Reagent-Free Immobilization of Industrial Lipases to Develop Lipolytic Membranes with Self-Cleaning Surfaces

Abstract

Biocatalytic membrane reactors combine the highly efficient biotransformation capability of enzymes with the selective filtration performance of membrane filters. Common strategies to immobilize enzymes on polymeric membranes are based on chemical coupling reactions. Still, they are associated with drawbacks such as long reaction times, high costs, and the use of potentially toxic or hazardous reagents. In this study, a reagent-free immobilization method based on electron beam irradiation was investigated, which allows much faster, cleaner, and cheaper fabrication of enzyme membrane reactors. Two industrial lipase enzymes were coupled onto a polyvinylidene fluoride (PVDF) flat sheet membrane to create self-cleaning surfaces. The response surface methodology (RSM) in the design-of-experiments approach was applied to investigate the effects of three numerical factors on enzyme activity, yielding a maximum activity of 823 ± 118 U m^-2 (enzyme concentration: 8.4 g L^-1, impregnation time: 5 min, irradiation dose: 80 kGy). The lipolytic membranes were used in fouling tests with olive oil (1 g L^-1 in 2 mM sodium dodecyl sulfate), resulting in 100% regeneration of filtration performance after 3 h of self-cleaning in an aqueous buffer (pH 8, 37 °C). Reusability with three consecutive cycles demonstrates regeneration of 95%. Comprehensive membrane characterization was performed by determining enzyme kinetic parameters, permeance monitoring, X-ray photoelectron spectroscopy, FTIR spectroscopy, scanning electron microscopy, and zeta potential, as well as water contact angle measurements.

Keywords: electron beam; enzyme membrane reactor; fouling; lipase; response surface methodology; self-cleaning surface.

Figure 1

Evaluation of V_max (I-optimal RSM design). Contour plots for enzyme concentration and irradiation dose at (a) 1 min; (b) 10 min; and (c) 3D surface plot at 5 min; as well as (d) progress curves of optimized PVDF-g-EL and pristine PVDF-Ref (n = 5).

Figure 2

Characterization of surface properties. (a) Zeta potential curves; (b) contact angles of the top and bottom site of the membrane samples.

Figure 3

First fouling and self-cleaning cycle for PVDF-Ref and lipolytic PVDF-g-EL. (a) Permeance curves as a function of total filtered volume, V_tot, during the fouling cycle, and as a function of time, t, during the self-cleaning cycle; (b) relative permeance P/P₀ at the beginning, after fouling, and after self-cleaning.

Figure 4

SEM images of pristine PVDF-Ref and modified PVDF-g-EL before and after the first fouling and self-cleaning cycle.

Figure 5

Characterization of samples before and after the first fouling and self-cleaning cycle by using (a) XPS; and (b) FTIR spectroscopy.

Figure 6

Reusability of lipolytic PVDF-g-EL within 3 consecutive fouling and self-cleaning cycles. Fouling cycles are highlighted with a yellow background, self-cleaning cycles (each 3 h) are highlighted with a blue background. PVDF-Ref was not used due to severe fouling after the first cycle.