Ultrafiltration Cleans Up Environmentally Friendly Workflow Design

Increasing environmental concerns are prompting many companies to rethink the biochemistry behind the manufacture of their products. Glucaric acid (GlucA) is a glucose-derived chemical that can potentially be used as starting material for a wide variety of more environmentally friendly, biodegradable plastic products.

The problem with ramping up usage of this chemical is that GlucA production is itself highly inefficient, involving toxic chemicals, and consuming large quantities of necessary co-factors. The Australian research group aimed to address these issues by devising an alternative, more sustainable GlucA production pathway.

Recent studies have focused on using microbial cell-based production of GlucA as a more cost-effective solution, but results have shown that these methods are still inefficient, producing low yields and requiring conditions that are toxic to most microbes.

For this study, the scientists set out to overcome the limitations of previous GlucA manufacturing pathways by using a cell-free biocatalysis approach to GlucA production. Biocatalysis uses biological enzymes in place of chemicals to create a more sustainable production workflow. Making the approach cell-free decouples the production pathway from cellular growth, allowing for more flexibility in the mixtures and concentrations of necessary enzymes, thus making the whole process more efficient.

The researchers first task was to immobilize the necessary enzymes on a synthetic substrate, and then validate that enzymatic activity was not affected by this process. Multi-enzyme conversion of GlucA from its precursor, glucose-1-phosphate, was then optimized by testing a panel of conditions which included varying the relative concentration of GlucA production enzymes, and the reaction duration.

After carrying out the enzymatic reactions, the scientists prepared to run an analysis of reaction metabolite results to evaluate the efficiency of each condition. The group used AcroPrep™ Advance ultrafiltration plates with an Omega™ 10K MWCO (molecular weight cutoff) membrane for this portion of their work (Fig 1.). Ultrafiltration plates were attached to a Pall vacuum manifold to purify the metabolite samples and stop the enzymatic reactions.