Bacillus pumilus Cyanide Dihydratase Mutants with Higher Catalytic Activity.

Cyanide degrading nitrilases are noted for their potential to detoxify industrial wastewater contaminated with cyanide. However, such application would benefit from an improvement to characteristics such as their catalytic activity and stability. Following error-prone PCR for random mutagenesis, several cyanide dihydratase mutants from Bacillus pumilus were isolated based on improved catalysis. Four point mutations, K93R, D172N, A202T, and E327K were characterized and their effects on kinetics, thermostability and pH tolerance were studied. K93R and D172N increased the enzyme's thermostability whereas E327K mutation had a less pronounced effect on stability. The D172N mutation also increased the affinity of the enzyme for its substrate at pH 7.7 but lowered its k cat. However, the A202T mutation, located in the dimerization or the A surface, destabilized the protein and abolished its activity. No significant effect on activity at alkaline pH was observed for any of the purified mutants. These mutations help confirm the model of CynD and are discussed in the context of the protein-protein interfaces leading to the protein quaternary structure.

Evaluation of the LIAISON chemiluminescence immunoassay for diagnosis of syphilis.

We report the results of an evaluation of the LIAISON Treponema pallidum-specific assay, a one-step sandwich chemiluminescence immunoassay (CLIA), as a screening test and as a confirmatory test for the diagnosis of syphilis. The assay was compared with the CAPTIA Syphilis-G enzyme immunoassay (EIA) and with a testing algorithm that also included the rapid plasma reagin (RPR) and T. pallidum particle agglutination (PA) assays. As a screening test, the CLIA showed levels of agreement with the EIA and with the algorithm, respectively, of 94.1 and 100% for 51 samples from patients with primary or secondary syphilis, 93.2 and 98.7% for 999 samples sent to the laboratory for routine syphilis testing, 84.5 and 94.0% for 200 samples from human immunodeficiency virus-positive patients, 98.0 and 100% for 200 samples from pregnant patients, and 94.3 and 98.3% for 992 samples from apparently healthy adults. As a confirmatory test, the CLIA showed 99% agreement with the EIA for 204 RPR-positive samples. After resolution with further T. pallidum PA testing and the discarding of one sample of insufficient quantity, there was 100% agreement for the remaining 203 samples. For the total group of 2,645 samples, the overall relative sensitivity was 95.8% and the relative specificity was 99.1%. We conclude that the LIAISON CLIA demonstrated excellent sensitivity and specificity when evaluated as a confirmatory test and as a screening test for syphilis among various patient populations, including specific populations with reportedly increased rates of false-positive nontreponemal test results.