Theron Louwrens Wiid, Bely Marina, Divol Benoit
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.
J Sci Food Agric. 2017 Aug;97(11):3584-3593. doi: 10.1002/jsfa.8217. Epub 2017 Feb 14.
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.
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 Cu and Ni . 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 (K and V ) 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.
Overall, the results suggest that MpAPr1 could be applied in food biotechnology processes such as winemaking. © 2017 Society of Chemical Industry.
MpAPr1编码来自酿酒酵母梅奇酵母IWBT Y1123的一种酸性蛋白酶，此前已被分离出来，并显示出对酪蛋白和葡萄蛋白具有潜在活性。然而，其特性仍不完整。
MpAPr1被克隆到pGAPZαA载体中，并转化到巴斯德毕赤酵母X33中进行异源表达。在验证活性后，对酶的特性进行了表征。浓缩上清液中的蛋白酶活性在pH值3.0至5.0以及10℃至50℃之间保持稳定。蛋白酶活性的最佳条件是40℃和pH 4.5。除了铜和镍之外，金属离子的存在对活性影响不大。此外，在糖和乙醇存在的情况下，蛋白水解活性仍然保留。优化了巴斯德毕赤酵母中MpAPr1表达的pH和温度条件。通过阳离子交换色谱法实现了纯化，并测定了动力学参数（K和V）。证实了MpAPr1对葡萄蛋白的活性，但降解程度取决于这些蛋白的性质和环境条件。
总体而言，结果表明MpAPr1可应用于食品生物技术过程，如酿酒。©2017化学工业协会。
