粗毛栓菌木聚糖酶的纯化及性质

以麦草粉为基质培养粗毛栓菌Trametes gallica,浸提固态培养物得浸提液,后经超滤浓缩、硫酸铵盐析、Phenyl-Sepharose CL-4B疏水层析、DEAE Sepharose fast flow阴离子交换层析和Sephadex G-150分子筛层析等分离与纯化步骤,获得部分纯化的木聚糖酶,其回收率和纯化倍数分别为1.45%和15.6。进一步经活性-PAGE回收,获得三种SDS-PAGE电泳纯级的木聚糖酶同工酶组分:XⅠ、XⅡ和XⅢ(按等电点从大到小排列)。三种组分分子量均约为19.0kDa;等电点分别为:5.6、4.7和4.0;含糖量分别为:0.25%、0.63%和3.4%;XⅠ既能降解木聚糖,又能降解纤维素;XⅡ的最适作用pH值为5.0,最适作用温度45℃;Mg2+、Fe2+对XⅡ有激活作用;Mn2+和Co2+有抑制作用;测得XⅡ的Km值为0.75mg/mL,Vmax为5,000mmoL/min·mg。

Purification and characterization of xylanases from white-rot fungus Trametes gallica

The white-rot fungus Trametes gallica was cultivated on wheat straw powder.Following protein precipitation with (NH_4)_2SO_4,the fractions showing xylanase activity were separated and purified by chromatographic analyses with Phenyl-Sepharose CL-4B,DEAE Sepharose FF and Sephadex G-150.The recovery rate and purification fold reached up to 1.45% and 15.6-fold,respectively.The enzymes were further recovered by native-PAGE and three isoenzyme xylanases (XⅠ,XⅡ and XⅢ) of SDS-PAGE purity were finally obtained.The three components had similar molecular weights (approximately 19.0kDa),but exhibited different isolectric points (5.6 fro XⅠ,4.7 for XⅡ and 4.0 for XⅢ) and carbohydrate content (0.25%for XⅠ,0.63% for XⅡ and 3.4% for XⅢ).XⅠ can degrade both xylan and cellulose,while XⅡ and XⅢ can only degrade xylan.The optimal temperature and pH for XⅡ were 45℃ and 5.0,respectively.Its activity was increased by Mg~(2+) and Fe~(2+),while inhibited by Mn~(2+)