Using skimmed milk agar to functionally screen a gut metagenomic library for proteases may lead to false positives

Abstract

Aims: Functional screens using skimmed‐milk agar to obtain protease activity is a common approach. The aim of this study was to determine the efficacy of this screen to obtain protease activity from a metagenomic library. Methods and Results: A distal gut metagenomic library was functionally screened using a skimmed‐milk agar. The functional screen provided 231 clones generating the characteristic clear halo indicative of protease production. Clone analysis revealed that they were not protease‐positive, but expressed glycosidic hydrolases and produced acid, which was responsible for the clear halos. Conclusions: The current skimmed‐milk agar method to obtain proteases is not sufficiently robust to provide a definitive screen. Other‐ non‐protease activities will also give the same clear halo and these would be interpreted as protease positive clones without further analysis. Hence a more robust buffered medium or a specific protein should be used. Significance and Impact of the Study: Functional screens are a powerful approach to obtaining enzymes from large metagenomic libraries and proteases are a particularly interesting target. The skimmed‐milk agar is not sufficiently robust to ensure that only proteases are isolated and in order to save time and money this study has shown that better designed media can aid in the process.

Queijos artesanais: fonte de bactérias ácido láticas selvagens para formulação de fermentos tradicionais

“…Para a determinação da atividade proteolítica extracelular qualitativa, as bactérias foram semeadas em meio sólido composto por 1% (v/v) de leite desnatado e 1,5% de ágar, como descrito por Jones et al, 13 sendo as temperaturas de incubação modificadas, 30ºC ou 37ºC de acordo com a temperatura de isolamento de cada bactéria, sendo observada em intervalos de 24h durante três dias. A atividade proteolítica foi indicada como uma zona clara ao redor das colônias.…”

Isolation and Characterization of Metalloproteases with a Novel Domain Structure by Construction and Screening of Metagenomic Libraries

“…In addition, a serine protease has been mentioned in reviews, but the original work was not published (13,29). In other cases, screening of metagenomic libraries for proteolytic activity was unsuccessful (19,44). Jones et al (19) found glycoside hydrolases instead of proteases during activitybased screening of the human gut microbiome on skim milkcontaining LB medium.…”

“…In other cases, screening of metagenomic libraries for proteolytic activity was unsuccessful (19,44). Jones et al (19) found glycoside hydrolases instead of proteases during activitybased screening of the human gut microbiome on skim milkcontaining LB medium. These authors concluded that this screen is not entirely suitable for detection of proteolytic enzymes.…”

“…In addition, one serine protease has been cited in reviews (13,29). In other metagenome projects, activity-based screening was unsuccessful (19,44), or the thereby recovered proteolytic clones were not characterized (47).…”

“…In addition, one serine protease has been cited in reviews (13,29). In other metagenome projects, activity-based screening was unsuccessful (19,44), or the thereby recovered proteolytic clones were not characterized (47).We report here on the identification and characterization of two metagenome-derived metalloproteases. We constructed metagenomic DNA libraries from environmental samples by a T4 DNA ligase-based and a topoisomerase-based cloning method.…”