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Ultrafiltration Cleans Up Environmentally Friendly Workflow Design

Scientists at Macquarie University in Australia use AcroPrep ultrafiltration plates in new approach to the production of glucaric acid, a biodegradable, biocompatible manufacturing staple.

May 20, 2021

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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.

The AcroPrep 96-well ultrafiltration plates used in this study are ideal for protein purification applications and are designed for use with sample volumes up to 300 µL. Pall also supplies AcroPrep plates designed to accommodate higher sample volumes of up to 7 ml.

 

AcroPrep ultrafiltration plates with Omega MWCO membranes are available in 1K, 3K, 10K, 30K, and 100K sizes.  

 

Once the products of the enzymatic reactions were purified, metabolite analysis of the Gluc A production pathway allowed the researchers to rapidly optimize enzyme concentrations and reaction timepoints such that they could minimize resource use, while maximizing productivity. The results demonstrated successful production of GlucA with “the highest productivities

so far reported for glucaric acid production using a synthetic enzyme pathway”. The group were also able to design their reaction strategy such that five of the six enzymatic co-factors they worked with could be recycled and reused.

 

This study is proof of concept that a cell-free strategy with an immobilized enzyme can be used in a cost-effective manner to produce GlucA. The group plans to continue their work to improve productivity and sustainability of the new manufacturing pathway.  

 

Pall is proud to support research into more sustainable biochemical products and pathways. Please visit our website for more information on our AcroPrep filter plates for protein purification products.  

 

Reference:

1. Petroll K., et al. A novel framework for the cell-free enzymatic production of glucaric acid. Metabolic Engineering 57; 162–173. 2020.

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