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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 method of extracting crude enzyme from fresh green onion and provide basis for further purification and clinical application. Methods: The mixed substrate extracted with light blue was used to measure the activity of the enzyme in the light blue by pyruvic acid method. The crude enzyme was extracted by phosphate buffered saline and the protein was determined by Coomassie brilliant blue G-250 method concentration. Results: The relative activity of crude enzyme extracted from the extraction solution with pyridoxal-5’-phosphate before use was precooling for 12 h in Na-K phosphate buffer solution of pH 6.5 + 10% glycerol + 0.2% EDTA The higher the degree, so the composition of the extract was light crude extract of the crude extract; PEG dosage was 15% supernatant enzyme activity decreased sharply, and then tends to a flat trend, the concentration of the supernatant The specific activity of the enzyme is relatively minimum, so the concentration of PEG8000 was selected as the concentration of the precipitated protein. With the extension of the dialysis time, the protein content and enzyme activity of the protein solution decreased, and the enzymatic activities in the protein solution at 6 h and 24 h Activity and protein content are not much difference between, we can see that when dialysis 6 h, dialysis basically reached equilibrium. Conclusion: The method is simple and can be used for the extraction of crude enzyme in fresh green onion.