双孢菇酪氨酸酶的提取纯化及其性质

Study on Isolation,Purification and Characterization of Tyrosianse from the Agaricus bisporus

以双孢菇为原料,通过硫酸铵盐溶、盐析、透析和反渗透方法提取双孢菇酪氨酸酶并进行纯化,探究其性质。结果表明,经丙酮沉淀和硫酸铵盐溶初步纯化的酪氨酸酶与粗酶相比较,酶活力(25 U/mL)提高5倍,比活力提高8.75倍;经60%饱和硫酸铵盐析、半透膜透析、反渗透进一步纯化后的酪氨酸酶与粗酶相比较,酶活力(25 U/mL)提高68.4倍,比活力提高501.2倍。通过测定双孢菇酪氨酸酶的性质发现,最适pH为6.5,最适反应温度为30℃,米氏常数为0.543 mmol/L,最大反应速度为41.5 U/min,反应速度与酶量、底物浓度的关系均符合酶促反应动力学。D-异抗坏血酸可有效抑制双孢菇酪氨酶的活性。

Tyrosinase from Agaricus bisporus was extracted and purified by ammonium sulfate solution,salting out,dialysis and reverse osmosis,and its properties was investigated.The results showed that the enzyme activity and specific activity were significantly affected by different extraction condition.The activity of tyrosinase(25 U/mL)increased 5 times and 8.75 times by acetone precipitation and ammonium sulfate solution method,while the activity of tyrosinase(25 U/mL)increased 68.4 times and 501.2 times by 60%saturated ammonium sulfate salting-out,semi-permeable membrane dialysis and reverse osmosis.By determining the properties of tyrosinase from Agaricus bisporus,it was found that the optimum pH was 6.5,the optimum reaction temperature was 30℃,the Michaelis constant was 0.543 mmol/L,and the maximum reaction rate was 41.5 U/min.The relationship between the reaction rate and the amount of enzyme and the concentration of substrate conformed to the enzymatic reaction kinetics.D-isoascorbic acid could effectively inhibit the activity of tyrosinase of Agaricus bisporus.