Biocatalytic oxidation reactions have the potential to substitute many chemically catalyzed oxidations in the pharmaceutical and fine chemical industry due to their superior regio-and stereoselectivity and low environmental impact. Galactose oxidase (GOase) has been shown to be a promising biocatalyst for the oxidation of primary and secondary alcohols to their corresponding aldehydes and ketones, respectively. However, GOase requires a number of additives to sustain its catalytic function, such as the enzyme catalase for degradation of the byproduct hydrogen peroxide as well as single-electron oxidants to reactivate the enzyme upon loss of the amino acid radical in its active site. In this work, the addition of catalase, single-electron oxidants, and copper ions was investigated systematically in order to find the minimum concentrations required to obtain a fully active GOase. Furthermore, it was found that the concentration and type of buffer is essential for the activity of GOase, which was significantly more active in sodium phosphate buffer than in other buffers investigated. Enzyme stability and oxygen requirements are of crucial importance for the implementation of oxidase based processes. GOase was shown to be completely stable for 120 h in buffer with stirring at 25 °C, and the activity even increased 30% if the enzyme solution was also aerated in a similar experiment. The high K m for oxygen of GOase (>5 mM) relative to the solubility of oxygen in water reveals a trade-off between supplying oxygen at a sufficiently high rate and ensuring a high degree of enzyme utilization (i.e., ensuring the highest possible specific rate of reaction). Nevertheless, the good stability and high activity of GOase bode well for its future application as an industrial biocatalyst.

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Abstract5-Hydroxymethylfurfural (HMF) has emerged as a crucial bio-based chemical building block in the drive towards developing materials from renewable resources, due to its direct preparation from sugars and its readily diversifiable scaffold. A key obstacle in transitioning to bio-based plastic production lies in meeting the necessary industrial production efficiency, particularly in the cost-effective conversion of HMF to valuable intermediates. Toward addressing the challenge of developing scalable technology for oxidizing crude HMF to more valuable chemicals, here we report coordinated reaction and enzyme engineering to provide a galactose oxidase (GOase) variant with remarkably high activity toward HMF, improved O2 binding and excellent productivity (>1,000,000 TTN). The biocatalyst and reaction conditions presented here for GOase catalysed selective oxidation of HMF to 2,5-diformylfuran offers a productive blueprint for further development, giving hope for the creation of a biocatalytic route to scalable production of furan-based chemical building blocks from sustainable feedstocks.