Directed Evolution and Predictive Modelling of Galactose Oxidase towards Bulky Benzylic and Unactivated Secondary Alcohols

17 February 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


The growth of industrial biocatalysis for sustainable chemical manufacturing has been limited by the narrow range of chemistries associated with natural enzymes and experiment-intensive regimes of enzyme engineering. Consequently, there has been deep interest to expand enzyme substrate scopes for broader synthetic utility, and to streamline the enzyme engineering process. In the field of alcohol oxidation, galactose oxidase (GOase) is one of the most established enzymes capable of this important chemical transformation under benign conditions. However, the applicability of GOase towards more complex molecules such as those frequently found in the pharmaceutical, or agrochemical industries remains restricted. Here, by employing a combined approach of directed evolution and predictive modelling, we have identified new GOases with significantly expanded substrate specificity toward both bulky benzylic and unactivated secondary alcohols, showing activity enhancements of up to 2,400-fold compared to the reported benchmark M3-5 mutant. Beneficial mutations conveying relaxed substrate enantioselectivity biases (R/S ratios down to 1.05) and higher thermostabilities (up to 20-fold versus benchmark) have also been identified. We have developed predictive models based on computational tools YASARA, FoldX, SCWRL and Glide that are well correlated with features related to enzyme structure, selectivity, protein stability and catalytic activity. The generated enzyme activity models based on Glide-MM/GBSA (r = -0.85) and YASARA (r = -0.89) have successfully predicted the activity trend of a family of related substrates based on the 1-phenyl-1-alkyl alcohol scaffold with varying alkyl chain lengths. It is envisioned that these in silico models can serve as valuable tools to explore desirable enzyme characteristics, establish enzyme substrate scopes, and accelerate biocatalyst development, thus promoting it as a competitive and competent solution for sustainable chemical manufacturing.


Galactose oxidase
alcohol oxidation
directed evolution
high throughput

Supplementary materials

Supplementary Infomation
Contains materials information, as well as analytical, assay development and modelling methods.


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