Influence of Lipase Immobilization Mode on Ethyl Acetate Hydrolysis in a Continuous Solid-Gas Biocatalytic Membrane Reactor.

Solid-gas biocatalysis was performed in a specially designed continuous biocatalytic membrane reactor (BMR). In this work, lipase from Candida rugosa (LCR) and ethyl acetate in vapor phase were selected as model enzyme and substrate, respectively, to produce acetic acid and ethanol. LCR was immobilized on functionalized PVDF membranes by using two different kinds of chemical bond: electrostatic and covalent. Electrostatic immobilization of LCR was carried out using a membrane functionalized with amino groups, while covalent immobilization was carried out using membrane, with or without surface-immobilized polyacrylamide (PAAm) microgels, functionalized with aldehyde groups. These biocatalytic membranes were tested in a solid-gas BMR and compared in terms of enzyme specific activity, catalytic activity, and volumetric reaction rate. Results indicated that lipase covalently immobilized is more effective only when the immobilization is mediated by microgels, showing catalytic activity doubled with respect to the other system with covalently bound enzyme (4.4 vs 2.2 µmol h-1). Enzyme immobilized by ionic bond, despite a lower catalytic activity (3.5 vs 4.4 µmol h-1), showed the same specific activity (1.5 mmol·h-1·g-1ENZ) of the system using microgels, due to a higher enzyme degree of freedom coupled with an analogously improved enzyme hydration. Using the optimized operating conditions regarding immobilized enzyme amount, ethyl acetate, and molar water flow rate, all three BMRs showed continuous catalytic activity for about 5 months. On the contrary, the free enzyme (in water/ethyl acetate emulsion) at 50 °C was completely inactive and at 30 °C (temperature optimum) has a specific activity 2 orders of magnitude lower (8.4 × 10-2 mmol h-1 g-1) than the solid-gas biocatalytic membrane reactor. To the best of our knowledge, this is the first example of solid-gas biocatalysis, working in the gaseous phase in which a biocatalytic membrane reactor, with the enzyme/substrate system lipase/ethyl acetate, was used.

Phosphotriesterase-Magnetic Nanoparticle Bioconjugates with Improved Enzyme Activity in a Biocatalytic Membrane Reactor.

The need to find alternative bioremediation solutions for organophosphate degradation pushed the research to develop technologies based on organophosphate degrading enzymes, such as phosphotriesterase. The use of free phosphotriesterase poses limits in terms of enzyme reuse, stability, and process development. The heterogenization of enzyme on a support and their use in bioreactors implemented by membranes seems a suitable strategy, thanks to the ability of membranes to compartmentalize, to govern mass transfer, and to provide a microenvironment with tuned physicochemical and structural properties. Usually, hydrophilic membranes are used since they easily guarantee the presence of water molecules needed for the enzyme catalytic activity. However, hydrophobic materials exhibit a larger shelf life and are preferred for the construction of filters and masks. Therefore, in this work, hydrophobic polyvinylidene fluoride (PVDF) porous membranes were used to develop biocatalytic membrane reactors (BMR). The phosphotriesterase-like lactonase (PLL) enzyme ( SsoPox triple mutant from S. solfataricus) endowed with thermostable phosphotriesterase activity was used as model biocatalyst. The enzyme was covalently bound directly to the PVDF hydrophobic membrane or it was bound to magnetic nanoparticles and then positioned on the hydrophobic membrane surface by means of an external magnetic field. Investigation of kinetic properties of the two BMRs and the influence of immobilized enzyme amount revealed that the performance of the BMR was mostly dependent on the amount of enzyme and its distribution on the immobilization support. Magnetic nanocomposite mediated immobilization showed a much better performance, with an observed specific activity higher than 90% compared to grafting of the enzyme on the membrane. Even though the present work focused on phosphotriesterase, it can be easily translated to other classes of enzymes and related applications.

Development of a Novel Immobilization Method by Using Microgels to Keep Enzyme in Hydrated Microenvironment in Porous Hydrophobic Membranes.

Using colloidal polyacrylamide (PAAm) microgels as carriers, a novel strategy for covalent immobilization of enzymes maintained in hydrated microenvironment on/in a macroporous surface-functionalized hydrophobic polyvinylidene fluoride (PVDF) membrane is developed. The PAAm microgels are synthesized by inverse miniemulsion polymerization, and first the parameters are investigated which are suited to obtain particles in the desired size range, 100-200 nm, with narrow size distribution. Amino functions are then imparted to the microgels applying the Hofmann reaction. The modification is confirmed by Fourier-transform infrared spectroscopy analysis, ninhydrin test, and elemental analysis. In addition, functionalized microgels are characterized by dynamic light scattering. The amino-functionalized PAAm microgels are then immobilized on pre-modified PVDF membrane having aldehyde functionalities on the surface. Afterward, unreacted aldehyde groups still present on the membrane where quenched by ethanolamine and the enzyme lipase from Candida rugosa (LCR) is subsequently immobilized on the microgels loaded PVDF membrane via glutaraldehyde cross-linking, exploiting the free amino groups on immobilized microgels. Catalytic efficiency of LCR immobilized by this strategy is evaluated using para-nitrophenyl palmitate as substrate and compared with LCR directly immobilized on PVDF membrane without microgels. Results show that LCR immobilized by means of microgels exhibits better performance with a 2.3-fold higher specific biocatalytic activity.