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
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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 phosphate( NADP+) to NADPH specifically,and the latter is a fluorescent molecule that can be visualized by fluorescence 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]
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 phosphate( NADP+) to NADPH specifically,and the latter is a fluorescent molecule that can be visualized by fluorescence 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]
引文
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[17]Denis A L,Denis N.Determination of pyridine nucleotide contents of cell monolayers by bioluminescence[J].J Biochem Bioph Meth,1988,17(2):107-117.
[2]Paul D M,John C,Shanda W.Pyruvate improves redox status and decreases indicators of hepatic apoptosis during hemorrhagic shock in swine[J].Am J Physiol,2002,283(4):1634-1644.
[3]王晶,王静雪,林洪.NADH荧光法快速检测细菌总数[J].微生物学通报,2009,36(5):773-779.
[4]梅册霞.细菌荧光素酶体外发光体系的建立及应用于NADH定量检测[J].微生物学报,2009,49(9):1223-1228.
[5]Warren D,Hasting J W.Flavin mononucleotide reductase of luminous bacteria[J].Mol Cell Biochem,1974,6(1):53-64.
[6]Watanabe H,Hastings J W.Specificities and properties of three reduced pyridine nucleotide-flavin mononucleotide reductases coupling to bacterial luciferase[J].Mol Cell Biochem,1982,44(3):181-187.
[7]Erik G,Josee C.Identification of NADH-specific and NADPH-specific FMN reductases in Beneckea harvey I[J].Eur J Biochem,1975,57(2):461-467.
[8]Ross C,Paul A I,Wolfgang G J.A novel method using fluorescence microscopy for real-time assessment of ATP release from individual cells[J].Am J Physiol Cell Physiol,2007,293:1420-1425.
[9]Aiping Z,Roberto R,Howard R P.An enzymatic fluorimetric assay for glucose-6-phosphate:application in an in vitro Warburg-like effect[J].Anal biochem,2009,388(1):97-101.
[10]Gibon Y,Vigeolas H,Tiessen A,et al.Sensitive and high throughput metabolite assays for inorganic pyrophosphate,ADPGlc,nucleotide phosphates and glycolytic intermediates based on a novel enzymic cycling system[J].Plant J,2002,30(2):221-235.
[11]刘昆元,周佐平,王璞.NADH荧光传感器研究[J].化学传感器,1992,12(1):1-8.
[12]Chenault H K,Whitesides G M.Regeneration of nicotinamide cofactors for use in organic synthesis[J].Appl Biochem Biotech,1987,14:147-197.
[13]吕陈秋,姜忠义,王娇,烟酰型辅酶NAD(P)+和NAD(P)H再生的研究进展[J].有机化学,2004,24:1366-1379.
[14]David W P,Susan M K.Real-time analysis of glucose metabolism by microscopy[J].TEM,1999,10:413-417.
[15]Charles O,Mary E G,Richard L.Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides[J].J Biol Chem,1975,246:2047-2057.
[16]安选,刘良忠,胡鹏.葡萄糖-6-磷酸脱氢酶的研究进展[J].中国生物制品学杂志,2011,24(6):745-748.
[17]Denis A L,Denis N.Determination of pyridine nucleotide contents of cell monolayers by bioluminescence[J].J Biochem Bioph Meth,1988,17(2):107-117.