可降解大豆过敏原蛋白酶的纯化与酶学性质

将前期筛选获得的芽孢杆菌Bacillus sp. BBE201108发酵培养后，以其发酵上清液作为蛋白酶的粗酶液，经硫酸铵分级沉淀、透析除盐、Q FF 阴离子交换层析和Superdex 75凝胶过滤层析，蛋白酶的比酶活达到2 575.45 U/mg，纯化倍数为10.55，回收率为6.48%，经SDS-PAGE电泳显示为单一条带，该蛋白酶的表观相对分子质量约为46 000。该酶与大豆分离蛋白在pH 8.0，50 ℃下反应30 min，可有效降解β-伴大豆球蛋白的α亚基和α'亚基。在pH 7.0~9.0以及30~40 ℃下的稳定较好；Ca2+存在的情况下，酶活提高了5%左右，而Mn2+、Fe2+和Cu2+在不同程度上对该蛋白酶有抑制作用；苯甲基磺酰氟（PMSF）对该蛋白酶的抑制作用较为显著。酶学性质分析表明，该蛋白酶的Km为4.19 g/L，Vmax为4.04 g/（L·s），Kcat为107.73 s-1，Kcat/Km为25.71 L/（g·s）。
The protease isolated from Bacillus sp. BBE201108 was purified using ammonium sulfate fractional precipitation, dialysis, Q FF anion exchange chromatography and Superdex 75 gel filtration chromatograph after fermentation. A purified protease with a recovery of 6.48% had an overall purification of 10.55 fold and a specific enzyme activity of 2 575.45 U/mg. The molecular weight was approximately 46 000 as determined by SDS-PAGE. The α-subunit and α'-subunit of β-conglycinin could be effectively degraded after reaction with the soybean protein isolate (SPI) at pH 8.0 under 50 ℃ for 30 min. The protease was relatively stable over the pH range of 7.0-9.0 at 30~40 ℃. The protease activity enhanced by 5%in the presence of Ca2+, and inhibited by Mn2+, Fe2+ and Cu2+. A significant negative effect of phenylmethanesulfonyl fluoride (PMSF) was observed on the protease activity. The values of Km, Vmax, Kcat and Kcat /Km for the protease were 4.19 g/L, 4.04 g/ (L·s), 107.73 s-1 and 25.71 L/(g·s), respectively