A two-enzyme system composed of immobilized ketoreductase (Hansenula polymorpha) and glucose dehydrogenase (Bacillus megaterium) was developed for the asymmetric reduction of keto esters to optically active hydroxy esters via immobilization in polyvinyl alcohol (PVA) gel particles. The concentration of enzymes was optimized, and the final particles were used 18 times in a row in a batch mode to achieve minimal loss of activity and complete conversion of the model substrate, β-ketoester ethyl-2-methylacetoacetate. Excellent stability was also achieved using new storage conditions of PVA particles, with 80% of activity being retained after almost 10 months.

The co-immobilization of ketoreductase (KRED) and glucose dehydrogenase (GDH) on highly cross-linked agarose (sepharose) was studied. Immobilization of these two enzymes was performed via affinity interaction between His-tagged enzymes (six histidine residues on the N-terminus of the protein) and agarose matrix charged with nickel (Ni²⁺ ions). Immobilized enzymes were applied in a semicontinuous flow reactor to convert the model substrate; α-hydroxy ketone. A series of biotransformation reactions with a substrate conversion of >95% were performed. Immobilization reduced the requirement for cofactor (NADP+) and allowed the use of higher substrate concentration in comparison with free enzymes. The immobilized system was also tested on bulky ketones and a significant enhancement in comparison with free enzymes was achieved.

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 Lifetech^TM 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.