Modulation of the properties of immobilized CALB by chemical modification with 2,3,4-trinitrobenzenesulfonate or ethylendiamine. Advantages of using adsorbed lipases on hydrophobic supports

Lipase B from Candida antarctica (CALB) has been adsorbed on octyl-agarose or covalently immobilized on cyanogen bromide agarose. Then, both biocatalysts have been modified with ethylenediamine (EDA) or 2,4,6-trinitrobenzensulfonic acid (TNBS) just using one reactive or using several modifications in a sequential way (the most complex preparation was CALB–TNBS–EDA–TNBS). Covalently immobilized enzyme decreased the activity by 40–60% after chemical modifications, while the adsorbed enzyme improved the activity on p-nitrophenylbutyrate (pNPB) by EDA modification (even by a 2-fold factor). These biocatalysts were further characterized. The results showed that the effects of the chemical modification on the enzyme features were strongly dependent on the immobilization protocol utilized, the experimental conditions where the catalyst will be utilized, and the substrate. Significant changes in the activity/pH profile were observed after the chemical modifications. The effect of the modifications on the enzyme activity depends on the substrate and the reaction conditions: enzyme specificity is strongly altered by the chemical modification. Moreover, enzyme activity versus pNPB (using octyl-CALB–EDA) or versus R methyl mandelate (using octyl-CALB–TNBS) increased by almost a 2-fold factor at pH 5. The stability of the modified enzymes at different pH and in the presence of organic solvents generally decreased after the modifications, usually by no more than a 2-fold factor. However, under some conditions, some stabilization was found. CALB enantioselectivity in the hydrolysis of R/S methyl mandelate could be also improved by these chemical modifications (e.g., E-value went from 11 to 16 using octyl-CALB–TNBS at pH 5). Therefore, solid phase chemical modification of immobilized lipases may become a powerful tool in the design of lipase libraries with very different properties, each immobilized preparation may be used to produce a variety of forms with altered properties.