3 research outputs found

    Amino acid supplementation improves the production of extracellular peptidases by aspergillus section flavi and their ionic immobilization

    No full text
    Bioprocess studies have been highlighted due to the importance of physiological processes and industrial applications of enzymes. The potential of peptidase production from Aspergillus section Flavi using different amino acids as a supplemental nitrogen source was investigated. A production profile revealed that amino acids had positive effects on peptidase production when compared to the control without amino acids. Optimal production (100 U/mL) was obtained with Arginine amino acid in 96 h of fermentation. Extracellular peptidase from Aspergillus section Flavi was identified in submerged bioprocesses by in situ activity. Biochemical studies revealed that the maximum activities of the enzyme extract were obtained at pH 6.5 and a temperature of 55°C. The inhibition by EDTA and PMSF suggests the presence of more than one peptidase while the Ni2+ and Cu2+ had a negative influence on the enzyme activity. When the crude extract was reversibly immobilized on ionic supports, DEAE-Agarose and MANAE-Agarose the derivative showed different profiles of thermal and pH stabilities. Hence, this study revealed the basic properties and biochemical characteristics that allowed the production improvement of this class of enzyme. Moreover, with known properties stabilization and immobilization process is required to further explore its biotechnological capacities.The authors would like to thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the financial support under Grant number 2011/06986-0; and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.Peer reviewe

    Purification and enzymatic characterization of a novel metalloprotease from Lachesis muta rhombeata snake venom

    No full text
    Abstract Background Lachesis muta rhombeata (Lmr) is the largest venomous snake in Latin America and its venom contains mainly enzymatic components, such as serine and metalloproteases, L-amino acid oxidase and phospholipases A2. Metalloproteases comprise a large group of zinc-dependent proteases that cleave basement membrane components such as fibronectin, laminin and collagen type IV. These enzymes are responsible for local and systemic changes, including haemorrhage, myonecrosis and inflammation. This study aimed the isolation and enzymatic characterization of the first metalloprotease (Lmr-MP) from Lmr venom (LmrV). Methods and results Lmr-MP was purified through two chromatographic steps and submitted to enzymatic characterization. It showed proteolytic activity on azocasein with maximum activity at pH 7.0–9.0. It was inhibited by EDTA (a metal chelator that removes zinc, which is essential for enzymatic activity) and no effect was observed with PMSF, iodoacetic acid or pepstatin (inhibitors of serine, cysteine and aspartyl proteases, respectively). Ca2+, Mg2+ and Ba2+ ions increased its activity, while Al3+, Cu2+, Ni2+ and Zn2+ inhibited it. Additionally, ZnCl2 showed a dose dependent inhibition of the enzyme. Lmr-MP activity was also evaluated upon chromogenic substrates for plasma kallikrein (S-2302), plasmin and streptokinase-activated plasminogen (S-2251) and Factor Xa (S-2222) showing the highest activity on S-2302. The activity in different solutions (5 mM or 50 mM ammonium bicarbonate, pH 7.8; 0.1% trifluoroacetic acid + 50% acetonitrile; phosphate buffer saline, pH 7.4; 50 mM sodium acetate, pH 4.0 or ammonium acetate pH 4.5) was also evaluated and the results showed that its activity was abolished at acidic pHs. Its molecular mass (22,858 Da) was determined by MALDI-TOF and about 90% of its primary structure was verified by high-resolution mass spectrometry using HCD and ETD fragmentations and database search against the sequence of closely related species. It is a novel enzyme which shared high identity with other snake venom metalloproteases (svMPs) belonging to the P-I group. Conclusion The purification procedure achieved a novel pure highly active metalloprotease from LmrV. This new molecule can help to understand the metalloproteases mechanisms of action, the Lachesis envenoming, as well as to open new perspectives for its use as therapeutic tools
    corecore