have become relevant resources following global industrial sustainability concerns. Galactaric acid is a promising renewable chemical building block, potentially replacing fossil-based terephthalic acid derivatives and adding value to pectin-rich biomass. However, novel biocatalysts able to produce galactaric acid from the abundant pectin building block, galacturonic acid, are needed. In this work, glucose oxidase from Aspergillus niger was subjected to protein engineering to modify its substrate specificity away from glucose and towards galacturonic acid for galactaric acid production. The resulting mutagenesis libraries were screened for activity against galacturonic acid, iden-tifying two variants having amino acid substitutions F414K and Y68W. These variants catalyze galacturonic acid conversion in the presence of catalase, with minimal glucose oxidation activity. In silico amino acid substitution analysis in the active site suggests changes in substrate orientation for galacturonic acid, resembling that of glucose in the parental glucose oxidase. Thermal performance and pH stability experiments on the identified variants suggest further structural and functional differences, potentially related to the observed changes in catalytic performance. Finally, in a one-pot reaction with pectinase, the release of galacturonic acid from citrus pectin and its oxidation to galactaric acid was measured by HPLC, showing that simultaneous pectin depoly-merization by pectinase and the production of galactaric acid from the released galacturonic acid by the engi-neered glucose oxidase is possible. This study provides a proof of concept for a potential enzymatic production of hexaric acid building blocks from complex macromolecules such as pectin, using well-established enzymes, such as pectinase preparations and engineered glucose oxidases.

Automated summary

This study demonstrates the potential enzymatic production of galactaric acid from galacturonic acid by protein engineering of glucose oxidase. Two variants of glucose oxidase with amino acid substitutions F414K and Y68W were identified and showed catalytic activity towards galacturonic acid with minimal glucose oxidation activity. Further experiments indicate structural and functional differences in the variants. Additionally, the study shows the possibility of simultaneous pectin depolymerization and galactaric acid production from citrus pectin using engineered glucose oxidases and pectinase.