新型α-半乳糖苷酶的稳定性研究 |
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引用本文: | 张雪,高红伟,李素波,鲍国强,檀英霞,王颖丽,季守平,官锋.新型α-半乳糖苷酶的稳定性研究[J].生物技术通讯,2013(6):850-852. |
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作者姓名: | 张雪 高红伟 李素波 鲍国强 檀英霞 王颖丽 季守平 官锋 |
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作者单位: | 军事医学科学院野战输血研究所,北京100850 |
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摘 要: | 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。
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关 键 词: | α-半乳糖苷酶 稳定性 血型转变 活性测定 |
Stability of a Novel α-Galactosidase from Bacteroides fragilis |
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Affiliation: | ZHANG Xue, GAO Hong-Wei, LI Su-Bo, BAO Guo-Qiang, TAN Ying-Xia, WANG Ying-Li, JI Shou-Ping, GONG Feng (Beijing Institute of Transfusion Medicine, Beijing 100850, China) |
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Abstract: | 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. |
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Keywords: | α-galactosidase stability blood group conversion activity assay |
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