摘要
采用乙醇分级沉淀、Superdex200凝胶过滤、Mono Q离子交换对Enterobactria spR-SYB082产酒用酸性脲酶进行了纯化。SDS-PAGE电泳结果表明,该酶已达到电泳纯,纯化倍数为21.1,酶活回收42%。Superdex200凝胶过滤测定其全酶相对分子质量约为430 000,SDS-PAGE电泳测定其亚基相对分子质量约为72 000,表明该酶为同源六聚体,并利用Edman降解法测定了蛋白质N端序列的8个氨基酸残基序列:S-F-K-M-D-R-K-Q。酶反应的最适pH为4.5,最适温度为35℃。以尿素为底物时,该酶表观Km及Vmax分别为19.5μmol/L和0.87μmol/min。Na+、Mn2+对酶活有一定的激活作用,Ca2+、Zn2+对酶活有一定的抑制作用,酒石酸、琥珀酸对酶活有一定的激活作用,草酸、乙酸对酶活有一定的抑制作用。
An acid urease from Enterobactria sp. R-SYB082 was purified to electrophoretic homogeneity by three successive steps including ethanol precipitation, superdex 200 and Mono Q, with a yield and purification-fold of 42% and 21.1, respectivily. The molecular weight of the enzyme was estimated to be 430 000 by gel filtration and 72 000 by SDS-PAGE , suggesting that it＇s a hexamer comprising of six identical subunits. Automated Edman degradation showed that the N-terminal sequence of acid urease was S-F-K-M-D-R-K-Q. The purified enzyme was optimally active at 35℃ and at pH 4. 5. The apparent Km and Vmax values for hydrolyze of urea were 19.5μmol/L and 0.87 μmol/min, respectivily. The activity can be activated by Na^＋, Mn^2＋ , tartaric acid and succinic acid but inhibited by Ca^2＋ , Zn^2＋ , oxalic acid and acetic acid.
出处
《食品与生物技术学报》
CAS
CSCD
北大核心
2008年第6期93-97,共5页
Journal of Food Science and Biotechnology
基金
国家"十一五"科技支撑计划项目(2007BAK36B02)
关键词
酸性脲酶
尿素
肠杆菌属细菌
纯化
N端序列
acid urease
urea
Enterobactria sp.
purification
N-terminal sequence
