葡萄糖-6-磷酸脱氢酶反应条件优化及在大肠杆菌生物发光检测法中的应用  

Optimization of conditions for glucose- 6- phosphate dehydrogenase and its application on bioluminescence assay of Escherichia coli

作　　者：刘慧慧[1] 王静雪[1] 林洪[1] 

机构地区：[1]中国海洋大学食品科学与工程学院,山东青岛266003

出　　处：《中国渔业质量与标准》2014年第3期12-17,共6页Chinese Fishery Quality and Standards

基　　金：山东省自然科学基金(ZR2011CQ024);国家自然科学基金(31071540)

摘　　要：利用还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)具有荧光性的原理,对葡萄糖-6-磷酸脱氢酶催化的反应条件进行了优化,目的是提高细菌生物发光法检测大肠杆菌的灵敏度。葡萄糖-6-磷酸脱氢酶可以专一性地将烟酰胺腺嘌呤二核苷酸磷酸(NADP+)转化为NADPH,在激发波长为340 nm、发射波长为460 nm的条件下可以测定反应生成的NADPH荧光强度。本研究确定了实验的最适反应pH为7.5,葡萄糖-6-磷酸的反应浓度为5 mmol/L,葡萄糖-6-磷酸脱氢酶的反应浓度为0.4 U/mL。同时将细菌生物发光法与酶促反应相结合,建立了大肠杆菌与NAD(P)H发光强度之间的关系,为酶促细菌生物发光法应用于大肠杆菌的检测提供基础资料。[中国渔业质量与标准,2014,4(3):12-17]Based on the fluorescent characteristic of reduced nicotinamide adenine dinucleotide phosphate （ NADPH）, the reaction conditions of glucose - 6 - phosphate dehydrogenase were optimized to increase the sensitivity of bacterial bioluminescence assay. Glucose - 6 - phosphate dehydrogenase could convert nicotinamide adenine dinucleotide phos- phate （NADP ＋ ） to NADPH specifically, and the latter is a fluorescent molecule that can be visualized by fluores- cence spectrophotometer using an excitation wavelength of 340 nm and an emission wavelength of 460 nm. The results showed that the optimum pH was 7.5, the concentration of glucose - 6 - phosphate was 5 mmol/L and concentration of glucose - 6 - phosphate dehydrogenase was 0.4 U/mL. Combining enzymatic reaction with bioluminescence, the luminescence intensity has a good correlation with bacterial count, which can be applied to the detection of Escherichia coli. [ Chinese Fishery Quality and Standards, 2014, 4 （3） ： 12 - 17 ]

关 键 词：NADPH NADP+ 荧光法 葡萄糖-6-磷酸脱氢酶 大肠杆菌 优化 

分 类 号：Q936[生物学—微生物学] S94[农业科学—水产养殖]