Immobilization and Catalytic Properties of Limonene-1,2-Epoxide Hydrolase

In this work, limonene-1,2-epoxide hydrolase (LEH) from Rhodococcus erythropolis was immobilized on Immobead 150P and various Purolite® methacrylate-based carriers, and subsequently applied in the hydrolysis of cis-/trans-(+)-limonene-1,2-epoxide. The immobilization efficiency was assessed based on the recovery of activity and the immobilization yield. The best results were observed when LEH was immobilized on Purolite LifetechTM ECR8309M modified with glutaraldehyde and 1,4-diaminobutane, yielding a specific activity of 60.5 U·g−1. The optimal temperature for enzymatic reaction increased from 40 °C (free LEH) to 60 °C following immobilization. Thermal inactivation studies revealed that the free LEH followed a simple aggregation mechanism, whereas the immobilized form conformed to a two-step deactivation model, involving an initial reversible equilibrium followed by an irreversible inactivation step. When applied in 10 reaction cycles at 30 °C the immobilized biocatalyst retained 62% of the initial activity in the presence of 10% (v/v) acetonitrile and 75% in its absence. At temperatures up to 60 °C, immobilized LEH appeared very stable, retaining more than 80% of activity at 60 °C after 6 reaction cycles. This study improves our understanding of the inactivation mechanism of LEH, and the results highlight covalent immobilization as a promising method for the thermal stabilization of LEH.