The lipase from Pseudomonas fluorescens (PFL) has been immobilized on octyl-agarose beads under 16 different conditions (varying pH, ionic strength, buffer, adding some additives) at two different loadings, 1 and 60 mg of enzyme/g of support with the objective of check if this can alter the biocatalyst features. The activity of the biocatalysts versus p-nitrophenyl butyrate and triacetin and their thermal stability were studied. The different immobilization conditions produced biocatalysts with very different features. Considering the extreme cases, using 1 mg/g preparations, PFL stability changed more than fourfolds, while their activities versus pNPB or triacetin varied a 50–60%. Curiously, PFL specific activity versus triacetin was higher using highly enzyme loaded biocatalysts than using lowly loaded biocatalysts (even by a twofold factor). Moreover, stability of the highly loaded preparations was higher than that of the lowly loaded preparations, in many instances even when using 5°C higher temperatures (e.g., immobilized in the presence of calcium, the highly loaded biocatalysts maintained after 24 h at 75°c a 85% of the initial activity, while the lowly loaded preparation maintained only 27% at 70°C). Using the highly loaded preparations, activity of the different biocatalysts versus pNPB varied almost 1.7-folds and versus triacetin 1.9-folds. In this instance, the changes in stability caused by the immobilization conditions were much more significant, some preparations were almost fully inactivated under conditions where the most stable one maintained more than 80% of the initial activity. Results suggested that immobilization conditions greatly affected the properties of the immobilized PFL, partially by individual molecule different conformation (observed using lowly loaded preparations) but much more relevantly using highly loaded preparations, very likely by altering some enzyme-enzyme intermolecular interactions. There is not an optimal biocatalyst considering all parameters. That way, preparation of biocatalysts using this support may be a powerful tool to tune enzyme features, if carefully controlled.

Ficin extract has been immobilized on different 4% aminated-agarose beads. Using just ion exchange, immobilization yield was poor and expressed activity did not surpass 10% of the offered enzyme, with no significant effects on enzyme stability. The treatment with glutaraldehyde of this ionically exchanged enzyme produced an almost full enzyme inactivation. Using aminated supports activated with glutaraldehyde, immobilization was optimal at pH 7 (at pH 5 immobilization yield was 80%, while at pH 9, the immobilized enzyme became inactivated). At pH 7, full immobilization was accomplished maintaining 40% activity versus a small synthetic substrate and 30% versus casein. Ficin stabilization upon immobilization could be observed but it depended on the inactivation pH and the substrate employed, suggesting the complexity of the mechanism of inactivation of the immobilized enzyme. The maximum enzyme loading on the support was determined to be around 70 mg/g. The loading has no significant effect on the enzyme stability or enzyme activity using the synthetic substrate but it had a significant effect on the activity using casein; the biocatalysts activity greatly decreased using more than 30 mg/g, suggesting that the near presence of other immobilized enzyme molecules may generate some steric hindrances for the casein hydrolysis.

Lipases are among the most utilized enzymes in biocatalysis. In many instances, the main reason for their use is their high specificity or selectivity. However, when full modification of a multifunctional and heterogeneous substrate is pursued, enzyme selectivity and specificity become a problem. This is the case of hydrolysis of oils and fats to produce free fatty acids or their alcoholysis to produce biodiesel, which can be considered cascade reactions. In these cases, to the original heterogeneity of the substrate, the presence of intermediate products, such as diglycerides or monoglycerides, can be an additional drawback. Using these heterogeneous substrates, enzyme specificity can promote that some substrates (initial substrates or intermediate products) may not be recognized as such (in the worst case scenario they may be acting as inhibitors) by the enzyme, causing yields and reaction rates to drop. To solve this situation, a mixture of lipases with different specificity, selectivity and differently affected by the reaction conditions can offer much better results than the use of a single lipase exhibiting a very high initial activity or even the best global reaction course. This mixture of lipases from different sources has been called “combilipases” and is becoming increasingly popular. They include the use of liquid lipase formulations or immobilized lipases. In some instances, the lipases have been coimmobilized. Some discussion is offered regarding the problems that this coimmobilization may give rise to, and some strategies to solve some of these problems are proposed. The use of combilipases in the future may be extended to other processes and enzymes.