| 摘要: |
| 为了充分探讨草鱼内脏的利用途径并深入了解草鱼消化生理学,将草鱼肠道用冰丙酮脱脂,用磷酸盐缓冲溶液提取,然后经过硫酸铵分级沉淀,再经过离子交换层析、凝胶过滤层析等纯化,分离得到一种碱性蛋白酶,并对其部分理化性质进行了检测。结果表明,该酶是一种胰蛋白酶(GT-A),其相对分子质量为30.75 ku;其最适温度为40 ℃,最适pH为8.0,在pH 6.0～10.0保持稳定,在65 ℃加热20 min其活性丧失95%。表明草鱼肠道中含有在低温下能很好发挥水解作用的胰蛋白酶。 |
| 关键词: 草鱼 碱性蛋白酶 胰蛋白酶 纯化 SDS-底物-PAGE |
| DOI： |
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| 基金项目:湖南省教育厅中青年教师计划项目（02C580） |
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| Purification and preliminary research of some properties of a Trypsin (GT-A) from the intestines of grass carp (Ctenopharyngodon idellus) |
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| Abstract: |
| In order to widely investigate ways of utilizing the gut of grass carp (Ctenopharyngodon idellus) and intensively learn its digestive physiology,intestines of the grass carp were defatted with cooled acetone (-20℃),and then extracted with 0.1 mol/L phosphate buffer,pH 6.8.And then,an alkaline protease was purified from the crude extract by ammonium sulfate fractionation of 10-40% saturation,Sepharose CL-6B gel chromatography,DEAE-Sepharose CL-6B ion exchange chromatography,and Sephadex G-75 and Sephacryl S-100 HR gel chromatography.SDS PAGE electrophoresis showed the enzyme was homogeneous.SDS-substrate-PAGE and inhibition assays showed that it was a trypsin,named as GT-A,with a relative molecular weight of 30,75 ku.GT-A had optimum temperature of 40 ℃ and optimum pH of 8.0 for α-N-bezoyl-L-arginine ethyl ester·HCl (BAEE).GT-A remained stable at range of pH 6.0-10.0,and lost 95% of its activity when heated for 20 min at 65℃,and exhibited some unique properties in comparison with those of trypsins from other fish species.These results showed that the intestines of grass carp contained an efficiently hydrolyzing trypsin at lower temperature. |
| Key words: grass carp,alkaline protease,trypsin,purification,SDS-substrate-PAGE |
