Catalytic behavior of lipase immobilized onto congo red and PEG-decorated particles

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.

Figure 1

Particle size distribution histograms determined for (a) PS/PEG and (b) PS/PEG/CR particles. The insets correspond to SEM images taken for dried dispersions.

Figure 2

(a) Adsorption isotherms determined at (24 ± 1) °C and pH 7.0 for lipase onto PS/PEG (blue circles) and PS/PEG/CR particles (red triangles). The lines are guides for the eyes. (b) Mean D, (c) PD and (d) ζ-potential values determined for PS/PEG or PS/PEG/CR particles after lipase adsorption as a function of Q_lipase. Particle size distribution histograms and SEM images (insets) obtained for (e) PS/PEG and (f) PS/PEG/CR particles after lipase adsorption, Q_lipase = 85 ± 3 nmol∙mg⁻¹ and Q_lipase = 105 ± 5 nmol∙mg⁻¹, respectively. Each point is the average of triplicates ± SD.

Figure 3

(a) Initial rates determined for the hydrolysis of p-NPB catalyzed by free lipase (black squares) at 0.4 × 10⁻⁶ mol/L and lipase immobilized onto PS/PEG (blue circles, Q_lipase = 85 ± 3 nmol∙mg⁻¹) or onto PS/PEG/CR (red triangles, Q_lipase = 105 ± 5 nmol∙mg⁻¹) as a function of p-NPB concentration. Each point is the average of triplicates ± SD. (b) Lineweaver-Burk fitting curves for free lipase (black squares) and lipase immobilized onto PS/PEG (blue circles) and PS/PEG/CR (red triangles).

Figure 4

(a) Circular dichroism (CD) and (b) electronic spectra obtained for mixtures of CR at 8.0 μmol∙L⁻¹ and lipase at increasing concentration from 3.3 µmol∙L⁻¹ to 33 µmol∙L⁻¹.

Figure 5

Concentration of p-NP formed from the hydrolysis of p-NPB catalyzed by free lipases (black squares), lipases immobilized onto PS/PEG/CR (red triangles, Q_lipase = 105 ± 5 nmol∙mg⁻¹) and PS/PEG (blue circles, Q_lipase = 85 ± 3 nmol∙mg⁻¹) particles at 25 °C, 37 °C, 40 °C, 45 °C and 60 °C. Each point is the average of triplicates ± SD.

Figure 6

Activity recovery determined at 25 °C and 40 °C for lipases immobilized onto PS/PEG/CR particles (Q_lipase = 105 ± 5 nmol∙mg⁻¹). Each point is the average of triplicates ± SD.

Scheme 1

Representation of chemical structures of (a) Tween-20 and (b) Congo red (CR).

Scheme 2

Representation of p-NPB hydrolysis catalyzed by lipase, yielding butyric acid and (p-NP).