RESUMEN
A modified colorimetric high-throughput screen based on pH changes combined with an amidase inhibitor capable of distinguishing between nitrilases and nitrile hydratases. This enzymatic screening is based on a binary response and is suitable for the first step of hierarchical screening projects.
Asunto(s)
Aminohidrolasas/análisis , Colorimetría/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Hidroliasas/análisis , Amidohidrolasas/antagonistas & inhibidores , Concentración de Iones de HidrógenoRESUMEN
A modified colorimetric high-throughput screen based on pH changes combined with an amidase inhibitor capable of distinguishing between nitrilases and nitrile hydratases. This enzymatic screening is based on a binary response and is suitable for the first step of hierarchical screening projects.
Asunto(s)
Aminohidrolasas/análisis , Colorimetría/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Hidroliasas/análisis , Amidohidrolasas/antagonistas & inhibidores , Concentración de Iones de HidrógenoRESUMEN
A modified colorimetric high-throughput screen based on pH changes combined with an amidase inhibitor capable of distinguishing between nitrilases and nitrile hydratases. This enzymatic screening is based on a binary response and is suitable for the first step of hierarchical screening projects.(AU)
Asunto(s)
Aminohidrolasas/análisis , Colorimetría/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Hidroliasas/análisis , Amidohidrolasas/antagonistas & inhibidores , Concentración de Iones de HidrógenoRESUMEN
Growth and nitrilase production by recombinant Escherichia coli cells harbouring pET 21 (b) plasmid, for the expression of Pseudomonas putida nitrilase were improved using response surface methodology. Central composite design was used for obtaining ideal concentration of critical medium components which include fructose, tryptone, yeast extract and lactose. The optimal values for the concentration of fructose, tryptone, yeast extract and lactose were found to be 1.13, 2.26, 3.25 and 0.9 percent (w/v), respectively. Here, fructose served as carbon source for the growth while lactose was preferably used as inducer for the expression of foreign protein. Yeast extract in the medium was used as a growth promoter while tryptone was added as a major nitrogen source. Using this optimized medium, an experimental growth of 6.67 (OD at 600 nm) and nitrilase activity of 27.13 U/ml was achieved. This approach for medium development led to an enhancement of the growth and enzyme activity by 1.4 and 2.2 times, respectively, as compared to the un-optimized medium.
Asunto(s)
Aminohidrolasas/análisis , Nitrilos/análisis , Péptido Hidrolasas/análisis , Proteínas Recombinantes/análisis , Proteínas de Escherichia coli/análisis , Catálisis , Activación Enzimática , Métodos , MétodosRESUMEN
Fifty-five children with CID and known ADA status were studies at a workshop held in Albany, New York. Erythrocyte ADA determinations were performed in 22 of the 55 patients, 13 of whom were ADA negative. The ADA defect appears to be transmitted as an autosomal recessive trait. Some patients with CID and ADA deficiency have characteristic radiologic abnormalities of the skeleton, which are not found in other illnesses. The thymus glands of all patients with CID and ADA deficiency who could be examined have evidence of thymic involution manifested by presence of Hassall's corpuscles and differentiated germinal epithelium; this is in contrast to "classic" thymus findings in CID with normal ADA. Adenosine deaminase probably plays an important, although as yet undefined, role in lymphocyte development and/or function. The deficiency of ADA in CID is the first enzyme defect observed in a deficiency disease of specific immunity.