色谱

色谱 ›› 2018, Vol. 36 ›› Issue (4): 413-415.DOI: 10.3724/SP.J.1123.2017.09039

• 技术与应用 • 上一篇    

2,3-吡啶二甲酰亚胺及其工程菌转化产物的高效液相色谱检测

钮利喜, 马师师   

  1. 山西大学生物技术研究所, 教育部化学生物学与分子工程重点实验室, 山西 太原 030006
  • 收稿日期:2017-10-09 出版日期:2018-04-08 发布日期:2014-02-26
  • 通讯作者: 钮利喜,E-mail:nlx@sxu.edu.cn

Analysis of 2, 3-pyridinedicarboximide and its products conversed by engineering strain with high performance liquid chromatography

NIU Lixi, MA Shishi   

  1. Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Shanxi University, Taiyuan 030006, China
  • Received:2017-10-09 Online:2018-04-08 Published:2014-02-26

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摘要：

建立了利用含D-海因酶的基因工程菌转化吡啶二甲酰亚胺（PDI）生成3-氨基甲酰基-α-吡啶甲酸（α-3CP）的高效液相色谱（HPLC）检测方法。将工程菌pET3a-hyd/BL21（DE3）诱导表达后收集菌体，以PDI为底物，37℃摇床反应30 min后，以13000 r/min离心，取上清液进行HPLC检测。色谱条件：HypersilTM GOLD C18色谱柱（250 mm×4.6 mm，5 μm）；流动相为H2O-乙腈（体积比为90：10，含0.1%（体积分数）三氟乙酸）；检测波长为254 nm。当底物PDI达到饱和浓度时，测得工程菌pET3a-hyd/BL21（DE3）的比活力为0.61 U/（mL·10OD600 nm）。该研究为今后利用生物法制备复杂半酰胺有机物提供了坚实的理论基础。

关键词: -吡啶甲酸, 2, 3-氨基甲酰基-&alpha, 3-吡啶二甲酰亚胺, 高效液相色谱法, 工程菌

Abstract:

A high performance liquid chromatography (HPLC) method was established for the detection of 2, 3-pyridinedicarboximide (PDI) and its enzyme reaction products, 3-carbamoyl-α-picolinic acid (α-3CP), using an engineering strain containing the D-hydantoinase gene expression box.The strain pET3a-hyd/BL21(DE3) was collected after induction and added to a PDI saturated aqueous solution.After reacting at 37℃ for 30 min with constant stirring, the supernatant was separated by centrifugation at 13000 r/min and detected by HPLC.The chromatographic conditions were as follows:HypersilTM GOLD C18 column (250 mm×4.6 mm, 5 μm), H2O-acetonitrile (90:10, v/v) containing 0.1%(v/v) trifluoroacetic acid as the mobile phase with a flow rate of 1 mL/min and a detection wavelength of 254 nm.The specific activity of pET3a-hyd/BL21(DE3) was found to be 0.61 U/(mL·10OD600 nm).This study provides a theoretical basis for the preparation of complicated half-amides using biological methods.

Key words: 2,3-pyridinedicarboximide, 3-carbamoyl-α-picolinic acid, engineering strain, high performance liquid chromatography (HPLC)

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