关键词: 多功能淀粉酶, 壳聚糖, 固定化, 底物, 凝胶微球

Abstract:

The catalytic site of the multifunctional amylase OPMA-N occupied a relatively large region in OPMA-N molecule. The optimal conditions to immobilize OPMA-N with hydrogel microspheres of chitosan as a carrier were established by orthogonal experiment as follows: 20 mg/mL glutaraldehyde served as cross-linking agent, at 25 ℃, pH=6.5 for 1.5 h, but as long as after the introduction of the substrate-mediated protective effect and conformational memory in order to obtain a stabilized immobilized OPMA-N(IOPMA-N). This highlighted the importance of the protective effect of the substrate in the immobilization process of the multifunctional enzymes such as OPMA-N in which the catalytic site-covering region generally occupies a larger region in OPMA-N molecule. Compared with the free OPMA-N, IOPMA-N showed more than 3 times apparent activity and more than 4 times catalytic efficiency(k_cat/K_m) at 50 ℃, pH=6.0—7.0, and had a significantly better tolerance to the pH from neutral to weakly acidic under moderate temperatures(30—50 ℃). IOPMA-N also exhibited a better operational stability and storage stability by its more than 75% of the apparent activity after 15 times repeated use and 31 d half-life when stored at 4 ℃, while OPMA-N would lose its activity completely after stored 5 d at 4 ℃. Comparative analysis of the catalytic product showed that IOPMA-N exhibited higher hydrolytic activity but slightly lower transglycosyl activity than OPMA-N, which is consistent with the fact that there is a functional(active) balance between hydrolytic and transglycosyl activities in OPMA-N molecule, and meanwhile, it also indicated that its substrate had a significant protective effect on its hydrolytic activity but hardly on its transglycosyl activity in its immobilization.

Key words: Multifunctional amylase, Chitosan, Immobilization, Substrate, Hydrogel microsphere