Purification and characterization of a novel extracellular tripeptidyl peptidase from Rhizopus oligosporus |
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Authors: | Lin Jia-Shin Lee Shuo-Kang Chen Yeh Lin Wei-De Kao Chao-Hung |
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Affiliation: | Department of Biotechnology, Hungkuang University, 34 Chung-Chie Road, Taichung 43302, Taiwan. |
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Abstract: | A novel extracellular tripeptidyl peptidase (TPP) was homogenously purified from the culture supernatant of Rhizopus oligosporus by sequential fast protein liquid chromatography. The purified enzyme was a 136.5 kDa dimer composed of identical subunits. The effects of inhibitors and metal ions indicated that TPP is a metallo- and serine protease. TPP was activated by divalent cations, such as Co(2+) and Mn(2+), and completely inhibited by Cu(2+). Enzyme activity was optimal at pH 7.0 and 45 °C with a specific activity of 281.9 units/mg for the substrate Ala-Ala-Phe-pNA. The purified enzyme catalyzed cleavage of various synthetic tripeptides but not when proline occupied the P1 position. Purified TPP cleaved the pentapeptide Ala-Ala-Phe-Tyr-Tyr and tripeptide Ala-Ala-Phe, confirming the TPP activity of the enzyme. |
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