PEPTIDASES SECRETADAS POR FUNGOS TERMOFILICOS: uma revisão sistemática DOI: http://dx.doi.org/10.5892/ruvrd.v13i1.1878

Peptidases são enzimas que atuam sobre as ligações peptídicas de proteínas e peptídeos, liberando peptídeos e/ ou aminoácidos. São comercializadas em todo o mundo e abrange cerca de 60% do comércio de enzimas, devido sua vasta aplicação em diferentes setores industriais.  As características desejáveis para uma peptidase aplicável a indústria são: termoestabilidade, estabilidade a variação de pH, solventes orgânicos entre outros. A busca por enzimas termoestáveis contribui para o aumento de estudos envolvendo fungos termofilos, pois estes micro-organismos possuem a termofilia como característica fisiológica, são de fácil cultivo, se comparado a bactérias, e secretam enzimas. O processo de obtenção não requer equipamentos refinados para a obtenção enzimática tornando a manufatura do produto mais barata. Esta revisão realizou o levantamento de estudos com fungos termófilos e peptidases nos últimos dez anos dez anos, a fim de relatar a importância desses fungos e as peptidases por eles produzidas bem como seu potencial de aplicação

Peptidases secretadas por fungos termofilicos: uma revisão sistemática

Peptidases são enzimas que atuam sobre as ligações peptídicas de proteínas e peptídeos, liberando peptídeos e/ ou aminoácidos. São comercializadas em todo o mundo e abrange cerca de 60% do comércio de enzimas, devido sua vasta aplicação em diferentes setores industriais.  As características desejáveis para uma peptidase aplicável a indústria são: termoestabilidade, estabilidade a variação de pH, solventes orgânicos entre outros. A busca por enzimas termoestáveis contribui para o aumento de estudos envolvendo fungos termofilos, pois estes micro-organismos possuem a termofilia como característica fisiológica, são de fácil cultivo, se comparado a bactérias, e secretam enzimas. O processo de obtenção não requer equipamentos refinados para a obtenção enzimática tornando a manufatura do produto mais barata. Esta revisão realizou o levantamento de estudos com fungos termófilos e peptidases nos últimos dez anos dez anos, a fim de relatar a importância desses fungos e as peptidases por eles produzidas bem como seu potencial de aplicação

Peptidase with Keratinolytic Activity Secreted by Aspergillus terreus During Solid-State Fermentation

The aim of this study was to evaluate peptidase production by Aspergillus terreus in solid-state bioprocess and evaluate its parameters. The best conditions were 5.0 g of wheat bran as substrate, incubation temperature 30°C, inoculum 2.0x105spores/g and 75% saline volume, with production reaching 677 U/mL (5400 U/g culture medium) after 72 h of fermentation. Biochemical characterization of the crude enzymatic extract showed the optimum pH and temperature of 6.5 and 55°C, respectively. The stability at different temperatures and pH values showed that the extract could endure different pH. The evaluation of the ions influence and inhibitors proved that the enzyme required an ion for better activity, which was corroborated with the inhibition of EDTA and PMSF, characterizing serine and/or metallo peptidase. The extract was also tested for specific activities and showed promising results for keratinolytic and collagenolytic activities (0.252 and 0.165 OD/mL, respectively)

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

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

SK-HEP cells and lentiviral vector for production of human recombinant factor VIII

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.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP

Bis(trisyl)oxadiborirane

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 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 S1. Subsites S2, S3, 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 S2, S3, 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. © 2012 Springer Science+Business Media New York