Characterization of a recombinant 7,8-linoleate diol synthase from Glomerella cingulate

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• 作者：Min-Ju Seo ; Kyung-Chul Shin ; Jung-Ung An…
• 关键词：Glomerella cingulate ; Linoleic acid ; 7S ; 8S ; Dihydroxy ; 9 ; 12(Z ; Z) ; octadecadienoic acid ; 7 ; 8 ; Linoleate diol synthase ; Enzyme characterization
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2016
• 出版时间：April 2016
• 年：2016
• 卷：100
• 期：7
• 页码：3087-3099
• 全文大小：1,844 KB
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• 作者单位：Min-Ju Seo (1)
  Kyung-Chul Shin (1)
  Jung-Ung An (1)
  Woo-Ri Kang (1)
  Yoon-Joo Ko (2)
  Deok-Kun Oh (1)
  
  1. Department of Bioscience and Biotechnology, Konkuk University, Seoul, 05029, Republic of Korea
  2. National Center for Inter-University Research Facilities (NCIRF), Seoul National University, Seoul, 08826, Republic of Korea
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Microbiology
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-0614

文摘

A putative diol synthase from the fungus Glomerella cingulate was cloned and expressed in Escherichia coli. The putative diol synthase from G. cingulate was purified by His-Trap affinity chromatography with a specific activity of 0.87 U mg−1, an eightfold purification, and a yield of 28 %. One unit of activity was defined as the amount of enzyme required to produce 1 μmol of 7,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid (7,8-DiHODE) per min. The purified enzyme was estimated as a 127-kDa tetramer with a molecular mass of 510 kDa by gel filtration chromatography. The enzyme converted linoleic acid to a product, identified as 7S,8S-DiHODE by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. The specific activity and catalytic efficiency (k _cat/K _m) of 7,8-diol synthase from G. cingulate for the conversion of fatty acid to dihydroxy fatty acid followed the order linoleic acid > α-linolenic acid > oleic acid > palmitoleic acid, indicating that the enzyme is a 7,8-linoleate diol synthase (7,8-LDS). The activity of the enzyme for the conversion of 7,8-DiHODE from linoleic acid was maximal at pH 6.5, 40 °C, and 2.5 % (v/v) dimethyl sulfoxide (DMSO). Under these conditions, 7,8-LDS from G. cingulate converted 1.0 mM linoleic acid to 0.62 mM 7,8-DiHODE for 30 min, with a conversion yield of 62 % (mol/mol), via 8-hydroperoxy-9,12(Z,Z)-octadecadienoic acid (8-HPODE) as an intermediate. The accumulation of 8-HPODE was due to a higher 8-dioxygenase activity in the N-terminal domain than hydroperoxide isomerase activity in the C-terminal domain.