大蒜蒜酶提取纯化及临床应用研究

Study of extraction and purification of alliinase from garlic and its clinical application

目的：研究从鲜蒜中分离纯化蒜酶的方法和临床常用剂对酶活性的影响,为今后工艺的进一步放大、优化和合理临床应用提供必要理论参数。方法：以天然蒜氨酸作底物,采用丙酮酸法测定酶浓度;以牛血清白蛋白为标准,采用考马斯亮兰G-250法测定蛋白质浓度。结果：从鲜蒜中提取纯化蒜酶,其粗酶浸提液的最佳组成为Na2HPO4-KH2PO4缓冲溶液;硫酸铵沉淀酶蛋白的最适饱和度范围为0%～35%;选用超滤技术脱盐;蒜酶最佳调节等电点为4.9。在临床常用溶剂中,含氯化钠的注射液（不含葡萄糖）对蒜酶活力有显著的激活作用,而含葡萄糖的注射液则较强地抑制了蒜酶活力;75%和95%乙醇具有强烈的杀酶作用。结论：本研究为今后工艺的进一步放大与优化和蒜酶合理临床应用提供了指导。

Objective ：The study of extraction and purification of alliinase from garlic and the effect of clinical solvent commonly used on enzyme activity were accomplished in this paper. It provides essential parameter for further purification procedure and clinical application. Methods：Protein concentrations of allinase preparations were determined according to the methods of bradford usint BSA as standards. Enzyme activity was assayed from the amount of enzymatically formed pyruvate using natural L-（＋）-alliin as substrate. Results ：The processing conditions for the extraction and purification of alliinase from garlic were optimized. It is in Na/K phosphate buffer that peeled garlic cloves were homogenized in a blender. Precipitation with solid ammonium sulfate to give 35% saturation was selected to isolate alliinase. It is by ultrafiltration that supernatant solution containing alliinase is desalted. The isoisnic point of alliinase was found to be 4.9. When injections with NaCl and without glucos was used as solvent ,there was a marked stimulation of enzyme activity,but injections with glucos gave a strong inhibition; The lyase solved in 75% or 95% al- cohol completely lost its activity rapidly. Conclusion：The present paper gave directions to furtherly amplify and optimize purification procedure of alliinase and its clinical application.