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

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.