title: Biochemical and milk-clotting properties and mapping of catalytic subsites of an extracellular aspartic peptidase from basidiomycete fungus Phanerochaete chrysosporium.
abstract: For a long time, proteolytic enzymes have been employed as key tools of industrial processes, especially in the dairy industry. In the present work, we used Phanerochaete chrysosporium for biochemical characterization and analysis of catalytic specificity of an aspartic peptidase. Our results revealed an aspartic peptidase with molecular mass ∼38kDa, maximal activity at pH 4.5 and 50°C, and stability above 80% in the pH range of 3-8 and temperature up to 55°C for 1h. In a milk-clotting assay, this peptidase showed maximal milk clotting activity at 60-65°C and maintenance of enzymatic activity above 80% in the presence of 20mM CaCl2. In a specificity assay, we observed stronger restriction of catalysis at the S1subsite, with a preference for lysine, arginine, leucine, tyrosine, and phenylalanine residues. The restricted proteolysis and milk-clotting potential are attractive properties for the use in cheese production.
authors.affiliation: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors.forename: Ronivaldo Rodrigues
authors.initials: RR
authors.lastname: da Silva
authors.affiliation: Universidade Federal de São Paulo, UNIFESP, São Paulo, Brazil.
authors.forename: Lilian Caroline Gonçalves
authors.initials: LC
authors.lastname: de Oliveira
authors.affiliation: Universidade Federal de São Paulo, UNIFESP, São Paulo, Brazil.
authors.forename: Maria Aparecida
authors.initials: MA
authors.lastname: Juliano
authors.affiliation: Universidade Federal de São Paulo, UNIFESP, São Paulo, Brazil.
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.affiliation: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors.forename: Arthur H C
authors.initials: AH
authors.lastname: de Oliveira
authors.affiliation: Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors.forename: Jose C
authors.initials: JC
authors.lastname: Rosa
authors.affiliation: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil. Electronic address: hamilton@fcfrp.usp.br.
authors.forename: Hamilton
authors.initials: H
authors.lastname: Cabral
mesh_terms: D000818:Animals; D057055:Aspartic Acid Proteases / Q000378:metabolism*; D002611:Cheese / Q000382:microbiology*; D005525:Food-Processing Industry; D005656:Fungal Proteins / Q000378:metabolism; D008892:Milk / Q000382:microbiology*; D020075:Phanerochaete / Q000201:enzymology*; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D057055:Aspartic Acid Proteases
keywords: Aspartic protease; FRET; Fungal enzyme; Milk-clotting; Phanerochaete chrysosporium