title: Biochemical and functional characterization of a metalloprotease from the thermophilic fungus Thermoascus aurantiacus.
abstract: 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)).
authors.affiliation: Instituto de Biociências Letras e Ciências Exatas, Universidade Estadual Paulista Júlio de Mesquita Filho, Sao Paulo, Brazil.
authors.forename: Carolina
authors.initials: C
authors.lastname: Merheb-Dini
authors.forename: Hamilton
authors.initials: H
authors.lastname: Cabral
authors.forename: Rodrigo S R
authors.initials: RS
authors.lastname: Leite
authors.forename: Letícia M
authors.initials: LM
authors.lastname: Zanphorlin
authors.forename: Débora N
authors.initials: DN
authors.lastname: Okamoto
authors.forename: Gustavo O Bonilla
authors.initials: GO
authors.lastname: Rodriguez
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.forename: Eliane C
authors.initials: EC
authors.lastname: Arantes
authors.forename: Eleni
authors.initials: E
authors.lastname: Gomes
authors.forename: Roberto
authors.initials: R
authors.lastname: da Silva
mesh_terms: D000595:Amino Acid Sequence; D002364:Caseins / Q000378:metabolism; D004795:Enzyme Stability; D031541:Fluorescence Resonance Energy Transfer; D006358:Hot Temperature; D007700:Kinetics; D045726:Metalloproteases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D055327:Thermoascus / Q000201:enzymology*
chemical_list: D002364:Caseins; D045726:Metalloproteases