假单胞菌胞外弹性蛋白酶的纯化研究  

Studies on Purification of Pseudomonas Extracellular Elastase

作　　者：贺稚非[1] 周泽扬[2] 李洪军[1] 高兆建[1] 宫春波[1] 

机构地区：[1]西南农业大学食品科学学院,重庆400716 [2]西南农业大学农业部蚕桑学重点实验室,重庆400716

出　　处：《中国食品学报》2005年第2期22-27,共6页Journal of Chinese Institute Of Food Science and Technology

摘　　要：假单胞菌的发酵液经过硫酸铵盐析、透析、DEA E-Sephadex A25阴离子交换层析、Sephadex G75凝胶过滤层析等纯化步骤,获得了较纯的酶蛋白。SDS-PAGE凝胶电泳:分离胶12%、压缩胶4%,15m A、30m A恒流电泳后,采用考马斯亮蓝染色和银染色,并用H PLC检测,将蛋白质转移到PV DF膜上,N-端测定10个氨基酸序列。结果表明:SDS-PAGE电泳得到单一条带,H PLC检测活性峰为单峰。该酶在SD S-PAG E上测得的分子质量为32000Da,N-端氨基酸测序结果为APPSNLM QLP。The purpose of this study is to make the research base of the recombination strain of secreting extracellular elastase. The purification of the elastase was achieved by the combination of(NH4)2SO4 fraction,dialyse,Anion-exchange chromatography on DEAE-Sephadex A25 and Gel-filtration chromatography on Sephadex G75. SDS-PAGE homogenous of preparation elastase was obtained. The purified enzyme was homogenous on polyacrylamide gel electrophoresis. After constant current electrophoresis at 15mA and 30mA,using coomassieblue staining and Ag-staining,HPLC detection and electrotransfer on PVDF membrane,ten amino acid was analysed by N-terminal sequential determination. Results showed that the molecular weight estimated by SDS-PAGE was 32 000Da. The elastase showed the single peak by HPLC. The N-terminal amino acid sequence of elastase showed APPSNLMQLP.

关 键 词：假单胞菌 酶的纯化 弹性蛋白 SEPHADEX 阴离子交换层析 考马斯亮蓝染色 凝胶过滤层析 PAGE电泳 N-端氨基酸 HPLC 硫酸铵盐析 PVDF膜 氨基酸序列 SDS DEAE 凝胶电泳 分子质量 发酵液 酶蛋白 分离胶 银染色 蛋白质 检测 测序 

分 类 号：TS201.25[轻工技术与工程—食品科学]