The lipases from Thermomyces lanuginosus (TLL) and Candida antarctica (B) (CALB) were immobilized on octyl-agarose beads at 1 mg/g (a loading under the capacity of the support) and by overloading the support with the enzymes. These biocatalysts were compared in their stabilities in 10 mM of sodium phosphate, HEPES, and Tris-HCl at pH 7. Lowly loaded CALB was more stable than highly loaded CALB preparation, while with TLL this effect was smaller. Phosphate was very negative for the stability of the CALB biocatalyst and moderately negative using TLL at both loadings. The stability of the enzymes in HEPES and Tris-HCl presented a different response as a function of the enzyme loading (e.g., using lowly loaded CALB, the stabilities were similar in both buffers, but it was clearly smaller in HEPES using the highly loaded biocatalysts). Moreover, the specific activity of the immobilized enzymes versus p-nitrophenol butyrate, triacetin and R- or S-methyl mandelate depended on the buffer, enzyme loading, and interaction between them. In some cases, almost twice the expected activity could be obtained using highly loaded octyl-CALB, depending on the buffer. A co-interaction between the effects on enzyme activity and the specificity of support enzyme loading and buffer nature was detected.

The objective of this paper was to analyze whether an interaction between the effects of the buffer nature and chemical modification on enzyme stability exists. For this, the lipase B from Candida antarctica (CALB) and the lipase from Thermomyces lanuginosus (TLL) were immobilized on octyl agarose beads and modified with picryl sulfonic acid (TNBS) and ethylenediamine via the carbodiimide route. The obtained biocatalysts were them inactivated in different buffers (2-amino-2-(hydroxymethyl)propane-1,3-diol ( Tris)-HCl, N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) or phosphate). A significant interaction was found between the chemical modification of the enzyme surfaces and their stabilities in these different buffers. While phosphate was the buffer where the lowest enzyme stabilities were found for both unmodified immobilized enzymes , the differences on enzyme stabilities become much smaller after some chemical modifications. In many instances, chemical modification improves enzyme stability when using a buffer and was negative when using the other one (e.g., TNBS modification of TLL was positive using Tris-HCl or negative using the other buffers, amination of CALB decreased its stability when inactivated in Tris-HCl or HEPES while it almost had no effect in phosphate). Thus, clear co-interactions of the effects on enzyme stability between chemical modification and buffer nature were established.

The lipase from Prunus dulcis almonds has been immobilized for the first time. For this purpose, two different supports, an octadecyl methacrylate particulate support, and aminated agarose (monoaminoethyl-N-aminoethyl) have been utilized. Both immobilized biocatalysts show improved enzyme stability, but great changes in enzyme specificity were detected. The enzyme immobilized via ion exchange maintained its activity intact versus p-nitrophenyl butyrate, while the enzyme immobilized on the hydrophobic support fully lost its activity versus this substrate, which was confirmed to be due to substrate adsorption on the support. However, this biocatalyst was much more active versus triacetin (more than 10-fold), R- or S- methyl mandelate at pH 7. At pH 9, a strong effect of using phosphate or bicarbonate as reaction buffers was detected. Using bicarbonate, the interfacially immobilized enzyme presented no activity versus R-isomer, but it was very active versus the S-isomer and triacetin. Using a phosphate buffer during the reaction, all compounds were recognized as substrates. The enzyme immobilized via ion exchange was significantly more active using phosphate; in fact, using bicarbonate, the enzyme was inactive versus both methyl mandelate isomers. This paper shows for the first time a great interaction between the effects of the immobilization protocol and buffer used during reaction on the enantiospecificity of lipases.

Alcalase was immobilized–stabilized on carrageenan beads following a previously described protocol. Then, the activities of free and immobilized enzymes were compared using different protein substrates (casein, (CS), bovine serum albumin (BSA), or hemoglobin (HG)) at different pH values and temperatures. The observed activity depended on the substrate protein and enzyme formulation used. The highest enzyme activity could be observed at pHs 5, 7, or 10, depending on the substrate protein and the Alcalase formulation. The effect of the temperature at these pHs on the activity versus the different substrate proteins showed a common pattern. At low temperatures, the immobilized enzyme presented higher (mainly at acidic-neutral pH values and using BSA) or similar specific activity than the free enzyme. At temperatures near the optimal for the free enzyme, it became the most active, while at higher temperatures, the immobilized enzyme recovered the lead, although differences in the optimal temperature were not very significant. This may be explained by the lower mobility of the immobilized–stabilized enzyme. The immobilized enzyme could be much more active than the free enzyme or slightly less active, even using mild conditions, depending on the substrate protein, pH, and temperature used to determine the enzyme activity.

It has been reported that the modification of immobilized glyoxyl–ficin with aldehyde dextran can promote steric hindrances that greatly reduce the activity of the immobilized protease against hemoglobin, while the protease still maintained a reasonable level of activity against casein. In this paper, we studied if this effect may be different depending on the amount of ficin loaded on the support. For this purpose, both the moderately loaded and the overloaded glyoxyl–ficin biocatalysts were prepared and modified with aldehyde dextran. While the moderately loaded biocatalyst had a significantly reduced activity, mainly against hemoglobin, the activity of the overloaded biocatalyst was almost maintained. This suggests that aldehyde dextran was able to modify areas of the moderately loaded enzyme that were not available when the enzyme was overloaded. This modification promoted a significant increase in biocatalyst stability for both biocatalysts, but the stability was higher for the overloaded biocatalyst (perhaps due to a combination of inter- and intramolecular crosslinking).