title: Evaluation of the catalytic specificity, biochemical properties, and milk clotting abilities of an aspartic peptidase from Rhizomucor miehei.
abstract: In this study, we detail the specificity of an aspartic peptidase from Rhizomucor miehei and evaluate the effects of this peptidase on clotting milk using the peptide sequence of k-casein (Abz-LSFMAIQ-EDDnp) and milk powder. Molecular mass of the peptidase was estimated at 37 kDa, and optimum activity was achieved at pH 5.5 and 55 °C. The peptidase was stable at pH values ranging from 3 to 5 and temperatures of up 45 °C for 60 min. Dramatic reductions in proteolytic activity were observed with exposure to sodium dodecyl sulfate, and aluminum and copper (II) chloride. Peptidase was inhibited by pepstatin A, and mass spectrometry analysis identified four peptide fragments (TWSISYGDGSSASGILAK, ASNGGGGEYIFGGYDSTK, GSLTTVPIDNSR, and GWWGITVDRA), similar to rhizopuspepsin. The analysis of catalytic specificity showed that the coagulant activity of the peptidase was higher than the proteolytic activity and that there was a preference for aromatic, basic, and nonpolar amino acids, particularly methionine, with specific cleavage of the peptide bond between phenylalanine and methionine. Thus, this peptidase may function as an important alternative enzyme in milk clotting during the preparation of cheese.
authors.affiliation: Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista "Júlio de Mesquita Filho", São José do Rio Preto, São Paulo, Brazil.
authors.forename: Ronivaldo Rodrigues
authors.initials: RR
authors.lastname: da Silva
authors.affiliation: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café, s/nº Campus Universitário da USP CEP, Ribeirão Preto, São Paulo, 14040-903, Brazil.
authors.forename: Tatiane Beltramini
authors.initials: TB
authors.lastname: Souto
authors.affiliation: Departamento de Bioquímica e Microbiologia, Universidade Estadual Paulista "Júlio de Mesquita Filho", Rio Claro, São Paulo, Brazil.
authors.forename: Tássio Brito
authors.initials: TB
authors.lastname: de Oliveira
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: Daniel
authors.initials: D
authors.lastname: Karcher
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: Departamento de Bioquímica e Microbiologia, Universidade Estadual Paulista "Júlio de Mesquita Filho", Rio Claro, São Paulo, Brazil.
authors.forename: André
authors.initials: A
authors.lastname: Rodrigues
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, Av. do Café, s/nº Campus Universitário da USP CEP, Ribeirão Preto, São Paulo, 14040-903, Brazil. hamilton@fcfrp.usp.br.
authors.forename: Hamilton
authors.initials: H
authors.lastname: Cabral
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry; D057055:Aspartic Acid Proteases / Q000737:chemistry* / Q000378:metabolism*; D055162:Biocatalysis; D002364:Caseins / Q000737:chemistry; D002611:Cheese; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008892:Milk / Q000737:chemistry*; D020103:Rhizomucor / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002364:Caseins; D057055:Aspartic Acid Proteases; D016282:Aspartic Acid Endopeptidases; C035794:rhizopuspepsin
keywords: Aspartic protease; Biochemical characterization; Intramolecularly quenched fluorogenic substrate; Rhizomucor miehei; Specificity