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


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.


CONCLUSIONS
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.
Jones
B V
BV
Alimentary Pharmabiotic Centre, Department of Microbiology, University College Cork, Cork, Ireland.
Sun
F
F
Marchesi
J R
JR
D000362:Agar / Q000378:metabolism; D000818:Animals; D001419:Bacteria / Q000201:enzymology* / Q000378:metabolism; D005243:Feces / Q000382:microbiology*; D015698:Genomic Library; D006801:Humans; D008892:Milk / Q000378:metabolism; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*
D000362:Agar; D010447:Peptide Hydrolases