生鲜葱白中粗酶的提取研究

Study on the extraction of crude enzyme from fistular onion stalk

目的:研究从生鲜葱白中提取粗酶的方法,为下一步分离纯化和临床应用提供依据。方法:采用葱白中提取的混合底物,用丙酮酸法测定葱白中酶的活力,采用磷酸盐缓冲液提取粗酶,以小牛血清白蛋白为标准,用考马斯亮蓝G-250法测定蛋白质的浓度。结果:pH6.5的Na-K磷酸盐缓冲液+10%甘油+0.2%EDTA,预冷12 h,临用前加5’-磷酸吡哆醛为浸提液所提取粗酶的相对比活度较高,故定该组分为葱白中粗酶提取的浸提液;PEG用量为15%时上清中酶活急剧减小,随后趋于平缓趋势,该浓度下的上清液中的酶的比活度相对最小,故选择该浓度为沉淀蛋白时PEG8000的浓度;随着透析时间的延长,蛋白质溶液中蛋白质含量及酶活性均在下降,透析6 h和24 h的蛋白质溶液中酶活性和蛋白质含量均相差不大,可以说明当透析6 h后,透析基本已经达到平衡。结论:该方法操作简便可以用于生鲜葱白中粗酶的提取。

OBJECTIVE To study the extraction of crude enzyme from fistular onion stalk, providing the basis for separation, purification and clinical application. METHODS Enzyme activity was assayed from the amount of enzymatically formed pyruvate using mixed substrate extracted from fistular onion stalk. Crude enzyme was extracted with phosphate buffer. Protein concentrations of crude enzyme were determined according to the methods of Bradford using BSA as standards. RESULTS U- sing the extracting solution precooled for 12 hours with pH6. 5 Na-K phosphate buffer, 10% glycerin, 0. 2% EDTA and pyridoxal 5＇-phosphate, we could get crude enzyme with hyperactivity. When the application amount of PEG was 15%, the enzyme activity declined rapidly, and supernatant relative to the minimum specific activity of the enzyme activity, so we chose 15% as the application amount of PEG8000. With the prolongation of dialysis time, the protein content and enzyme activity of the the solution the declined. There was no difference of the protein content and enzyme activity when the dialysis time between 6 h. CONCLUSION The method is simple, which can he used for the extraction of crude enzyme from fistular onion stalk.