Monitoring the impact of an aspartic protease (MpAPr1) on grape proteins and wine properties.

The perception of haze in wine is brought about when pathogenesis-related proteins become unstable and aggregate, subsequently resulting in crosslinking until it develops into light-dispersing particles. Elimination of these proteins is usually achieved via bentonite fining, which, although effective, suffers from several drawbacks. The utilization of proteases has been proposed as an ideal alternative. In a previous study, an aspartic protease (MpAPr1) from the yeast Metschnikowia pulcherrima was purified and shown to be partially active against grape proteins in synthetic medium. In this study, the effects of pure MpAPr1 supplemented to Sauvignon Blanc juice on subsequent fermentation were investigated. The juice was incubated for 48 h and thereafter inoculated with Saccharomyces cerevisiae. Results revealed that the enzyme had no observable effects on fermentation performance and retained activity throughout. Protein degradation could be detected and resulted in a significant modification of the wine composition and an increase in the presence of certain volatile compounds, especially those linked to amino acid metabolism.

Characterisation of the enzymatic properties of MpAPr1, an aspartic protease secreted by the wine yeast Metschnikowia pulcherrima.

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 (Km and 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. CONCLUSION: Overall, the results suggest that MpAPr1 could be applied in food biotechnology processes such as winemaking. © 2017 Society of Chemical Industry.