In vitro Ca(2+)-dependent maturation of milk-clotting recombinant Epr: minor extracellular protease: from Bacillus licheniformis.
The minor extracellular protease (Epr) is secreted into the culture medium during Bacillus licheniformis, strain USC13, stationary phase of growth. Whereas, B. subtilis Epr has been reported to be involved in swarming; the B. licheniformis protease is also involved in milk-clotting as shown by the curd forming ability of culture broths expressing this protein. The objectives of this study are the characterization of recombinant B. licheniformis Epr (minor extracellular protease) and the determination of its calcium-dependent activation process. In this work, we have cloned and expressed B. licheniformis Epr in Escherichia coli. We were also able to construct a tridimensional model for Epr based on its homology to Thermococcus kodakarensis pro-tk-subtilisin 2e1p, fervidolysin from Fervidobacterium pennivorans 1rv6, and B. lentus 1GCI subtilisin. Recombinant Epr was accumulated into inclusion bodies; after protein renaturation, Epr undergoes an in vitro calcium-dependent activation, similar to that described for tk protease. The recombinant Epr is capable of producing milk curds with the same clotting activity previously described for the native B. licheniformis Epr enzyme although further rheological and industrial studies should be carried out to confirm its real applicability. This work represents for the first time that Epr may be successfully expressed in a non-bacilli microorganism.
Department of Microbiology and Parasitology, University of Santiago de Compostela, Santiago de Compostela, Spain.
José Manuel
JM
Ageitos
Juan Andrés
JA
Vallejo
Manuel
M
Serrat
Angeles
A
Sánchez-Pérez
Tomás G
TG
Villa
D000818:Animals; D001407:Bacillus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism; D008892:Milk / Q000382:microbiology*; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019712:Thermococcus / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010447:Peptide Hydrolases; D002118:Calcium