Abstract
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.
Highlights
Lipases are among the most used enzymes at both academic and industrial level (Jaeger and Eggert, 2002; Salihu and Alam, 2015)
The main characteristic of lipases is that they are interfacial enzymes, that is, enzymes that are able to perform their function at the interface of drops of their natural substrates (Schmid and Verger, 1998; Reis et al, 2009). This is possible by their special structure: they have one conformation where the active center is isolated from the medium by a polypeptide, in equilibrium with another form where this lid moves and exposes the active center to the medium (Brzozowski et al, 1991; Van Tilbeurgh et al, 1993; Verger, 1997)
It was checked that the Pseudomonas fluorescens (PFL) activity remained unaltered after 10 h of incubation under those conditions
Summary
Lipases are among the most used enzymes at both academic and industrial level (Jaeger and Eggert, 2002; Salihu and Alam, 2015). The main characteristic of lipases is that they are interfacial enzymes, that is, enzymes that are able to perform their function at the interface of drops of their natural substrates (triglycerides) (Schmid and Verger, 1998; Reis et al, 2009) This is possible by their special structure: they have one conformation where the active center is isolated from the medium by a polypeptide (lid or flat), in equilibrium with another form where this lid moves and exposes the active center to the medium (Brzozowski et al, 1991; Van Tilbeurgh et al, 1993; Verger, 1997). This adsorbed lipase form is more stable than the enzyme in conformational equilibrium (Derewenda et al, 1994)
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