Pore size of macroporous polystyrene microspheres affects lipase immobilization

Polystyrene (PST) microspheres are commonly chosen as immobilization carriers due to their unique advantages such as ideal mechanical strength, adjustable particle size, and favorable chemical stability. However, there were few reports on immobilization by using microspheres with large pore sizes (>100 nm). We have successfully prepared the PST microspheres with macropores and gigapores by a novel method. In this study, giga-/macro-/meso-porous PST microspheres (314 nm, 104 nm, and 14.7 nm in pore sizes) were employed to immobilize lipase (from Burkholderia cepacia) by strong hydrophobic interactions, and the effects of pore sizes on lipase distribution, relative activity, kinetic behavior, thermal stability, storage stability, and reusability were also investigated in detail. According to laser scanning confocal microscope (LSCM) observation, lipase penetrated into the center of those giga-/macro-porous microspheres. With regard to the mesoporous microspheres, lipase only adsorbed to the external shell. The relative activities of immobilized lipase were 146%, 126%, and 50.9% for giga-/macro-/meso-porous PST microspheres, respectively. Comparing with the kinetic constants of free lipase (0.441 mM), the Km value for mesoporous PST-lipase (0.532 mM) was higher, whereas for giga-/macro-porous PST-lipase (0.402 mM and 0.411 mM), the Km values were comparatively lower suggesting the accessibility of substrate to the enzyme active sites was unlimited. The thermal stability, storage stability, and reusability were all improved significantly with the increase of pore sizes. In stimulant system, even after 100 times of recycling, the activity of lipase immobilized on gigaporous and macroporous PST microspheres remained nearly 100% and 93%, respectively, while that of lipase-PST with 14.7 nm pores could only keep 64.1%. In real system, 73.5% and 68.8% activity of gigaporous PST-lipase and macroporous PST-lipase, respectively, were retained after being used 30 times, whereas only 49.4% activity was remained for mesoporous PST-lipase. Therefore, compared with other microspheres, the gigaporous PST microsphere exhibited obvious advantages as a potential enzyme support in industry.