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

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

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Amer Chemical Soc


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)).



Thermoascus aurantiacus, Solid state fermentation (SSF), metalloprotease, Thermostability, fluorescent peptides, N-terminal sequence

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Journal of Agricultural and Food Chemistry. Washington: Amer Chemical Soc, v. 57, n. 19, p. 9210-9217, 2009.