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

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• 作者：Carolina Merheb-Dini ; Hamilton Cabral ; Rodrigo S. R. Leite ; Letcia M. Zanphorlin ; Dbora N. Okamoto ; Gustavo O. Bonilla Rodriguez ; Luiz Juliano ; Eliane C. Arantes ; Eleni Gomes ; Roberto da Silva
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2009
• 出版时间：October 14, 2009
• 年：2009
• 卷：57
• 期：19
• 页码：9210-9217
• 全文大小：848K
• 年卷期：v.57,no.19(October 14, 2009)
• ISSN：1520-5118

文摘

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 Fe²⁺ revealing to be a metalloprotease activated by iron. Optimum pH was 5.5, optimum temperature was 75 °C, and it was thermostable at 65 °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 P₁ (k_cat/K_m = 30.1 mM⁻¹ s⁻¹).