Copolymeric Hydrogel-Based Immobilization of Yeast Cells for Continuous Biotransformation of Fumaric Acid in a Microreactor

Although enzymatic microbioreactors have recently gained lots of attention, reports on the use of whole cells as biocatalysts in microreactors have been rather modest. In this work, an efficient microreactor with permeabilized Saccharomyces cerevisiae cells was developed and used for continuous biotransformation of fumaric into industrially relevant L-malic acid. The immobilization of yeast cells was achieved by entrapment in a porous structure of various hydrogels. Copolymers based on different ratios of sodium alginate (SA) and polyvinyl alcohol (PVA) were used for hydrogel formation, while calcium chloride and boric or phenylboronic acid were tested as crosslinking agents for SA and PVA, respectively. The influence of hydrogel composition on physico-chemical properties of hydrogels prepared in the form of thin films was evaluated. Immobilization of permeabilized S. cerevisiae cells in the selected copolymeric hydrogel resulted in up to 72% retained fumarase activity. The continuous biotransformation process using two layers of hydrogels integrated into a two-plate microreactor revealed high space time yield of 2.86 g/(L·h) while no activity loss was recorded during 7 days of continuous operation.

Real Time Nuclear Magnetic Resonance Detection of Fumarase Activity using Parahydrogen-Hyperpolarized [1-13C]fumarate

Hyperpolarized fumarate can be used as a probe of real-time metabolism in vivo, using carbon-13 magnetic resonance imaging. Dissolution dynamic nuclear polarization is commonly used to produce hyperpolarized fumarate, but a cheaper and faster alternative is to produce hyperpolarized fumarate via PHIP (parahydrogen induced polarization). In this work we <i>trans</i>-hydrogenate [1-¹³C]acetylene dicarboxylate with <i>para</i>-enriched hydrogen using a commercially available Ru catalyst in water to produce hyperpolarized [1-¹³C]fumarate. We show that fumarate is produced in 89% yield, with succinate as a side product in 11% yield. The proton polarization is converted into ¹³C magnetization using a constant adiabaticity field cycle, and a polarization level of 25% is achieved using 86% <i>para</i>-enriched hydrogen gas. We inject the hyperpolarized [1-¹³C]fumarate into cell suspensions and track the metabolism. This work opens the path to greatly accelerated preclinical studies using fumarate as a biomarker.

Real Time Nuclear Magnetic Resonance Detection of Fumarase Activity using Parahydrogen-Hyperpolarized [1-13C]fumarate

Hyperpolarized fumarate can be used as a probe of real-time metabolism in vivo, using carbon-13 magnetic resonance imaging. Dissolution dynamic nuclear polarization is commonly used to produce hyperpolarized fumarate, but a cheaper and faster alternative is to produce hyperpolarized fumarate via PHIP (parahydrogen induced polarization). In this work we <i>trans</i>-hydrogenate [1-¹³C]acetylene dicarboxylate with <i>para</i>-enriched hydrogen using a commercially available Ru catalyst in water to produce hyperpolarized [1-¹³C]fumarate. We show that fumarate is produced in 89% yield, with succinate as a side product in 11% yield. The proton polarization is converted into ¹³C magnetization using a constant adiabaticity field cycle, and a polarization level of 25% is achieved using 86% <i>para</i>-enriched hydrogen gas. We inject the hyperpolarized [1-¹³C]fumarate into cell suspensions and track the metabolism. This work opens the path to greatly accelerated preclinical studies using fumarate as a biomarker.