CATALYSTS
enzymatic cascade process with appropriate process control.
Combined with process intensification using batch reactors in lab scale, this cascade already achieves a space-time yield of 200 g L- 1 / day- 1. Samples of GA in free acid form or in different salt formats are now available to test for potential applications.
Figure 2- Enzyme engineering by BioEngine platform for sugar acid production
Figure 3- FDCA production via HMF route
Figure 4- FDCA production via D-glucaric acid route
process stability using Enzymaster’ s BioEngine * platform.
A UDH variant showing activity in oxidising L-guluronic acid to GA was identified by screening Enzymaster’ s in-house enzyme panel. Subsequently, this variant was subjected to six rounds of directed evolution to improve its catalytic activity and process stability, also using the BioEngine * platform.
During evolution campaigns, mutagenesis libraries or variants were designed based on the consensus of our in-house in silico models consisting of sequence- and structure-based AI models( EnzyAI platform) and mechanistic computational chemistry models( BioNavigator * platform). All of the in silico models of evolving backbones were iteratively refined with
References: 1: T. Werpy et al., US Department of Energy, 2004. https:// www. osti. gov / biblio / 15008859
2: J. V. Machado et al., Catalysis Communications 2023, 182, 106740
the screening data and process data generated in the wet lab to improve the models’ quality.
With the enhancement of the enzymes’ catalytic performance, the process conditions were also under iterative optimisation to achieve commercial viability. This is a systematic process where in silico models, screening assays and process conditions co-evolve altogether, orchestrated by the BioEngine * platform( Figure 2).
Once the evolution campaigns were finished, the production of final enzyme variants via recombinant expression using E. coli in high-celldensity fermentation was scaled up and optimised. The enzymes produced from fermenters were used to develop
3: G. Totaro et al., ChemSusChem 2022, 15( 13), e202200501. Published 7 July 2022
Application of GA
Scaling up this cascade is expected to drastically reduce GA costs, unlocking its applications in personal care, healthcare, agriculture and plant protection, industrial water treatment, etc.
A particularly promising use is in the production of 2,5-furandicarboxylic acid( FDCA), a long-sought biobased material building block. Current FDCA development by start-ups relies on fructose as the starting material. Acidic dehydration produces 5-hydroxymethylfurfural( HMF), which is unstable and hard to oxidise to FDCA with noble metal catalysts( Figure 3). 3
In contrast, GA is stable: acidic dehydration directly yields FDCA( Figure 4). This route uses cheaper glucose instead of fructose( e. g. derived from non-grain biomass) as the starting material and enzymatic oxidation generates far fewer by-products, boosting overall yield and simplifying( lowering the cost of) DSP for isolation of the target compound.
The enzyme cascade’ s modular design enables broader application to other molecules’ oxidations, positioning it as a versatile platform for biobased manufacturing. Future work will focus on optimising scale-up economics and expanding substrate scope via combinatorial enzyme evolution and process intensification. ●
*- BioEngine and BioNavigator are both trademarks of Enzymaster
Dr Haibin Chen
CO-FOUNDER & VP R & D
ENZYMASTER( NINGBO) BIOENGINEERING
J haibin. chen @ enzymaster. com j https:// enzymaster. de /
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