期刊
JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC
卷 99, 期 -, 页码 26-33出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.molcatb.2013.10.015
关键词
Nitrite hydratase; Recombinant expression; Substrate specificity; Activator gene; Genome mining
资金
- Key Project of Chinese National Programs for Fundamental Research and Development [2011CB710800]
- Hi-Tech Research and Development Program of China [2011AA02A209]
- National Natural Science Foundation of China [21206024, 20936002]
- Zhejiang Provincial Natural Science Foundation of China [LQ12B06007]
Microbial nitrile hydratases (NHases) are important industrial catalysts to produce valuable amides. However, only some NHase genes have been reported and studied at the molecular level. In this study, we developed a genome mining method to discover Fe-type NHases from GenBank. The putative NHase gene from Pseudomottas putida F1 was cloned and functionally expressed in Escherichia coli BL21 (DE3) by assisting of a putative activator gene adjacent to beta-subunit region. Three recombinant plasmids containing NHase gene or the activator gene were designed and constructed. Maximal enzyme activity was obtained when the structural and activator genes were transcribed as one unit in plasmid pCDFDuet-1 at 18 degrees C. However, the expressed product did not show any NHase activity when the downstream activator gene was ignored, and the product completely existed in insoluble inclusion body. The activator gene might be involved in protein folding of the alpha- and beta-subunits of NHase. In addition, the Fe-type NHase exhibited broad substrate specificity. The enzyme can efficiently hydrate aromatic nitriles, such as 3-cyanopyridine, 4-cyanopyridine, and benzonitrile, asides from aliphatic nitriles preferentially. Therefore, the recombinant NHase shows potential applications in some amides preparation. (C) 2013 Elsevier B.V. All rights reserved.
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