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Galactose oxidase has long captured the interest of the biocatalysis and biotechnology communities due to its unique catalytic characteristics and versatility with various substrates. In our previous studies, we demonstrated that galactose oxidase GAO-5F from Fusarium odoratissimum can oxidize agarose to produce a polyaldehyde polymer, which can be further crosslinked with gelatin to produce food packaging films. Despite its commendable catalytic performance, GAO-5F falls short of meeting the requirements for industrial applications. In this study, we employed a combination of multiple sequence comparisons and site-directed mutagenesis to pinpoint key amino acid sites crucial for enhancing the enzyme's catalytic activity, resulting in the creation of the double mutant GAO-5F/AR (D403A/Q484R), showing a six-fold increase in catalytic activity. The catalytic mechanism of mutant was further elucidated through homology modeling and molecular docking. Results highlighted the significance of increased hydrogen bonding interactions between the enzyme and substrate in enhancing catalytic activity. Then, agarose was transformed into a polyaldehyde polymer by oxidation catalyzed by GAO-5F mutant. The resulting polyaldehyde polymer was crosslinked with gelatin to prepare an edible packaging film; the properties and structure of the film were characterized. In this study, we successfully obtained mutants with increased catalytic activity through a semi-rational-driven site-directed mutagenesis strategy. This approach, which combines rational design with targeted mutagenesis, holds promise for furthering our understanding of enzyme function and may find widespread use in comparative functional genomics studies of other natural enzymes. This study provides valuable insights for the improvement of galactose oxidase, and new ideas for the preparation of edible packaging films for use in the food industry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The SCO2837 open-reading frame is located within the conserved central core region of the Streptomyces coelicolor A3(2) genome, which contains genes required for essential cellular functions. SCO2837 protein (SCO2837p) expressed by Pichia pastoris is a copper metalloenzyme, catalyzing the oxidation of simple alcohols to aldehydes and reduction of dioxygen to hydrogen peroxide. Distinct optical absorption spectra are observed for oxidized and one-electron reduced holoenzyme, and a free radical EPR signal is present in the oxidized apoprotein, characteristic of the Tyr-Cys redox cofactor previously reported for fungal secretory radical copper oxidases, galactose oxidase and glyoxal oxidase, with which it shares weak sequence similarity. SCO2837p was detected in the growth medium of both S. coelicolor and a recombinant expression host (Streptomyces lividans TK64) by Western blotting, with the expression level dependent on the nature of the carbon source. This represents the first characterized example of a prokaryotic radical copper oxidase.

The effect of ethanol and tunicamycin on synthesis and secretion of galactose oxidase was studied in resting cells of Dactylium dendroides. Ethanol promoted an overall decrease in both intra- and extracellular enzyme levels to the same extent that it inhibited [14C]glucosamine incorporation into total protein. The carbohydrate content of the intracellular enzyme was also depressed (44%) with a simultaneous decrease in O-Ser linked oligosaccharides. The intracellular galactose oxidase obtained after exposure of mycelia to ethanol plus tunicamycin lost 86% of its carbohydrate moieties, whereas the extracellular form lost only 35%. In both cases, residual sugar moieties were not eliminated by mild alkaline treatment. These data suggest that ethanol affects O-glycosylation of galactose oxidase. O-Underglycosylation did not affect the S0.5 values for galactose but diminished the molar catalytic activity. The absence of O-Ser/Thr-linked saccharides turned the intracellular enzyme into a form more susceptible to proteolysis than that devoid of N-linked sugars (tunicamycin-treated). O-Underglycosylation had a significant effect on the renaturation-reactivation of the enzyme after denaturation with 2.4 M Gdn-HCl.