可降解大豆过敏原蛋白酶的纯化与酶学性质  

Purification and Enzyme Characterization of Protease with Anti-Allergic Activity of Soy Sauce

作　　者：付欧 张娟[1,3] 堵国成[4,3] 陈坚[5,3] 

机构地区：[1]江南大学工业生物技术教育部重点实验室,江苏无锡214122 [2]江南大学食品安全与营养协同创新中心,江苏无锡214122 [3]江南大学生物工程学院,江苏无锡214122 [4]江南大学糖化学与生物技术教育部重点实验室,江苏无锡214122 [5]江南大学粮食发酵工艺与技术国家工程实验室,江苏无锡214122

出　　处：《食品与生物技术学报》2016年第4期350-356,共7页Journal of Food Science and Biotechnology

基　　金：国家"十二五"科技支撑计划项目(2011BAK10B03);国家自然科学基金项目(31470160);教育部长江学者和创新团队发展计划项目(IRT1135)

摘　　要：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p H8.0,50℃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p H 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 sulthte 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 a-subunit and a ＇-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 , Kcut and Kcut /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.

关 键 词：大豆过敏原 芽孢杆菌 蛋白酶 分离纯化 酶学性质 

分 类 号：TS201.25[轻工技术与工程—食品科学]