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

DOI：10.3969/j.issn.1673-1689.2017.02.002

中文关键词: 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

英文关键词: pyrroloquinoline quinone,D-glucose dehydrogenase,affinity purification,enzymatic measurement,high-throughput measurement

基金项目:

摘要点击次数: 123

全文下载次数: 556

中文摘要:

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30 ?滋L纯化后酶液、2 ?滋L PQQ样品配合1.2 mL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。

英文摘要:

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 mL 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.

查看全文 查看/发表评论 下载PDF阅读器