Hochschule Offenburg, Fakultät Maschinenbau und Verfahrenstechnik, Offenburg, Germany.
Mass Spectrometry Unit, Universität Hohenheim, Stuttgart, Germany.
J Sci Food Agric. 2022 Sep;102(12):5190-5199. doi: 10.1002/jsfa.11871. Epub 2022 Apr 20.
Various neutral and alkaline peptidases are commercially available for use in protein hydrolysis under neutral to alkaline conditions. However, the hydrolysis of proteins under acidic conditions by applying fungal aspartic peptidases (FAPs) has not been investigated in depth so far. The aim of this study, thus, was to purify a FAP from the commercial enzyme preparation, ROHALASE® BXL, determine its biochemical characteristics, and investigate its application for the hydrolysis of food and animal feed proteins under acidic conditions.
A Trichoderma reesei derived FAP, with an apparent molecular mass of 45.8 kDa (sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SDS-PAGE) was purified 13.8-fold with a yield of 37% from ROHALASE® BXL. The FAP was identified as an aspartate protease (UniProt ID: G0R8T0) by inhibition and nano-LC-ESI-MS/MS studies. The FAP showed the highest activity at 50°C and pH 4.0. Monovalent cations, organic solvents, and reducing agents were tolerated well by the FAP. The FAP underwent an apparent competitive product inhibition by soy protein hydrolysate and whey protein hydrolysate with apparent K -values of 1.75 and 30.2 mg*mL , respectively. The FAP showed promising results in food (soy protein isolate and whey protein isolate) and animal feed protein hydrolyses. For the latter, an increase in the soluble protein content of 109% was noted after 30 min.
Our results demonstrate the applicability of fungal aspartic endopeptidases in the food and animal feed industry. Efficient protein hydrolysis of industrially relevant substrates such as acidic whey or animal feed proteins could be conducted by applying fungal aspartic peptidases. © 2022 Society of Chemical Industry.
各种中性和碱性肽酶可商购用于在中性至碱性条件下进行蛋白质水解。然而，迄今为止，尚未深入研究应用真菌天冬氨酸肽酶（FAP）在酸性条件下水解蛋白质。因此，本研究的目的是从商业酶制剂 ROHALASE® BXL 中纯化一种 FAP，确定其生化特性，并研究其在酸性条件下用于水解食品和动物饲料蛋白质的应用。
从 ROHALASE® BXL 中纯化得到一种源自里氏木霉的 FAP，其表观分子量为 45.8 kDa（十二烷基硫酸钠-聚丙烯酰胺凝胶电泳；SDS-PAGE），得率为 37%，纯化倍数为 13.8 倍。通过抑制和纳米 LC-ESI-MS/MS 研究，该 FAP 被鉴定为天冬氨酸蛋白酶（UniProt ID：G0R8T0）。FAP 在 50°C 和 pH 4.0 时表现出最高的活性。单价阳离子、有机溶剂和还原剂对 FAP 耐受良好。FAP 对大豆蛋白水解物和乳清蛋白水解物表现出明显的竞争性产物抑制，表观 K 值分别为 1.75 和 30.2 mg*mL-1。FAP 在食品（大豆蛋白分离物和乳清蛋白分离物）和动物饲料蛋白水解中表现出良好的效果。对于后者，在 30 分钟后，可观察到可溶性蛋白质含量增加了 109%。
我们的结果表明真菌天冬氨酸内肽酶在食品和动物饲料工业中的适用性。通过应用真菌天冬氨酸肽酶，可以有效地水解工业相关的底物，如酸性乳清或动物饲料蛋白质。© 2022 化学工业协会。
