EC Number   |
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   3.5.1.B15 | combined whole-cell high-throughput functional screening for identification of nicotinamidases/pyrazinamidases in metagenomic/polygenomic libraries. A fosmid polygenomic expression library obtained from deep-sea mesophilic bacteria is screened, discovering several positive clones with the ammonium ferrous sulfate method. Quantitative rescreening with the SNP method allowing the finding of the first nicotinamidase with balanced catalytic efficiency toward nicotinamidase activity (EC 3.5.1.19) and pyrazinamide (pyrazinamidase activity). Escherichia coli EPI300-T1 is used for the screening of the genomic libraries cloned in pCC1FOS fosmids library. Escherichia coli strain Rosetta2 (DE3) is used as the host for pET46Ek/LIC expression vector. The fosmid library contains inserts of mixed genomic DNA of 194 mesophilic bacteria (MB) isolated from deep-sea hydrothermal vents sampled at various sites in Mid Atlantic Ridge, genotyping, DNA and amino acid sequence determination and analysis |
| 3.5.1.B15 | expressed in a methionine auxotroph, Escherichia coli strain B834 |
| 3.5.1.B15 | expressed in Escherichia coli |
| 3.5.1.B15 | gene pncA, DNA and amino acid sequence determination and analysis of enzyme mutants, genotyping |
| 3.5.1.B15 | gene pncA, DNA and amino acid sequence determination and analysis, recombinant enzyme expression in Escherichia coli strain BL21(DE3)pLysS |
| 3.5.1.B15 | gene pncA, genotyping, DNA and amino acid sequence determination and analysis of wild-type and mutants |
| 3.5.1.B15 | gene pncA, genotyping, global statistics for 641 mutations in 171 (of 187) codons from 2760 resistant strains and 96 mutations from 3329 susceptible strains reported in 61 studies, overview |
| 3.5.1.B15 | gene pncA, PCR-based in vitro expression, and recombinant expression of His-tagged mutant enzyme in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain TOP10 |
| 3.5.1.B15 | gene pncA, PCR-based in vitro expression, and recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain TOP10 |
| 3.5.1.B15 | gene pncA, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3) |
