RESUMO
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.
Assuntos
Bactérias/enzimologia , Bactérias/isolamento & purificação , Proteínas de Bactérias/genética , Microbiologia Ambiental , Hidroliases/genética , Reação em Cadeia da Polimerase/métodos , Bactérias/classificação , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Biocatálise , Ácidos Carboxílicos/metabolismo , Clonagem Molecular , Hidroliases/química , Hidroliases/metabolismo , Nitrilas/metabolismo , Filogenia , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
Aldoxime dehydratase catalyses the conversion of aldoximes to their corresponding nitriles. Utilization of the aldoxime-nitrile metabolising enzyme pathway can facilitate the move towards a greener chemistry. In this work, a real-time PCR assay was developed for the detection of aldoxime dehydratase genes in aldoxime/nitrile metabolising microorganisms which have been purified from environmental sources. A conventional PCR assay was also designed allowing gene confirmation via sequencing. Aldoxime dehydratase genes were identified in 30 microorganisms across 11 genera including some not previously shown to harbour the gene. The assay displayed a limit of detection of 1 pg/µL DNA or 7 CFU/reaction. This real-time PCR assay should prove valuable in the high-throughput screening of micro-organisms for novel aldoxime dehydratase genes towards pharmaceutical and industrial applications.
