摘要
比较了用乙醇沉淀法和硫酸铵盐析法从“东一号”剑麻中提取蛋白酶的效果.用前者制得的粗酶经Sephadex G-25柱层析得两个蛋白峰,其中峰Ⅰ与酶活性重叠。峰Ⅰ再经SP—Sephadex C—50离子交换柱层析,pH.离子强度线性梯度洗脱得4个蛋白峰.其中峰4与酶活性重叠。此酶液经聚丙烯酰胺凝胶圆盘电泳.在每条胶柱上呈单一蛋白带;量4℃下2d后出现六角形结晶.结晶酶液在pH5.5-10.0.60℃范围内稳定性较好;其最适pH为7.5;最适温度为40℃;Km(酪蛋白)值为0.114％(w/v)用SDS—聚丙烯酰胺凝胶电泳(SDS—PAGE)测得其分子量66KD;用Sephadex G—100柱层析测得的分子量为69.9KD.用Ellman’s试剂测得其游离巯基含量为4.3 mol—SH/10~5g蛋白质。
A proteinase was extracted from the leaves of Agms hybrid No. 11648 by precipitation with ethanol. The crude enzyme wai fractionated with Sephadex G-25 into two fractiom (Ⅰ and Ⅱ). Only fraction Ⅰ had hydrolytic activity on casein. The peak of fraction Ⅰ coincides with the peak of proteinaae activity. Fraction Ⅰ protein wat further fractionated with SP-Sephadex C-50 Into four four fraction* (Ⅰ'. Ⅱ'. Ⅲ', and Ⅳ'). and only fraction Ⅳ' had hydrolytic activity on casein. Enzyme crystals of hexagonal ahape wete formed after placing the enzyme solution in 4 ℃ for two days, and a single band was found in the PAGE with the crystais. The optimum pH was 7. 5. and the optimum temperature was 40℃. It was relatively stable at temperature blsow 60℃ and at pH value ranting from 5. 5 to 10. 0. Its Km value for casein was 0.114 % (w/v). Its mokcular weight determined by SDS-PAGE and chro-matography on Sephadex G- 100 with atandard protein* were found to be 66KD and 69. 9KD respectively. Its content of free sulfhydryl groups determined by Ellman's reagent was 4. 3 mol-SH/105 g protein, approximately equal to 2. 9 mol free sulfhydryl in 1 mol proteinase.
出处
《华南农业大学学报》
CAS
CSCD
1993年第1期133-139,共7页
Journal of South China Agricultural University
关键词
东一号
剑麻
巯基蛋白酶
Agme hybrid No. 11648: Suifhydryl proteinass
