Toward scalable biocatalytic conversion of 5-hydroxymethylfurfural by galactose oxidase using coordinated reaction and enzyme engineering.

5-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.

Considerations when Measuring Biocatalyst Performance.

As biocatalysis matures, it becomes increasingly important to establish methods with which to measure biocatalyst performance. Such measurements are important to assess immobilization strategies, different operating modes, and reactor configurations, aside from comparing protein engineered variants and benchmarking against economic targets. While conventional measurement techniques focus on a single performance metric (such as the total turnover number), here, it is argued that three metrics (achievable product concentration, productivity, and enzyme stability) are required for an accurate assessment of scalability.

Use of image analysis to understand enzyme stability in an aerated stirred reactor.

Efficient regeneration of NAD(P)+ cofactors is essential for large-scale application of alcohol dehydrogenases due to the high cost and chemical instability of these cofactors. NAD(P)+ can be regenerated effectively using NAD(P)H oxidases (NOXs) that require molecular oxygen as a cosubstrate. In large-scale biocatalytic processes, agitation and aeration are needed for sufficient oxygen transfer into the liquid phase, both of which have been shown to significantly increase the rate of enzyme deactivation. As such, the aim of this study was to identify the existence of a correlation between enzyme stability and gas-liquid interfacial area inside the bioreactor. This was done by measuring gas-liquid interfacial areas inside an aerated stirred reactor, using an in situ optical probe, and simultaneously measuring the kinetic stability of NOXs. Following enzyme incubation at various power inputs and gas-phase compositions, the residual activity was assessed and video samples were analyzed through an image processing algorithm. Enzyme deactivation was found to be proportional to an increase in interfacial area up to a certain limit, where power input appears to have a higher impact. Furthermore, the presence of oxygen increased enzyme deactivation rates at low interfacial areas. The areas were validated with defined glass beads and found to be in the range of those in large-scale bioreactors. Finally, a correlation between the enzyme half-life and specific interfacial area was obtained. Therefore, we conclude that the method developed in this contribution can help to predict the behavior of biocatalyst stability under industrially relevant conditions, concerning specific gas-liquid interfacial areas.