Purification, Characterization, and Specificity Determination of a New Serine Protease Secreted by Penicillium waksmanii

The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 °C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl 2 , KCl, and BaCl, and partially inhibited by CuCl 2 , CoCl 2 and totally inhibited by AlCl 3 and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k cat / K m of 10,666 mM −1 s −1 , followed by the peptide Abz-GLRSSKQ-EDDnp with a k cat / K m of 7,500 mM −1 s −1 . Basic and acidic side chain-containing amino acids performed best at subsite S 1 . Subsites S 2 , S 3 , S ′ 2 , and S ′ 1 , S ′ 3 showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain, respectively. High values of k cat / K m were observed for the subsites S 2 , S 3 , and S ′ 2. The sequence of the N-terminus (ANVVQSNVPSWGLARLSSKKTGTTDYTYD) showed high similarity to the fungi Penicillium citrinum and Penicillium chrysogenum , with 89 % of identity at the amino acid level.