PEI-crosslinked lipase on the surface of magnetic microspheres and its characteristics.

Here, PEI@PMMA microspheres were prepared by grafting polyethyleneimine (PEI) on poly(methyl methacrylate) (PMMA) magnetic microspheres and successfully used to immobilize lipase. The results showed that PEI@PMMA microspheres had strongly adsorbed lipase (49.1 mg/g microsphere) via electrostatic attraction. To prevent lipase shedding, the adsorbed lipase was further crosslinked with PEI on microspheres using glutaraldehyde as crosslinker. Consequently, PEI-crosslinked lipase (2.14 U/mg) exhibited 2.6 times and 1.4 times higher activity respectively than the directly covalent lipase (0.82 U/mg) and the crosslinked lipase aggregates (1.57 U/mg), which was close to the activity of adsorbed lipase (2.20 U/mg). Conformational analysis from FTIR spectroscopy showed that PEI-crosslinked lipase retained its natural structure well. And the α-helix structure seemed to play a key role in enhancing lipase activity. Furthermore, the effects of various parameters on crosslinking reaction were investigated. Also, PEI-crosslinked lipase revealed higher pH and thermal stability. The Michaelis constant (Km) was increased and the optimum temperature of lipase was widened observably after crosslinking with PEI on PEI@PMMA magnetic microspheres.

Lipase immobilization on epoxy-activated poly(vinyl acetate-acrylamide) microspheres.

Poly(vinyl acetate-acrylamide) microspheres with an average diameter of 2-4µm were successfully prepared and characterized via SEM and FTIR. Then the microspheres were modified with epoxy groups through reacting with epichlorohydrin and used as carriers to covalently immobilize Candida rugosa lipase. The results revealed that agitation played an important role on epoxy activation and the immobilization ratio increased with the increase of the epoxy density. On the other hand, the specific activity of the immobilized lipase as well as the activity recovery declined gradually with the increase in the immobilization ratio from 72% to 93%, which were attributed to the steric hindrance effects caused by enzyme overloading. When epoxy density was 76µmol/g microsphere, the activity recovery reached the maximum at 47.5%, and the activity of the immobilized lipase was 261.3U/g microsphere. Moreover, the thermal stability of the immobilized lipase was much better than that of the free one, which indicated potential applications of the immobilized lipase.

Effect of poly(vinyl acetate-acrylamide) microspheres properties and steric hindrance on the immobilization of Candida rugosa lipase.

Poly(vinyl acetate-acrylamide) microspheres were synthesized in the absence or presence of isooctane via suspension polymerization and utilized as carriers to immobilize Candida rugosa lipase. When the hydrophobic/hydrophilic surface characteristics of the microspheres were modified by changing the ratio of vinyl acetate (hydrophobic monomer) to acrylamide (hydrophilic monomer) from 50:50 to 86:24, the immobilization ratio changed from 45% to 92% and the activity of the immobilized lipase increased from 202.5 to 598.0 U/g microsphere. Excessive lipase loading caused intermolecular steric hindrance, which resulted in a decline in lipase activity. The maximum specific activity of the immobilized lipase (4.65 U/mg lipase) was higher than that of free lipase (3.00 U/mg lipase), indicating a high activity recovery during immobilization.

Effect of hydrophobic/hydrophilic characteristics of magnetic microspheres on the immobilization of BSA.

Core-shell magnetic poly(styrene-acrylamide-acrylic acid) microspheres with carboxyl groups were successfully synthesized via dispersion copolymerization in the presence of nano-particle of Fe(3)O(4). The microspheres were characterized by FTIR spectra. They were used as carrier to immobilize bovine serum albumin (BSA). To investigate the effect of the microsphere surface properties on the immobilization of BSA, a series of microspheres with different hydrophobic/hydrophilic surface characteristics were prepared by adjusting molar percentages of monomers. The results showed that microspheres with different hydrophobicities/hydrophilicities had different immobilized ratios of BSA. In comparison with microspheres having hydrophilic characteristics those with hydrophobic characteristics had a much higher immobilized ratio. The possible reasons for these observations are discussed. In addition, ester activation and coupling times were optimized with respect to immobilized ratio.