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. 2019 Apr;8(4):e00700.
doi: 10.1002/mbo3.700. Epub 2018 Dec 30.

The use of clade-specific PCR assays to identify novel nitrilase genes from environmental isolates

Tríona-Marie Dooley-Cullinane  1 , Catherine O'Reilly  2 , Bilal Aslam  1 , David P Weiner  3 , David O'Neill  1 , Erica Owens  1 , Denise O'Meara  1 , Lee Coffey  1
Affiliations

The use of clade-specific PCR assays to identify novel nitrilase genes from environmental isolates

Tríona-Marie Dooley-Cullinane et al. Microbiologyopen. 2019 Apr.

Abstract

Nitrilase enzymes (EC 3.5.5.1) are responsible for the direct hydration of nitriles to their corresponding carboxylic acids and ammonia. The utilization of nitrilase enzymes in biocatalysis toward bio-pharmaceuticals and industrial applications facilitates the move towards green chemistry. The body of research presented describes a novel clade-specific touchdown PCR protocol for the detection of novel nitrilase genes. The presented study identified partial sequences of 15 novel nitrilase genes across 7 genera, with partial DNA sequence homology (%) displayed across an additional 16 genera. This research will prove valuable in the screening of microorganisms for the identification of novel clade-specific nitrilase genes, with predicted enantioselective profiles as determined by their clade characterizations.

Keywords: clade specific; degenerate primers; environmental isolates; gene detection; nitrilase; touchdown PCR.

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Conflict of interest statement

No conflict of interest to declare.

Figures

Figure 1
Figure 1
Positive control uncultured clones 1A1 (BD7032), 1A3 (BD5273), 1A21 (BD5344), 1A23 (BD6901) and, 1A23 (BD5156) were used in the contruction of the 1A phylogenetic tree. The uncultured organism clone 5B1 (BD6902) gene was use to root the phylogenetic tree
Figure 2
Figure 2
Positive control uncultured clones 1B15 (BD5265), 1B7 (BD5266) and, 1B10 (BD7031) were used in the contruction of the 1B phylogenetic tree.The uncultured organism clone 6A3 (BD7050) gene was use to root the phylogenetic tree
Figure 3
Figure 3
Positive control uncultured clones 2A24 (BD5332), 2A25 (BD5313), 2A20 (BD7753), 2A32 (BD5282), 2A7 (BD5275), 2A13 (BD5086) and, 2A14 (BD7757) were used in the contruction of the 2A phylogenetic tree.The uncultured organism clone 6A2 (BD7547) gene was use to root the phylogenetic tree
Figure 4
Figure 4
Positive control uncultured clones 4A8 (BD7626), 4A2 (BD5261) and, 4A14 (BD5070) were used in the contruction of the 4A phylogenetic tree.The nitrilase from Rhodococcus erythropolis AJ270 (EU234513.1) gene was use to root the phylogenetic tree
Figure 5
Figure 5
Positive control uncultured clones 5A2 (BD7269), 5A5 (BD7756) and, 5A8 (BD5222) were used in the contruction of the 5A phylogenetic tree. The nitrilase from Rhodococcus erythropolis AJ270 (EU234513.1) gene was use to root the phylogenetic tree
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