Abstract
Poly(ethylene glycol) (PEG)-decorated polystyrene (PS) nanoparticles with mean hydrodynamic diameter (D) and zeta–potential (ζ) of (286 ± 15) nm and (−50 ± 5) mV, respectively, were modified by the adsorption of Congo red (CR). The PS/PEG/CR particles presented D and ζ values of (290 ± 19) nm and (−36 ± 5) mV, respectively. The adsorption of lipase onto PS/PEG or PS/PEG/CR particles at (24 ± 1) °C and pH 7 changed the mean D value to (380 ± 20) and (405 ± 11) nm, respectively, and ζ value to (−32 ± 4) mV and (−25 ± 2) mV, respectively. The kinetic parameters of the hydrolysis of p-nitrophenyl butyrate were determined for free lipase, lipase immobilized onto PS/PEG and PS/PEG/CR particles. Lipase on PS/PEG/CR presented the largest Michaelis-Menten constant (KM), but also the highest Vmax and kcat values. Moreover, it could be recycled seven times, losing a maximum 10% or 30% of the original enzymatic activity at 40 °C or 25 °C, respectively. Although lipases immobilized onto PS/PEG particles presented the smallest KM values, the reactions were comparatively the slowest and recycling was not possible. Hydrolysis reactions performed in the temperature range of 25 °C to 60 °C with free lipases and lipases immobilized onto PS/PEG/CR particles presented an optimal temperature at 40 °C. At 60 °C free lipases and lipases immobilized onto PS/PEG/CR presented ~80% and ~50% of the activity measured at 40 °C, indicating good thermal stability. Bioconjugation effects between CR and lipase were evidenced by circular dichroism spectroscopy and spectrophotometry. CR molecules mediate the open state conformation of the lipase lid and favor the substrate approaching.
Highlights
Immobilized enzymes are “green” catalysts for many reactions of industrial interest
The kinetic parameters determined at 25 °C for the hydrolysis of p-nitrophenyl butyrate (p-NPB) catalyzed by free lipases and for lipases immobilized onto PS/Poly(ethylene glycol) (PEG)/Congo red (CR) particles were comparable, and they were superior to those determined for lipases immobilized onto PS/PEG
The activity of free lipase and lipase immobilized onto PS/PEG or PS/PEG/CR particles was determined by measuring the formation of para-nitrophenol (p-NP), as a result of enzymatic hydrolysis of (p-NPB), as indicated in Scheme 2
Summary
Immobilized enzymes are “green” catalysts for many reactions of industrial interest. They can be advantageous compared to free enzymes because: (i) immobilization improves their stability;(ii) subsequent reuse is possible; (iii) inhibition by products or sub-products is avoided; (iv) optimum pHs and temperatures might shift to more convenient values. Immobilized enzymes are “green” catalysts for many reactions of industrial interest. They can be advantageous compared to free enzymes because: (i) immobilization improves their stability;. The immobilized enzymes must maintain their active sites in a suitable orientation in order to retain their catalytic properties and the costs related to the support and to the immobilization process should be as low as possible [1]. Lipases are probably the most used enzymes in hydrolysis, esterification, acidolysis and alcoholysis reactions. Some of the reasons for this are their low cost, compared to other enzymes, high stability in organic media or emulsions or under extreme reactions conditions (pH, ionic strength and temperature), broad substrate specificity and the catalysis of regio- and enantioselective reactions [2].
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