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 A degrees C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl2, KCl, and BaCl, and partially inhibited by CuCl2, CoCl2 and totally inhibited by AlCl3 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.