title: Characterisation of the enzymatic properties of MpAPr1, an aspartic protease secreted by the wine yeast Metschnikowia pulcherrima.
abstract: BACKGROUND
  MpAPr1, encoding an acid protease from the wine yeast Metschnikowia pulcherrima IWBT Y1123, was previously isolated and shown to display potential activity against casein and grape proteins. However, its characterisation remained partial.
  
  
  RESULTS
  MpAPr1 was cloned into the pGAPZαA vector and transformed into Komagataella pastoris X33 for heterologous expression. After verification of activity, the enzyme properties were characterised. Protease activity within the concentrated supernatant was retained over a pH range of 3.0 to 5.0 and between 10 °C and 50 °C. Optimal conditions for protease activity were found at 40 °C and pH 4.5. Activity was mostly unaffected by the presence of metal ions with the exception of Cu2+and Ni2+. Furthermore, proteolytic activity was retained in the presence of sugar and ethanol. pH and temperature conditions for MpAPr1 expression in K. pastoris were optimised. Purification was achieved by means of cation exchange chromatography and kinetic parameters (Kmand Vmax) were determined. MpAPr1 activity against grape proteins was confirmed, but the extent of the degradation was dependent on the nature of these proteins and the environmental conditions.
  
  
  CONCLUSIONS
  Overall, the results suggest that MpAPr1 could be applied in food biotechnology processes such as winemaking. © 2017 Society of Chemical Industry.
authors.affiliation: Institute for Wine Biotechnology, Stellenbosch University, Private Bag X1, Matieland, South Africa.|Université de Bordeaux, ISVV, EA 4577, Unité de Recherche Œnologie, Villenave d'Ornon, France.
authors.forename: Louwrens Wiid
authors.initials: LW
authors.lastname: Theron
authors.affiliation: Université de Bordeaux, ISVV, EA 4577, Unité de Recherche Œnologie, Villenave d'Ornon, France.
authors.forename: Marina
authors.initials: M
authors.lastname: Bely
authors.affiliation: Institute for Wine Biotechnology, Stellenbosch University, Private Bag X1, Matieland, South Africa.
authors.forename: Benoit
authors.initials: B
authors.lastname: Divol
mesh_terms: D057055:Aspartic Acid Proteases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D000431:Ethanol / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D055163:Metschnikowia / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D027843:Vitis / Q000378:metabolism / Q000382:microbiology; D014920:Wine / Q000032:analysis / Q000382:microbiology
chemical_list: D005656:Fungal Proteins; D000431:Ethanol; D057055:Aspartic Acid Proteases
keywords: Metschnikowia pulcherrima; MpAPr1; aspartic protease; enzyme characterisation; protein purification; wine