Open AccessThe ywad gene from Bacillus subtilis encodes a double‐zinc aminopeptidase
Author(s) -
FundoianoHershcovitz Yifat,
Rabinovitch Larisa,
Shulami Smadar,
Reiland Vera,
Shoham Gil,
Shoham Yuval
Publication year - 2005
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1016/j.femsle.2004.12.001
Subject(s) - bacillus subtilis , aminopeptidase , serine protease , enzyme , escherichia coli , zinc , biochemistry , protease , biology , enzyme kinetics , serine , gene , bacillales , chemistry , microbiology and biotechnology , amino acid , leucine , bacteria , active site , genetics , organic chemistry
The yet uncharacterized ywad gene from Bacillus subtilis has been cloned and overexpressed in Escherichia coli . The gene product (BSAP) was purified and shown to be an aminopeptidase. The activity of BSAP was optimal at pH 8.4, the enzyme was stable for 20 min at 80 °C and its activity was not affected by serine protease and aspartic protease inhibitors, but was completely diminished by the Zn‐chelator 1,10‐phenanthroline. ZnCl 2 was able to restore activity, and the binding stochiometry of zinc to apo‐BSAP indicated two Zn ions per protein molecule. BSAP exhibited high preference toward p ‐nitroanilide derived Arg, Lys, and Leu synthetic substrates resulting in k cat / K m values of 1–5 × 10 1 s −1 mM −1 .