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.