目的探讨三嗪活性染料Cibacron Blue F3GA和KE-3B艳红制备的亲和层析柱用于建立分离纯化面包酵母乙醇脱氢酶和葡萄糖-6-磷酸脱氢酶的方法。方法在琼脂糖凝胶Sepharose 6B上,分别偶联三嗪活性染料Cibacron Blue F3GA和KE-3B艳红,制成染...目的探讨三嗪活性染料Cibacron Blue F3GA和KE-3B艳红制备的亲和层析柱用于建立分离纯化面包酵母乙醇脱氢酶和葡萄糖-6-磷酸脱氢酶的方法。方法在琼脂糖凝胶Sepharose 6B上,分别偶联三嗪活性染料Cibacron Blue F3GA和KE-3B艳红,制成染料亲和层析柱。将面包酵母经超声破碎、高速离心、硫酸铵分级沉淀、透析、冰冻干燥等步骤得到脱氢酶粗品,再经染料亲和层析柱进一步纯化。结果经过固定有Cibacron Blue F3GA的亲和层析柱,乙醇脱氢酶的纯化倍数为2.0,酶活性回收率为30.8%;经过固定有KE-3B艳红的亲和层析柱,葡萄糖-6-磷酸脱氢酶的纯化倍数为23.2,酶活性回收率为66.0%。所得的乙醇脱氢酶在SDS-聚丙烯酰胺凝胶电泳为1条带。结论染料配基亲和层析可作为面包酵母乙醇脱氢酶和葡萄糖-6-磷酸脱氢酶的精制纯化方法。展开更多
Alkaline phosphatase(AKP)was isolated and purified from the skin of Tylototriton taliangensis Liu and its kinetic property was examined.The partially purified alkaline phosphatase was purified by salting\|out,CM\|cell...Alkaline phosphatase(AKP)was isolated and purified from the skin of Tylototriton taliangensis Liu and its kinetic property was examined.The partially purified alkaline phosphatase was purified by salting\|out,CM\|cellulose ion\|exchange column and gel filtration with Sephadex G\|150.The purified enzyme from skin moved as a single electrophoretic band in PAGE.The specific activity was 90.26 units/mg protein.Its subunit weight was 42.0 kD as determined with SDS\|PAGE.The optimum pH value for the enzyme was pH9.0.The optimum temperature was about 34℃.The Michealis\|Menton constant( K \-m)was 0.83 mmol/L on the disodium phenyl phosphate.Activity differences of the enzymes were determined when metal ions effected on the AKP.The results showed that K + was found to have no inhibition of AKP activity.Mg 2+ ,Ca 2+ ,Cu 2+ could activate the AKP and the higher the metal ions concentration were,the more the activity of AKP increased.When 3.0 mmol/L Cu 2+ was used,the activity of AKP could rise to 187%.Ni 2+ ,Zn 2+ and Ag + could inhibit the AKP and the higher the metal ions concentration were,the more the activity of AKP decreased.When 3.0 mmol/L Ag + was used,the activity of AKP retained 23% only.展开更多
文摘目的探讨三嗪活性染料Cibacron Blue F3GA和KE-3B艳红制备的亲和层析柱用于建立分离纯化面包酵母乙醇脱氢酶和葡萄糖-6-磷酸脱氢酶的方法。方法在琼脂糖凝胶Sepharose 6B上,分别偶联三嗪活性染料Cibacron Blue F3GA和KE-3B艳红,制成染料亲和层析柱。将面包酵母经超声破碎、高速离心、硫酸铵分级沉淀、透析、冰冻干燥等步骤得到脱氢酶粗品,再经染料亲和层析柱进一步纯化。结果经过固定有Cibacron Blue F3GA的亲和层析柱,乙醇脱氢酶的纯化倍数为2.0,酶活性回收率为30.8%;经过固定有KE-3B艳红的亲和层析柱,葡萄糖-6-磷酸脱氢酶的纯化倍数为23.2,酶活性回收率为66.0%。所得的乙醇脱氢酶在SDS-聚丙烯酰胺凝胶电泳为1条带。结论染料配基亲和层析可作为面包酵母乙醇脱氢酶和葡萄糖-6-磷酸脱氢酶的精制纯化方法。
文摘为了对菠萝蛋白酶的抗过敏效果进行研究,采用透明质酸酶体外抑制试验考察菠萝蛋白酶在水解状态和未水解状态下对透明质酸酶的抑制效果,并运用小鼠被动过敏模型试验评价菠萝蛋白酶的抗过敏效果。结果表明,菠萝蛋白酶质量浓度为0.6~3.6 mg/m L时,对透明质酸酶的抑制效果呈正相关;继续增大菠萝蛋白酶质量浓度,对透明质酸酶的抑制效果反而下降。菠萝蛋白酶经水解后发现,当菠萝蛋白酶的水解度为12.3%时,对透明质酸酶的抑制率达到最大为53.5%,对此水解产物进行超滤,发现其抑制效果主要由分子量在8~10 k Da的组分贡献,此组分对透明质酸酶的抑制率达到最大59.6%。经蓝斑抑制试验表明,相比中、高剂量组,低剂量组的菠萝蛋白酶对小鼠皮肤蓝斑的抑制效果最好,抑制率为55.87%,是阳性药物息斯敏的1.06倍。菠萝蛋白酶具有良好的抗过敏效果,为其进一步开发成为抗过敏剂应用于临床提供理论基础。
文摘Alkaline phosphatase(AKP)was isolated and purified from the skin of Tylototriton taliangensis Liu and its kinetic property was examined.The partially purified alkaline phosphatase was purified by salting\|out,CM\|cellulose ion\|exchange column and gel filtration with Sephadex G\|150.The purified enzyme from skin moved as a single electrophoretic band in PAGE.The specific activity was 90.26 units/mg protein.Its subunit weight was 42.0 kD as determined with SDS\|PAGE.The optimum pH value for the enzyme was pH9.0.The optimum temperature was about 34℃.The Michealis\|Menton constant( K \-m)was 0.83 mmol/L on the disodium phenyl phosphate.Activity differences of the enzymes were determined when metal ions effected on the AKP.The results showed that K + was found to have no inhibition of AKP activity.Mg 2+ ,Ca 2+ ,Cu 2+ could activate the AKP and the higher the metal ions concentration were,the more the activity of AKP increased.When 3.0 mmol/L Cu 2+ was used,the activity of AKP could rise to 187%.Ni 2+ ,Zn 2+ and Ag + could inhibit the AKP and the higher the metal ions concentration were,the more the activity of AKP decreased.When 3.0 mmol/L Ag + was used,the activity of AKP retained 23% only.
