Repurposing Inflatable Packaging Pillows as Bioreactors: a Convenient Synthesis of Glucosone by Whole-Cell Catalysis Under Oxygen

Michael D Mozuch,Thomas J Schwartz,Philip J Kersten,Kolby C Hirth

doi:10.1007/s12010-020-03448-x

Michael D Mozuch, Thomas J Schwartz + Show 2 more

Open Access

https://doi.org/10.1007/s12010-020-03448-x

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Abstract

Biocatalysis using molecular oxygen as the electron acceptor has significant potential for selective oxidations at low cost. However, oxygen is poorly soluble in water, and its slow rate of mass transfer in the aqueous phase is a major obstacle, even for laboratory-scale syntheses. Oxygen transfer can be accelerated by vigorous mechanical methods, but these are often incompatible with biological catalysts. Gentler conditions can be achieved with shallow, high surface area bag reactors that are designed for single use and generally for specialized cell culture applications. As a less-expensive alternative to these high-end bioreactors, we describe repurposing inflatable shipping pillows with resealable valves to provide high surface area mixing under oxygen for preparative synthesis of glucosone (D-arabino-hexos-2-ulose) from D-glucose using non-growing Escherichia coli whole cells containing recombinant pyranose 2-oxidase (POX) as catalyst. Parallel reactions permitted systematic study of the effects of headspace composition (i.e., air vs 100% oxygen), cell density, exogenous catalase, and reaction volume in the oxidation of 10% glucose. Importantly, only a single charge of 100% oxygen is required for stoichiometric conversion on a multi-gram scale in 18 h with resting cells, and the conversion was successfully repeated with recycled cells.

Highlights

Molecular oxygen is considered the “greenest” oxidant in organic synthesis, and it has garnered increasing interest both in large industrial-scale syntheses and in fine chemical manufacture [1,2,3]
One challenge with the use of oxygen in chemical synthesis is its limited solubility in solvents, water, which leads to reaction rates that are controlled by the rate of mass transfer of oxygen from gas to liquid phase rather than on intrinsic kinetics [4]
Aggressive mechanical dispersive methods to increase oxygen transfer often inactivate enzymes and whole-cell catalysts, thereby making reactor design to address these issues an active area of research [7, 8]. To address this challenge and demonstrate the utility of an inexpensive bag reactor, we focus on the synthesis of glucosone from glucose on a multi-gram scale with molecular oxygen as the two-electron acceptor for oxidation of glucose, and with pyranose 2-oxidase as the regio-selective catalyst (Fig. 1)

Summary

Introduction

Molecular oxygen is considered the “greenest” oxidant in organic synthesis, and it has garnered increasing interest both in large industrial-scale syntheses and in fine chemical manufacture [1,2,3]. One challenge with the use of oxygen in chemical synthesis is its limited solubility in solvents, water, which leads to reaction rates that are controlled by the rate of mass transfer of oxygen from gas to liquid phase rather than on intrinsic kinetics [4]. Oxygen-dependent enzymes provide an alternative to chemical catalysts and have potential advantages in achieving selective oxidations in an environmentally sound manner [5, 6]. These reactions suffer the same reaction rate limitations associated with oxygen transfer from gas to liquid phase.

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