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KMID : 0380219990320030239
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Journal of Biochemistry and Molecular Biology
1999 Volume.32 No. 3 p.239 ~ p.246
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The Purification and Characterization of Bacillus subtilis Tripeptidase (PepT)
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Park Yong-Seek
Cha Myung-Hoon
Yong Whan-Mi
Kim Hyo-Joon
Chung Il-Yup
Lee Young-Seek
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Abstract
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A tripeptidase (PepT) was purified to homogeneity from Bacillus subtilis through four sequential chromatographies including DEAE-Sepharose ion exchange, hydroxylapatite, mono-Q FPLC ion exchange, and Superose-12 FPLC gel filtration. The apparent molecular mass of the enzyme was 49,200 Da and 51,400 Da as determined by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography, respectively, and the enzyme exists in a monomeric form. The physicochemical properties of the enzyme were as follows: optimum pH at 7.5, optimum temperature at 60℃, and pI at 4.9. The Km and Vmax values of the enzyme were 4.3 mM and 2.5 mmol/min/mg, respectively, with MetAla-Ser as substrate. The B. subtilis PepT requires Co2+ ion(s) for activation, while it is inactivated by EOTA and 1,10-phenanthroline, suggesting that it is a metalloprotein. The enzyme was not inhibited by any of serine protease, aspartic protease, or leucine aminopeptidase inhibitors. The enzyme showed comparable activities towards four different substrates including Met-Ala-Ser, Leu-Gly-Gly, Leu-Ser-Phe, and Leu-Leu-Tyr. The amino terminal sequence of PepT determined by Edman degradation was found to be MKEEIIERFTTYVXV and turned out to be identical to that of PepT deduced from a cloned B. subtilis pepT.
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KEYWORD
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Bacillus subtilis, Co2+ ion, Metalloenzyme, Tripeptidase (PepT)
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