摘要
从全国土壤腐蚀网站长辛店分离纯化到一株氢化酶活性较高的菌株 D-2,经鉴定为普通脱硫弧菌(Desulfovibrio oulgaris)。该菌氢化酶主要位于菌体的周质空间。用 pH9.0的0.1mmol/L Tris-EDTA 缓冲液将酶洗脱,经硫酸铵沉淀、DEAE-纤维素和 Sephadex G-150柱层析纯化,酶活力达到205μmolH_2·min^(-1)·mg 蛋白^(-1),得率为17.6%,纯化33.1倍。经 SDS-PAGE 和梯度-PAGE 测定,该酶为一条肽链,分子量49000道尔顿。该酶吸收光谱具有铁硫蛋白特征,ε_(400nm)和ε_(220nm)分别为34.8mM^(-1)·Cm^(-1)和120mM^(-1)cm^(-1),每个酶分子含有12个铁原子和11个硫原子。N-溴代丁二酰胺完全抑制酶活力,Hg^(2+)的抑制率也达54%,似该酶对巯基封闭性试剂具有抗性。
A strain of Desulfovibrio vulgaris D-2possessed the higher hydrogenase activity wasselected as the test strain from six strainsisolated in the national corrosion testing nets.The hydrogenase of D.vulgaris D-2 wasmainly located in the periplasmic space.Through(NH_4)_2SO_4 precipitation,DEAE-cellulose ion-exchange column chromatogra-phy,Sephadex G-150 molecular exclusionchromatography,the enzyme has been purifiedto homogeneity,with an overall 33.1-fold pu-rification,17.6% yeild.The purified enzymeshowed a specific activity of 205μ mol H_2·min^(-1)·mg protein^(-1).The enzyme has onlyone subunit and a relative M_r of 49000 asdetermined by SDS-PAGE and Gradient-PAGE.The pl value was 4.6.The absorp-tion spectrum of the enzyme was characteristicof an iron-sulfur protein.The molecularcoefficiency at 400 nm and 280 nm were 34.8mM^(-1)·cm^(-1) and 120mM^(-1)·cm^(-1),respectively.Moreover,the enzyme contained 12 atom Feand 11 atom S and was less inhibited by thio-blocking reagents.
出处
《微生物学报》
CAS
CSCD
北大核心
1990年第4期267-272,共6页
Acta Microbiologica Sinica
基金
国家自然科学基金
关键词
脱硫弧菌
氢化酶
Sulfate Reducing Bacteria
Hydrogenase
