Substrate developped to test amidase activity of esterases and lipase. corresponding amine released is detected by the formation of a fluorescent dye by coupling with 4-nitro-7-chloro-benzo-2-oxa-1,3-diazole (NBD-CI)
Title: Bioinformatic analysis of alpha/beta-hydrolase fold enzymes reveals subfamily-specific positions responsible for discrimination of amidase and lipase activities Suplatov DA, Besenmatter W, Svedas VK, Svendsen A Ref: Protein Engineering Des Sel, 25:689, 2012 : PubMed
Superfamily of alpha-beta hydrolases is one of the largest groups of structurally related enzymes with diverse catalytic functions. Bioinformatic analysis was used to study how lipase and amidase catalytic activities are implemented into the same structural framework. Subfamily-specific positions-conserved within lipases and peptidases but different between them-that were supposed to be responsible for functional discrimination have been identified. Mutations at subfamily-specific positions were used to introduce amidase activity into Candida antarctica lipase B (CALB). Molecular modeling was implemented to evaluate influence of selected residues on binding and catalytic conversion of amide substrate by corresponding library of mutants. In silico screening was applied to select reactive enzyme-substrate complexes that satisfy knowledge-based criteria of amidase catalytic activity. Selected CALB variants with substitutions at subfamily-specific positions Gly39, Thr103, Trp104, and Leu278 were produced and showed significant improvement of experimentally measured amidase activity. Based on these results, we suggest that value of subfamily-specific positions should be further explored in order to develop a systematic tool to study structure-function relationship in enzymes and to use this information for rational enzyme engineering.
Title: Fluorophoric assay for the high-throughput determination of amidase activity Henke E, Bornscheuer UT Ref: Analytical Chemistry, 75:255, 2003 : PubMed
An assay has been developed for the high-throughput identification of amidase activity. Amines released from the enzyme-catalyzed hydrolysis of corresponding amides were detected by the formation of a fluorescent dye by coupling with 4-nitro-7-chloro-benzo-2-oxa-1,3-diazole (NBD-CI). Using this format, 22 lipases and esterases were tested for their ability to hydrolyze aromatic substituted N-acylamines in a microtiter plate format. Identified active enzymes were further characterized toward a broad range of compounds to determine the influence of substrate structure on activity. For recombinantly produced esterases, it could be shown that the assay works with high reproducibility and sensitivity, even in the presence of amino acids and proteins present in culture media and cell debris.
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