新型α-半乳糖苷酶的稳定性研究

Stability of a Novel α-Galactosidase from Bacteroides fragilis

目的：观察脆弱类杆菌来源的α-半乳糖苷酶（GAL）在不同pH缓冲液、不同温度下的稳定性，以及不同离子及还原剂对酶活性的影响。方法：以GAL对单糖底物对硝基-苯基-α-D-吡喃半乳糖苷（PNPG）的活性为主要检测指标，观察不同离子及还原剂等对酶活性的影响；观察GAL在不同pH缓冲液中和不同温度下的稳定性。结果：钙离子、锌离子、钴离子和高浓度的锰离子增强酶的活性，DTT抑制酶的活性，螯合剂EDTA的加入提高了酶活性。GAL在pH4.6～7.5时保存1 h后稳定性很好，能保持最高活性的90％以上；在4℃～45℃下保存的稳定性最好，45℃开始活性下降。结论：GAL具有很好的温度稳定性和pH稳定性，使其适用于血型转变和异种移植。

Objective： To determine the stability and activity of recombinant Bacteroides fragilis α-galaetosidase in different pH buffer, different temperature and the influence of different additives on the enzyme. Methods： The activity of purified recombinant Bfragilis α-galactosidase was measured in the presence of divalent metals, metal chelating and reducing agents, at two concentrations（1 and 10 mmol/L respectively）. The enzyme was incubated with these reagents at 26℃ for 1 h and the activity was detected. The thermal stability was determined by incubating the enzyme solution at temperatures ranging from 4 to 70℃ for 1 h, and the stability was also determined by incubating the enzyme in different pH buffer（pH2.0-8.5） for 1 h at 26℃. Results： Some metals such as Ca^2＋, Zn^2＋ and Co^2＋, showed enhancement effects on enzyme activity. Inaddition, higher concentration Mn^2＋ showed an enhancement effects, and EDTA showed an enhancement effects too. But DTT showed an inhibitory effects. Cu2＋, Ni^2＋, Mg^2＋ and β-Me had no significantly effect on the activity. The results indicated that the enzyme was stable at a broad pH range of 4.6-7.5 and thermostability up to 45℃. Conclusion： Recombinant Bfragilis α-galactosidase could be kept its activity in an appropriate condition. This character makes it suitable for blood conversion and the application in xenotransplantation.