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From sequence to function: a new workflow for nitrilase identification.

ABSTRACT: Nitrilases are industrially important biocatalysts due to their ability to degrade nitriles to carboxylic acids and ammonia. In this study, a workflow for simple and fast recovery of nitrilase candidates from metagenomes is presented. For identification of active enzymes, a NADH-coupled high-throughput assay was established. Purification of enzymes could be omitted as the assay is based on crude extract containing the expressed putative nitrilases. In addition, long incubation times were avoided by combining nitrile and NADH conversion in a single reaction. This allowed the direct measurement of nitrile degradation and provided not only insights into substrate spectrum and specificity but also in degradation efficiency. The novel assay was used for investigation of candidate nitrilase-encoding genes. Seventy putative nitrilase-encoding gene and the corresponding deduced protein sequences identified during sequence-based screens of metagenomes derived from nitrile-treated microbial communities were analyzed. Subsequently, the assay was applied to 13 selected candidate genes and proteins. Six of the generated corresponding Escherichia coli clones produced nitrilases that showed activity and one unusual nitrilase was purified and analyzed. The activity of the novel arylacetonitrilase Nit09 exhibited a broad pH range and a high long-term stability. The enzyme showed high activity for arylacetonitriles with a KM of 1.29 mM and a Vmax of 13.85 U/mg protein for phenylacetonitrile. In conclusion, we provided a setup for simple and rapid analysis of putative nitrilase-encoding genes from sequence to function. The suitability was demonstrated by identification, isolation, and characterization of the arylacetonitrilase. KEY POINTS: • A simple and fast high-throughput nitrilase screening was developed. • A set of putative nitrilases was successfully screened with the assay. • A novel arylacetonitrilase was identified, purified, and characterized in detail.

SUBMITTER: Egelkamp R

PROVIDER: S-EPMC7228900 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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From sequence to function: a new workflow for nitrilase identification.

Egelkamp Richard R Friedrich Ines I Hertel Robert R Daniel Rolf R

Applied microbiology and biotechnology 20200414 11

Nitrilases are industrially important biocatalysts due to their ability to degrade nitriles to carboxylic acids and ammonia. In this study, a workflow for simple and fast recovery of nitrilase candidates from metagenomes is presented. For identification of active enzymes, a NADH-coupled high-throughput assay was established. Purification of enzymes could be omitted as the assay is based on crude extract containing the expressed putative nitrilases. In addition, long incubation times were avoided ...[more]

PMID: 32291488

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Project description:BackgroundOver the last few years, great progress has been made in the development of key technologies to detect peripheral blood-based, tumor-specific biomarkers, such as circulating tumor cells (CTCs) and circulating cell free tumor DNA (ctDNA). Despite the considerable advances and their multiple clinical values, liquid biopsies are challenged by the very low concentrations of CTCs and ctDNA in blood samples. Additionally, blood biomarkers which were found using data-driven methods may only be effective in few datasets.MethodsWe firstly collected the genes which have expression correlations between blood and the other tissues/organs using Genotype-Tissue Expression (GTEx). Survival hazard genes and differential expression genes of each cancer type in The Cancer Genome Atlas (TCGA) were then selected by Cox regression model and Wilcoxon rank sum test, respectively. By combining the P values of two steps, several blood biomarkers can be inferred for each cancer type. After applying these potential blood biomarker sets to 13 datasets of blood samples from solid tumor patients using single sample gene set enrichment analyses (ssGSEA), we got an enrichment score (ES) for each sample.ResultsThe inferred blood biomarker (BB infer) genes showed reliable predictive value in various malignancies. In all the blood samples that were analyzed, the ESs of positive BB Infer genes in cancer patients are higher than healthy people. Conversely, the ESs of negative BB Infer genes in cancer patients are lower than healthy people. Furthermore, lower ES of negative BB infer genes signify the dismal outcome of patients.ConclusionsWe developed a novel solid tumor blood biomarker inference workflow for cancer screening and diagnosis. Moreover, we demonstrated the utility of this inference method in a series of blood sample datasets of solid tumor patients. These results suggested the potential value of this method in the screening, diagnosis and prognosis of cancers.

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From sequence to function: a new workflow for nitrilase identification.

Publications

From sequence to function: a new workflow for nitrilase identification.

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