# A Cofactor Regeneration System for 2‐Aminobutyric Acid Production Based on Combined Cross‐Linked Enzyme Aggregates: Utilizing His‐Tagged Enzymes With Low‐Concentration Calcium Ions as Precipitant

## TL;DR

Researchers developed a new method to produce 2-aminobutyric acid using enzyme aggregates that are stable and reusable.

## Contribution

A novel, eco-friendly method for creating enzyme aggregates using low calcium concentrations and histidine-tagged enzymes.

## Key findings

- Combi-CLEAs showed enhanced thermal and pH tolerance compared to free enzymes.
- The combi-CLEAs retained 40% of initial activity after seven reuse cycles.
- Optimal reaction conditions were found to be 37°C and pH 7.5.

## Abstract

Combined cross‐linked enzyme aggregates (combi‐CLEAs) represent a promising carrier‐free immobilized enzyme technology. This study describes the preparation of combi‐CLEAs comprising leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH) for the regeneration of cofactor nicotinamide adenine dinucleotide necessary for 2‐aminobutyric acid production. Different from traditional methods using ammonium sulfate or organic reagents as precipitant, this work utilized low concentrations of calcium ions to purify and precipitate the histidine‐tagged enzymes. We developed a simple and environmentally friendly protocol for combi‐CLEAs formation, involving precipitation with 10 mM calcium ions at an enzyme activity ratio of 1:2 for LeuDH and FDH, respectively, followed by cross‐linking with 0.15% (w/v) glutaraldehyde at 20°C for 2 h at pH 7.5. The optimal catalytic reaction temperature and pH value for the combi‐CLEAs were determined to be a temperature of 37°C and a pH of 7.5. The combi‐CLEAs demonstrated enhanced thermal and pH tolerance compared to the free enzyme mixture. Moreover, the combi‐CLEAs showed good operational stability, retaining 40% of its initial activity after seven cycles of reuse. These findings suggest that the combi‐CLEAs of LeuDH and FDH are an efficient and cost‐effective option for 2‐aminobutyric acid production.