Immobilization of hydroaminase-expressing recombinant Escherichia coli whole-cell biocatalysts for the production of β-alanine.

Developing stable and easy-to-operate biocatalysts is crucial for their use as industrial catalysts. Here immobilized whole-cell catalysts were used for β-alanine production by immobilizing recombinant Escherichia coli cells (containing hydroaminase) with diatomite. E. coli BL21 (DE3)/pET-30a ( +)-HAMase was genetically engineered for the efficient synthesis of β-alanine from acrylic acid and aqueous ammonia. Using glutaraldehyde as a cross-linking agent, polyethyleneimine (PEI) as a flocculant, and diatomite as the immobilization carrier, optimal immobilization was achieved with 8% (w/v) PEI solution, 5% (w/v) glutaraldehyde, and 100 mg wet cell/mL cell suspension, along with a PEI flocculation time of 2.5 h and glutaraldehyde cross-linking time of 1.5 h. The enzyme activity recovery rate reached 70.72%. Remarkably, the immobilized whole-cell catalysts exhibited excellent stability, retaining over 90% of initial enzyme activity after 12 h incubation at 45 °C and maintaining over 72% enzyme activity after storage for 60 days at 4 °C. Additionally, the immobilized cells demonstrated enhanced reusability, maintaining consistent β-alanine yield even after ten consecutive reaction batches with an average yield of approximately 80%.