目的表征金属β-内酰胺酶(MBL)L1的结构和动力学特性,以其发展MBL的通用型抑制剂。方法在大肠埃希菌BL21(DE3)中表达并纯化野生型L1(Wild type L1,WT-L1),以SDS-PAGE电泳确认;用EDTA除去WT-L1中的Zn(Ⅱ)离子得Apo-L1,再用Co(II)滴定制得...目的表征金属β-内酰胺酶(MBL)L1的结构和动力学特性,以其发展MBL的通用型抑制剂。方法在大肠埃希菌BL21(DE3)中表达并纯化野生型L1(Wild type L1,WT-L1),以SDS-PAGE电泳确认;用EDTA除去WT-L1中的Zn(Ⅱ)离子得Apo-L1,再用Co(II)滴定制得Co(Ⅱ)-L1;通过UV-Vis,CD及荧光光谱表征WT-L1及Co(Ⅱ)-L1活性中心的结构;采用稳态动力学方法测定WT-L1及Co(Ⅱ)-L1催化3类9种β-内酰胺类抗生素水解反应的酶动力学参数,确定抗生素对L1的稳定性。结果获得纯化的WT-L1和重组的Co(Ⅱ)-L1;光谱表征表明L1具有两个金属活性中心,Co(II)-L1的二级结构发生显著变化;WT-L1对碳青霉烯类抗生素的酶促活性最强,青霉素类次之,头孢类抗生素较弱,Co(Ⅱ)-L1比WT-L1的酶催活性弱。结论 Co(Ⅱ)-L1的重组实现了对MBL-L1的结构表征,L1对青霉素类抗生素呈现强的催化活性,对头孢类抗生素稳定。展开更多
A simple, convenient and on-site determination method and the corresponding handheld analyzer for monitoring toxicity of organophosphates and carbamates in vegetables were developed based on enzyme catalytic dynamic p...A simple, convenient and on-site determination method and the corresponding handheld analyzer for monitoring toxicity of organophosphates and carbamates in vegetables were developed based on enzyme catalytic dynamic photometry. Variousexperimental parameters were studied and optimized. The toxicity level can be judged according to the enzyme inhibition rate directly displayed on the analyzer. The method is rapid, with an average analysis time of 6-8 min for per sample. It is of practicality and great value, and can effectively be applied for monitoring the toxicity of pesticides, such as organophosphates and carbamates, commonly used for vegetables.展开更多
文摘目的表征金属β-内酰胺酶(MBL)L1的结构和动力学特性,以其发展MBL的通用型抑制剂。方法在大肠埃希菌BL21(DE3)中表达并纯化野生型L1(Wild type L1,WT-L1),以SDS-PAGE电泳确认;用EDTA除去WT-L1中的Zn(Ⅱ)离子得Apo-L1,再用Co(II)滴定制得Co(Ⅱ)-L1;通过UV-Vis,CD及荧光光谱表征WT-L1及Co(Ⅱ)-L1活性中心的结构;采用稳态动力学方法测定WT-L1及Co(Ⅱ)-L1催化3类9种β-内酰胺类抗生素水解反应的酶动力学参数,确定抗生素对L1的稳定性。结果获得纯化的WT-L1和重组的Co(Ⅱ)-L1;光谱表征表明L1具有两个金属活性中心,Co(II)-L1的二级结构发生显著变化;WT-L1对碳青霉烯类抗生素的酶促活性最强,青霉素类次之,头孢类抗生素较弱,Co(Ⅱ)-L1比WT-L1的酶催活性弱。结论 Co(Ⅱ)-L1的重组实现了对MBL-L1的结构表征,L1对青霉素类抗生素呈现强的催化活性,对头孢类抗生素稳定。
文摘A simple, convenient and on-site determination method and the corresponding handheld analyzer for monitoring toxicity of organophosphates and carbamates in vegetables were developed based on enzyme catalytic dynamic photometry. Variousexperimental parameters were studied and optimized. The toxicity level can be judged according to the enzyme inhibition rate directly displayed on the analyzer. The method is rapid, with an average analysis time of 6-8 min for per sample. It is of practicality and great value, and can effectively be applied for monitoring the toxicity of pesticides, such as organophosphates and carbamates, commonly used for vegetables.
