吡咯喹啉醌高通量检测方法的建立与优化  

Establishment and Optimization of the High-Throughput Measurement of Pyrroloquinoline Quinone

作　　者：夏雨[1,2] 周景文[1,2] 陈坚[1,2] 

机构地区：[1]江南大学工业生物技术教育部重点实验室,江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与生物技术学报》2017年第2期122-128,共7页Journal of Food Science and Biotechnology

基　　金：国家863计划项目(2012AA022103)

摘　　要：吡咯喹啉醌(Pyrroloquinoline quinone,PQQ)是一种新型辅酶,具有广阔的应用前景。传统PQQ检测方法在检测发酵液中PQQ浓度时都存在一定的缺陷,且检测效率偏低。本研究在大肠杆菌(Escherichia coli BL21(DE3))中表达E.coli K-12来源的葡萄糖脱氢酶(PQQGDH),并通过亲和层析分离纯化PQQGDH,得到了高浓度、高纯度的PQQGDH。通过酶液和PQQ样品用量的双因素正交实验对传统重组酶法的反应体系加以改进,得到适用于96孔板高通量检测的反应体系。结果表明30μL纯化后酶液、2μL PQQ样品配合1.2 m L的显色剂组成的显色体系检测效果最佳。该方法线性范围大且具有较高的精确度和重现性。本研究中利用高浓度、高纯度的PQQGDH配合96孔板和酶标仪,所建立的PQQ高通量检测方法,不仅降低了实验误差,而且大幅提高了PQQ发酵液样品的检测效率。Pyrroloquinoline quinone（PQQ） is a new coenzyme that has broad application prospect.Previous methods used for measuring PQQ in fermentation broth are defective and low-efficient. In this study, D-glucose dehydrogenase（PQQGDH） from Escherichia coli K-12 was expressed in E. coli BL21 and then purified by the nickel-affinity chromatography column. For the high-throughput measurement of PQQ the optimal reaction system was found by orthogonal experiment to contain 30 μL PQQGDH,2 μL PQQ sample and 1.2 m L chromogenic reagent. The method was linear within a wide range and had high precision and good reproducibility. Compared with traditional methods,the high-throughput measurement of PQQ using purified PQQGDH,96-well plate and microplate reader are herein more accurate and efficient.

关 键 词：吡咯喹啉醌 葡萄糖脱氢酶 亲和纯化 重组酶法检测 高通量检测 

分 类 号：Q503[生物学—生物化学]