Abstract
Purpose of work
Our aim is to describe new fungal nitrilases whose sequences were published but whose catalytic properties were unknown. We adapted for expression in E. coli three of the genes and confirmed that the enzymes acted on organic nitriles.
The genome mining approach was used to search for nitrilases in filamentous fungi. Synthetic genes encoding nitrilases in Aspergillus niger, Gibberella moniliformis and Neurospora crassa were expressed in Escherichia coli. This is the first heterologous expression of fungal enzymes of this type. The recombinant enzyme derived from G. moniliformis was an aromatic nitrilase with an activity of 390 U l−1 culture with benzonitrile as substrate. This was much less than the activities of the recombinant enzymes derived from A. niger and N. crassa that had activities of 2500 and 2700 U l−1 culture, respectively, with phenylacetonitrile as substrate.
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Acknowledgments
Financial support via projects IAA500200708 (Grant Agency of the Academy of Sciences of the Czech Republic), LC06010, OC09046 (Ministry of Education of the Czech Republic), European Cooperation in Science and Technology (COST) Action CM0701 (short-term scientific mission fellowships COST-STSM-CM0701-4765 and -4766 to A. Malandra), FT-TA5/043 (Ministry of Industry and Trade of the Czech Republic), 305/09/H008 (Czech Science Foundation) and Institutional Research Concept AV0Z50200510 (Institute of Microbiology) is gratefully acknowledged.
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Online resource 1
Multiple sequence alignment of nitrilases from Aspergillus niger CBS 513.88 (AsNig Nit; GenBank: XP_001397369), Neurospora crassa OR74A (NeCra Nit; GenBank: CAD70472) and Gibberella moniliformis (GiMon Nit; GenBank: ABF83489) (DOC 28 kb)
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Nucleotide sequences of nitrilase genes adapted for expression in Escherichia coli using GeneArt (Regensburg, Germany) software (DOC 31 kb)
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Kaplan, O., Bezouška, K., Malandra, A. et al. Genome mining for the discovery of new nitrilases in filamentous fungi. Biotechnol Lett 33, 309–312 (2011). https://doi.org/10.1007/s10529-010-0421-7
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DOI: https://doi.org/10.1007/s10529-010-0421-7