摘要
为了给胆盐水解酶结构分析及降低血液胆固醇的机理研究提供理论基础,采用硫酸铵分级沉淀、透析、阴离子交换树脂层析等方法对分离自我国传统开菲尔粒中的1株干酪乳杆菌KTx(lactobillus casei KTx)产生的胆盐水解酶进行了纯化,并对其部分特性进行了研究。结果表明:在70%的硫酸铵条件下酶蛋白能最大程度地沉淀。采用DEAE-Sepharose Fast Flow阴离子交换树脂层析,流速1.0mL/min,用0.1mol/L NaCl-50mmol/L磷酸缓冲液(pH6.5)洗脱,比活力可达到115(AU/mg),是原粗酶液的17倍。纯化后的样品经SDS-PAGE电泳,初步确定了胆盐水解酶的分子质量约30kDa。该酶的最适反应温度35℃,最适pH6.0,最适反应底物浓度6mmol/L,酶促反应动力学常数4.57mmol/L。抑制剂对此酶的抑制作用强弱顺序依次为:尿素>SDS>EDTA。Al3+对此酶的激活能力最强,其次是Fe3+,Zn2+对酶活无激活作用。
The bile salt hydrolase produced by lactobillus casei KTx isolated from traditional Kefir grains was purified by ammonium sulphate precipitation,dialysis followed by DEAE-Sepharose FF cation exchange chromatography and its partial characteristics was studied in order to provide a theoretical basis for the structure analysis of bile salt hydrolase and the mechanism of reducing blood cholesterol.Results showed that the maximum precipitation of enzymic protein was obtained at condition of 70%(NH4)2SO4,the bile salt hydrolase was washed with 0.1mol/L NaCl(in phosphate buffer,pH6.5),at 1.0 ml/min.And the specific activity was 115 AU/mg.17 times as high as crude enzymic solution.The molecular size of bile salt hydrolase purified was estimated to be about 30 kDa,according to SDS-PAGE.the optimum reaction temperature of bile salt hydrolse was 30 to 40 ℃,the optimum reaction pH was 6.0.6 mmol/L was optimal substrate concentration.The Km value was 4.570 mmol/L.the inhibiting effect of inhibitor was carbamide > SDS > EDTA.Al3+ and Fe3+ activate the enzymatic activity,while Zn2+ has none any effect.
出处
《中国食品学报》
EI
CAS
CSCD
2007年第5期35-41,共7页
Journal of Chinese Institute Of Food Science and Technology
基金
"863"重大项目(2006AA10A208)
"863"项目(2006AA10Z343)
"十一五"科技支撑项目(2006BAD04A06)
关键词
胆盐水解酶
分离
纯化
特性
Bile salt hydrolase Isolation Purification Characteristics
