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.
Hemophilia A is caused by a deficiency in coagulation factor VIII. Recombinant factor VIII can be used as an alternative although it is unavailable for most patients. Here, we describe the production of a human recombinant B-domain-deleted FVIII (rBDDFVIII) by the human cell line SK-HEP-1, modified by a lentiviral vector rBDDFVIII was produced by recombinant SK-HEP cells (rSK-HEP) at 1.5-2.1 IU/10(6) in 24 h. The recombinant factor had increased in vitro stability when compared to commercial pdFVIII. The functionality of rBDDFVIII was shown by its biological activity and by tail-clip challenge in hemophilia A mice. The rSK-HEP cells grew in a scalable system and produced active rBDDFVIII, indicating that this platform production can be optimized to meet the commercial production scale needs.
Peptidases are important because they play a central role in pharmaceutical, food, environmental, and other industrial processes. A serine peptidase from Aspergillus terreus was isolated after two chromatography steps that showed a yield of 15.5%. Its molecular mass was determined to be 43 kD, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This peptidase was active between pH 5.0 to 8.0 and had maximum activity at pH 7.0, at 45°C. When exposited with 1 M of urea, the enzyme maintained 100% activity and used azocasein as substrate. The N-terminal (first 15 residues) showed 33% identity with the serine peptidase of Aspergillus clavatus ES1. The kinetics assays showed that subsite S2 did not bind polar basic amino acids (His and Arg) nonpolar acidic amino acids (Asp and Glu). The subsite S1 showed higher catalytic efficiency than the S2 and S3 subsites.
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