The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.
Microb Cell Fact. 2023 Mar 3;22(1):42. doi: 10.1186/s12934-023-02047-9.
Adding acid protease to feed can enhance protein digestibility, boost feed utilization, and stimulate the growth of animals in breading industry. In order to obtain an acid protease with high hydrolysis efficiency to plant protein, in this study, an aspartic protease from Aspergillus niger was heterologous expressed in Pichia pastoris (P. pastoris). The enzymatic properties and application in soybean protein degradation were also studied.
In our investigation, the high aspartic protease (Apa1) activity level of 1500 U/mL was achieved in 3 L bioreactor. After dialysis and anion exchange chromatography, the total enzyme activity and specific enzyme activity were 9412 U and 4852 U/mg, respectively. The molecular weight of the purified protease was 50 kDa, while the optimal pH and temperature were 3.0 and 50 °C, respectively. It was stable at pH 2.0-5.0 and 30-60 °C. Apa1 was used to hydrolyze soybean isolate protein (SPI) at 40 °C and pH 3.0, and a high hydrolysis degree (DH) of 61.65% was achieved. In addition, the molecular weight distribution of SPI hydrolysis products was studied, the result showed that the hydrolysis products were primarily oligopeptides with molecular weights of 189 Da or below.
In this study, Apa1 was successfully expressed in P. pastoris and high expression level was obtained. In addition, the highest protein hydrolysis rate to SPI degradation so far was achieved. The acid protease in this study provides a new protease that is suitable for the feed industry, which will be very helpful to improve the feed utilization and promote the development of the breeding industry.
在饲料中添加酸性蛋白酶可以提高蛋白质的消化率，提高饲料利用率，刺激养殖行业动物的生长。为了获得一种对植物蛋白具有高水解效率的酸性蛋白酶，本研究在毕赤酵母（Pichia pastoris）中异源表达了黑曲霉来源的天冬氨酸蛋白酶（Apa1）。同时还研究了该酶的酶学性质及其在大豆蛋白降解中的应用。
在 3 L 生物反应器中，获得了高达 1500 U/mL 的高活力天冬氨酸蛋白酶（Apa1）。经过透析和阴离子交换层析后，总酶活和比酶活分别达到 9412 U 和 4852 U/mg。纯化后的蛋白酶分子量为 50 kDa，最适 pH 和温度分别为 3.0 和 50°C，在 pH 2.0-5.0 和 30-60°C 下稳定。Apa1 用于在 40°C 和 pH 3.0 下水解大豆分离蛋白（SPI），水解度（DH）达到 61.65%。此外，还研究了 SPI 水解产物的分子量分布，结果表明水解产物主要是分子量为 189 Da 或以下的寡肽。
本研究成功在毕赤酵母中表达了 Apa1，并获得了高表达水平。此外，实现了迄今为止 SPI 降解的最高蛋白质水解率。本研究中的酸性蛋白酶为饲料工业提供了一种适合的新型蛋白酶，这将非常有助于提高饲料利用率，促进养殖行业的发展。
