Quorum sensing， the bacterial cell-cell communication by small molecules， controls important processes such as infection and biofilm formation. Therefore， it is a promising target with several therapeutic and technical applications besides its significant ecological relevance. Enzymes inactivating N-acyl-l-homoserine lactones， the most common class of communication molecules among Gram-negative proteobacteria， mainly belong to the groups of quorum-quenching lactonases or quorum-quenching acylases. However， identification， characterization， and optimization of these valuable biocatalysts are based on a very limited number of fundamentally different methods with their respective strengths and weak... More
Quorum sensing， the bacterial cell-cell communication by small molecules， controls important processes such as infection and biofilm formation. Therefore， it is a promising target with several therapeutic and technical applications besides its significant ecological relevance. Enzymes inactivating N-acyl-l-homoserine lactones， the most common class of communication molecules among Gram-negative proteobacteria， mainly belong to the groups of quorum-quenching lactonases or quorum-quenching acylases. However， identification， characterization， and optimization of these valuable biocatalysts are based on a very limited number of fundamentally different methods with their respective strengths and weaknesses. Here， a (bio)chemical activity assay is described， which perfectly complements the other methods in this field. It enables continuous and high-throughput activity measurements of purified and unpurified quorum-quenching enzymes within several minutes. For this， the reaction products released by quorum-quenching lactonases and quorum-quenching acylases are converted either by a secondary enzyme or by autohydrolysis to l-homoserine. In turn， l-homoserine is detected by the previously described calcein assay， which is sensitive to α-amino acids with free N and C termini. Besides its establishment， the method was applied to the characterization of three previously undescribed quorum-quenching lactonases and variants thereof and to the identification of quorum-quenching acylase-expressing Escherichia coli clones in an artificial library. Furthermore， this study indicates that porcine aminoacylase 1 is not active toward N-acyl-l-homoserine lactones as published previously but instead converts the autohydrolysis product N-acyl-l-homoserine.
