假单胞菌碱性木聚糖酶的纯化及性质

PURIFICATION AND CHARACTERIZATION OF ALKALINE XYLANASES FROM PSEUDOMONAS G6 2

假单胞菌（Ｐｓｅｕｄｏｍｏｎａｓ）Ｇ６２可产生两种胞外木聚糖酶，即ＸｙｎＡ和ＸｙｎＢ。经过硫酸铵沉淀、阴离子和阳离子交换层析、分子筛色谱，最终得到两种电泳纯酶。ＸｙｎＡ的分子量及等电点分别为４２ｋＤ和９１，ＸｙｎＢ的分子量和等电点分别是２０ｋＤ和８８。经薄层色谱分析证明，两酶以不同的方式水解木聚糖，但都不产生木糖，即两酶都为内切酶，它们的最适作用温度均为５０℃。ＸｙｎＡ的最适作用ｐＨ为７０～９８，而ＸｙｎＢ的为７０～７５。在６５℃时的半寿期ＸｙｎＡ为６ｍｉｎ，ＸｙｎＢ为１４０ｍｉｎ。ＸｙｎＡ的Ｋｍ和Ｖｍａｘ分别是５５６ｍｇ·ｍｌ－１和５４３μｍｏｌ·ｍｉｎ－１·ｍｇ－１，ＸｙｎＢ的Ｋｍ和Ｖｍａｘ分别是７７２ｍｇ·ｍｌ－１和８１９μｍｏｌ·ｍｉｎ－１·ｍｇ－１。两酶受Ｃｕ２＋、Ｆｅ３＋、Ｐｂ２＋、Ｚｎ２＋和Ｈｇ２＋强烈抑制。化学修饰的初步结果表明，两酶的活性位点氨基酸均含有色氨酸和羧基氨基酸。

Pseudomonas G6 2 produced two extracellular xylanases,named XynA and XynB.The enzymes were purified by amonium sulfate fractionation,Sephadex G 100,DEAE Sephadex,CM Sephadex and Bio gel P 10 chromatographies.Both enzymes were indicated to be endoxylanases,which produced oligomers of xylose from xylan and did not hydrolyze it to xylose.They had same temperature optimum(50℃) and different pH optimum(pH 7 0～9 8 for XynA and pH 7 0～8 0 for XynB).At pH 7 6 and 65℃,XynA and XynB possessed the half life of 6 min and 140 min,respectively.Their activities were strongly inhibited by Cu 2+ ,Fe 3+ ,Pb 2+ ,Zn 2+ and Hg 2+ .The results of chemical modification indicated that tryptophan and carboxy group were related to active center.