大蒜蒜酶/蒜氨酸催化动力学及临床应用研究

Enzymatic Properties and Kinetic Charactristics of Homogeneous Alliinase from Garlic

目的从鲜蒜中分离纯化蒜酶,并研究其酶学性质、动力学特性以及临床常用溶剂对酶活力的影响,为今后进一步优化蒜酶的提取纯化工艺和临床应用提供必要理论参数。方法采用盐析、超滤、冷冻干燥等方法提取纯化蒜酶,SDS-Page凝胶电泳法测定纯化蒜酶分子量,丙酮酸法和考马斯亮蓝G-250法测定蒜酶活力。结果分离纯化所得蒜酶的分子量为97.88KDa,纯度大于60%;蒜酶催化裂解天然蒜氨酸的最适温度为35℃,最适pH为7.00;随着温度的升高,蒜酶的稳定性下降;蒜酶在pH6.5的缓冲体系中最稳定;以天然蒜氨酸为底物测得米氏常数Km=2.139mmol.L-1,最大反应速度Vmax=41.67U.mg-1(蛋白);临床常用溶剂0.9%氯化钠注射液、复方氯化钠注射液(含0.85%NaCl、0.03%KCl、0.033%CaCl2)对蒜酶具有显著的激活作用,而5%葡萄糖注射液、10%葡萄糖注射液、葡萄糖氯化钠注射液(含5%葡萄糖、0.9%氯化钠)则较强的抑制了蒜酶活力,75%和95%乙醇具有强烈的杀酶作用。结论蒜酶具有热不稳定性和pH不稳定性;其酶解反应选择在35℃和pH7.00的缓冲体系中最佳;而蒜酶溶解在pH6.5的缓冲体系中能较长时间保持稳定;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,而含有葡萄糖的注射液强烈抑制蒜酶活力;75%和95%乙醇作为消毒液使用时也应避免其对蒜酶的杀伤作用。实验结果对进一步优化蒜酶的提取纯化工艺研究以及蒜酶的临床应用具有指导意义。

Objective To isolate alliinase （ EC 4.4.1.4） from its natural source, garlic and to study the effect, enzymatic properties, kinetic charactristics as well as dissolvant commonly used in clinic on enzyme activity. Methods The purification of the enzyme consisted of salting - out step, uhrafihration and freeze drying, etc. The relative molecular masses of this garlic enzyme was estimated by SDS - PAGE. Protein concentrations of allinase preparations were determined according to the method of Bradford with BSA as standard. Enzyme activity was assayed from the amount of enzymatically formed pyruvate using natural L - （ ＋ ） - alliin as substrate. Results SDS - PAGE gave evidence that alliinase obtained from garlic consisted of two identical subunits with molecular weights of 97.88 KDa and 60% of purity. The highest enzyme activity for the purified alliinase was found to be at 35℃. The enzyme pH optimal had at pH7.0, but it was the most stable at pH6.5. The Km value for alliinase from garlic was estimated to be 2. 139mmol · L^-1 for natural L - （ ＋ ） - alliin. Vmax using natural L - （ ＋ ） - alliin as substrate was at 41.67 U · mg^-1 protein）. When Sodium Chloride Injections containing 0.9% NaCl or complex prescription（0.85% NaCl, 0.03% KCl, 0.033% CaCl2 ） was used as solvent, there was a stimulation of enzyme activity, but Glucos Injections respectively containing 5% glucos, 10% glucos,5% glucose and 0.9% NaCl gave a strong inhibition. The lyase solved in 75% or 95% alcohol completely lost its activity rapidly. Conclusion It has been demonstrated that alliinase is sensitive to pH or temperatures. Injections containing glucos used as solvent is not suitable for this enzyme. Using 75% or 95% alcohol as disinfectant should avoid lowering the enzyme activity. The present paper gave directions to optimizing purification procedure of enzyme and its clinical application.