豆壳过氧化物酶的分离纯化及其性质研究

从豆壳抽提液经硫酸铵分级沉淀，ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０离子交换层析，ＣｏｎＡ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲合层析和Ｂｉｏ－ＧｅｌＰ－６０凝胶过滤，纯化了豆壳过氧化物酶（ｓｏｙｂｅａｎｈｕｌｐｅｒ－ｏｘｉｄａｓｅ，ＳｈＰ）．纯化酶的比活力为７０７７Ｕ／ｍｇ，在ＳＤＳ－ＰＡＧＥ上显示出一条蛋白质带．ＳｈＰ分子量为３８０００，等电点为３．９；ＳｈＰ为一含血红素的糖蛋白，含糖量为１８．７％，光谱学分析揭示，在４０６ｎｍ处有一典型的Ｓｏｒｅｔ带，在５１０ｎｍ和６４０ｎｍ处有特征吸收峰．酶反应的最适ｐＨ在４．０附近，最适温度为４５℃；在ｐＨ２．５～１２．０之间较稳定，７５℃，保温６０ｍｉｎ，酶活力残余６８％，ＳｈＰ是一种良好的耐酸碱、耐热过氧化物酶．动力学分析求得ＳｈＰ的表观Ｋｍ（愈创木酚）为１．６２ｍｍｏｌ／Ｌ，表现Ｋｍ（Ｈ２Ｏ２）为０．３４ｍｍｏｌ／Ｌ．在所测定的化学试剂中，Ｎ－３、ＣＮ－、Ｆｅ３＋、Ｆｅ２＋和Ｓｎ２＋对酶有较强烈的抑制作用，而重金属离子Ａｇ＋、Ｈｇ２＋、Ｐｂ２＋、Ｃｕ２＋、Ｃｒ３＋以及ＳＤＳ和ＥＤＴＡ对酶活力无显著影响

Purification and Characterization of Soybean Hull Peroxidase

Soybean hull peroxidase(ShP) was purified to electrophoretic homogeneity by combined consecutive treatments consisting of ammonium sulfate fractionation,ion exchange chromatography on DEAE Sephadex A 50,affinity chromatography on Con A Sepharose 4B and gel filtration on Bio Gel P 60.The specific activity of purified peroxidase was 7077 U/mg.The molecular weight of the enzyme was 38 000 and the isoelectric point was pH 3 9.The enzyme was a glycoprotein(carbohydrate content 18 7%) containing a heme as prosthetic group and showed optimum activity and optimum temperature at pH 4 0 and 45℃,respectively.The enzyme was stable in the pH range from 2 5 to 12 At 90℃,it took 15 min to inactivate 50% of the enzyme.Kinetic studies showed that the reaction catalyzed by the enzyme was in accordance with a Ping Pong mechanism,and a K m for H 2O 2 of 0 34 mmol/L at optimum guaiacol concentration and a K m for guaiacol of 1 62 mmol/L at optimum H 2O 2 concentration were obtained.Peroxidase activity was seriously inhibited by Fe 2+ 、Fe 3+ 、Sn 2+ 、CN - and N 3- ,while no obvious inhibition was observed with Hg 2+ 、Ag +、Pb 2+ 、Cr 3+ 、EDTA and SDS.