Lipase from Candida rugosa has been immobilized in different formulations of calcium alginate beads, prepared by ionotropic gelation, which differ from each other in CaCl2 concentration and hardening time, to...

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BackgroundThe addition of organic solvents to an aqueous medium for enzymatic reactions offers several advantages, as they can increase the solubility of substrates but can also lead to enzyme inactivation and/or aggregation.ResultsThe effect of adding 30% of several water‐soluble organic solvents on the catalytic activity of lipase B from Candida antarctica (CalB) was studied and the results showed that the highest activity was obtained with the addition of t‐butanol. t‐Butanol and acetonitrile were selected and the kinetic parameters, determined to deepen their effect on CalB activity, showed that the addition of acetonitrile improved the enzyme–substrate affinity, while water–t‐butanol mixtures led to a more than ninefold increase in kcat. To rationalize at a molecular level the kinetic results, molecular dynamic simulations were performed. Analysis of the accessibility of the active‐site cavity, solvent occupancy in the site and in the oxyanion hole, and the stability of the catalytic triad in the two solvent mixtures, provided insight into their effects on the catalytic properties of CalB.ConclusionThe lower occupancy in the oxyanion hole of water molecules and a shorter residence time in the active site of acetonitrile molecules in the acetonitrile–water mixture contribute to the higher enzyme–substrate affinity found experimentally. Conversely, the higher kcat in the t‐butanol mixture is explained by the higher stability of the catalytic triad and by an increase in the nucleophilicity of the catalytic serine due to the persistent presence of t‐butanol molecules in the active site. © 2023 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

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Horse Liver Alcohol Dehydrogenase (HLADH) has been immobilized on calcium‐alginate beads and used for both oxidation and reduction reactions. To avoid swelling of the beads and their subsequent breakage, calcium ions were added to both reaction and storage solutions, allowing the beads to maintain the initial structural features. The techniques used for this purpose revealed that 2 mM Ca2+ is the optimal concentration, which does not significantly change the weight of the beads, the amount of water in them, and their external and internal structure. The optimized experimental procedure has been used to verify the properties of the enzyme in terms of reusability, storage, and thermal stability. The addition of calcium ions allows the enzyme to retain more than 80 % of its initial activity for fourteen cycles and approximately 50 % at the twentieth cycle. Moreover, when the biocatalyst has been stored in a buffer solution containing 2 mM Ca2+, the retention of enzyme activity after 30 days was 100 %, compared to that measured before incubation. The encapsulated enzyme exhibits greater thermal stability than free HLADH up to at least 60 °C, preventing dimer dissociation into the two subunits.