Evaluation of the milk clotting properties of an aspartic peptidase secreted by Rhizopus microsporus

Author(s)

da Silva, Ronivaldo Rodrigues ; Souto, Tatiane Beltramini ; da Rosa, Nathalia Gonsales ; Goncalves de Oliveira, Lilian Caroline ; Juliano, Maria Aparecida ; Juliano, Luiz ; Rosa, Jose C. ; Cabral, Hamilton

• Document type: Journal article
• Source: PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY; v. 50, n. 3, p. 8-pg., 2019-10-26.

Abstract

Traditionally, chymosin has been used for milk-clotting, but this naturally occurring enzyme is in short supply and its use has raised religious and ethical concerns. Because milk-clotting peptidases are a promising substitute for chymosin in cheese preparation, there is a need to find and test the specificity of these enzymes. Here, we evaluated the milk-clotting properties of an aspartic peptidase secreted by Rhizopus microsporus. The molecular mass of this enzyme was estimated at 36 kDa and Pepstatin A was determined to be an inhibitor. Optimal activity occurred at a pH of 5.5 and a temperature range of 50-60 degrees C, but the peptidase was stable in the pH range of 4-7 and a temperature as low as 45 degrees C. Proteolytic activity was significantly reduced in the presence of Cu2+ and Al3+. When enzyme substrates based on FRET were used, this peptidase exhibited the highest catalytic efficiency for Abz-KNRSSKQ-EDDnp (4,644 +/- 155 mM(-1).s(-1)), Abz-KLRSSNQ-EDDnp (3,514 +/- 130 mM(-1).s(-1)), and Abz-KLRQSKQ-EDDnp (3,068 +/- 386 mM(-1).s(-1)). This study presents a promising peptidase for use in cheese making, due to its high stability in the presence of Ca2+ and broad pH range of 4-7, in addition to its ability to efficiently clot milk. (AU)

• FAPESP's process: 11/06986-0 - Determination of the specificity of peptidases isolated from fungi using fluorescence resonance energy transfer (FRET) peptides as substrates
• Grantee: Hamilton Cabral
• Support Opportunities: Regular Research Grants
• FAPESP's process: 12/24703-8 - Proteomics analyses of meso and thermophilic filamentous fungi exposed to physical and chemical factors
• Grantee: Hamilton Cabral
• Support Opportunities: Regular Research Grants