Biochemical and Functional Characterization of a Metalloprotease from the Thermophilic Fungus Thermoascus aurantiacus

Autor(es)

Merheb-Dini, Carolina ^[1] ; Cabral, Hamilton ^[2] ; Leite, Rodrigo S. R. ^[1] ; Zanphorlin, Leticia M. ^[1] ; Okamoto, Debora N. ^[1] ; Bonilla Rodriguez, Gustavo O. ^[1] ; Juliano, Luiz ^[3] ; Arantes, Eliane C. ^[2] ; Gomes, Eleni ^[1] ; da Silva, Roberto ^[1]

Afiliação do(s) autor(es)

^[1] Univ Estadual Paulista, Lab Bioquim & Microbiol Aplicada, Inst Biociencias Letras & Ciencias Exatas, BR-15054000 Sao Paulo - Brazil

^[2] Univ Sao Paulo, Fac Ciencias Farmaceut Ribeirao Preto, BR-14040903 Sao Paulo - Brazil

^[3] Univ Fed Sao Paulo, Dept Biofis, Escola Paulista Med, BR-04044020 Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Journal of Agricultural and Food Chemistry; v. 57, n. 19, p. 9210-9217, OCT 14 2009.
• Citações Web of Science: 18
• Assunto(s): Metaloproteases Fermentação Thermoascus

Resumo

Protease production was carried out in solid state fermentation. The enzyme was purified through precipitation with ethanol at 72% followed by chromatographies in columns of Sephadex G75 and Sephacryl S100. It was purified 80-fold and exhibited recovery of total activity of 0.4%. SDS-PAGE analysis indicated an estimated molecular mass of 24.5 kDa and the N-terminal sequence of the first 22 residues was APYSGYQCSMQLCLTCALMNCA. Purified protease was only inhibited by EDTA (96.7%) and stimulated by Fe(2+) revealing to be a metalloprotease activated by iron. Optimum pH was 5.5, optimum temperature was 75 degrees C, and it was thermostable at 65 degrees C for 1 h maintaining more than 70% of original activity. Through enzyme kinetic studies, protease better hydrolyzed casein than azocasein. The screening of fluorescence resonance energy transfer (FRET) peptide series derived from Abz-KLXSSKQ-EDDnp revealed that the enzyme exhibited preference for Arg in P(1) (k(cat)/K(m) = 30.1 mM(-1) s(-1)). (AU)

• Processo FAPESP: 06/06573-9 - Produção, purificação e caracterização da protease de Rhizomucor sp. e avaliação de sua aplicação na fabricação de queijo maturado
• Beneficiário: Carolina Merheb Dini
• Modalidade de apoio: Bolsas no Brasil - Doutorado