Immobilization of trypsin by thermo-responsive hydrogel for the affinity separation of trypsin inhibitor

A series of thermo-responsive hydrogels were synthesized from N-isopropylacrylamide (NIPAAm), glycidyl methacrylate (GMA), and N, N-dimethyl acrylamide (DMA). The effect of the different monomer ratios of GMA/DMA in the copolymeric hydrogel on the swelling behaviors, physical properties, and drug release behavior was investigated. Results showed that the swelling ratio in deionized water increased with an increase of content of DMA in the copolymeric hydrogel. However, the compressive modulus, crosslinking density, and mechanical strength decreased with increasing DMA content in the copolymeric hydrogel. In addition, the influence of the copolymeric composition on the immobilization of enzyme on the poly(NIPAAm-co-GMA-co-DMA) hydrogel which was used as a support to immobilize trypsin was also investigated. The result showed that the copolymeric hydrogel showed a good immobilized capacity of trypsin. The immobilized trypsin as a ligand of affinity chromatography having fifty three percent activity of trypsin inhibitor in the crude extract of sweet potato can be recovered by affinity chromatography. The molecular weights of the purified trypsin inhibitors are more than 20 kDa by SDS-PAGE.

A series of poly(NIPAAm-co-GMA-coDMA) copolymeric hydrogels were successfully synthesized through photo-polymerization. The various properties of the present hydrogels were characterized. The results exhibited that the gel strength and crosslinking density increased with increase in the molar ratio of GMA/DMA, but their swelling ratios decreased with increasing of the molar ratio of GMA/DMA. This is because the hydrophobic GMA moiety made the gel structure become tighter and made the water hard to penetrate into the network of the gels. However, adding hydrophilic DMA moiety into the gel can make the gel absorb more water content and diminish the deswelling rate of the gel under higher temperature. Hence, the present gels copolymerized with higher molar ratio GMA/DMA under low temperature can increase the mechanical strength of normal hydrogels. The other objective of this study is to separate the sporamin from sweet potato tuber use this series gels. A detailed investigation of trypsin immobilization on the poly(NIPAAm-co-GMAco-DMA) copolymeric hydrogel was performed and the sweet potato storage protein (primarily sporamin) which comprises about 53% of the total protein in sweet potato tuber can be recovered from this separation process. The obtained sporamin had a molecular weight of about 21 kDa that is the very close to results for trypsin inhibitors of sweet potato.