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Evaluation of the catalytic specificity, biochemical properties, and milk clotting abilities of an aspartic peptidase from Rhizomucor miehei

  • Biocatalysis
  • Published:
Journal of Industrial Microbiology & Biotechnology

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.

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Acknowledgments

The authors would like to acknowledge the financial support provided by Fundação de Amparo à Pesquisa do Estado de São Paulo-FAPESP (2011/06986-0 and 2012/24703-8) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (308078/2012-8).

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Authors and Affiliations

  1. Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista “Júlio de Mesquita Filho”, São José do Rio Preto, São Paulo, Brazil

    Ronivaldo Rodrigues da Silva

  2. 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

    Tatiane Beltramini Souto & Hamilton Cabral

  3. Departamento de Bioquímica e Microbiologia, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Rio Claro, São Paulo, Brazil

    Tássio Brito de Oliveira & André Rodrigues

  4. Universidade Federal de São Paulo, UNIFESP, São Paulo, Brazil

    Lilian Caroline Gonçalves de Oliveira, Daniel Karcher, Maria Aparecida Juliano & Luiz Juliano

  5. Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil

    Arthur H. C. de Oliveira

  6. Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil

    Jose C. Rosa

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  1. Ronivaldo Rodrigues da Silva
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  2. Tatiane Beltramini Souto
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  3. Tássio Brito de Oliveira
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  6. Maria Aparecida Juliano
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  7. Luiz Juliano
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  8. Arthur H. C. de Oliveira
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  9. André Rodrigues
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  10. Jose C. Rosa
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  11. Hamilton Cabral
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Corresponding author

Correspondence to Hamilton Cabral.

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da Silva, R.R., Souto, T.B., de Oliveira, T.B. et al. Evaluation of the catalytic specificity, biochemical properties, and milk clotting abilities of an aspartic peptidase from Rhizomucor miehei . J Ind Microbiol Biotechnol 43, 1059–1069 (2016). https://doi.org/10.1007/s10295-016-1780-4

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  • DOI: https://doi.org/10.1007/s10295-016-1780-4

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