摘要
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。
The white-rot fungus Trametes gallica was cultivated on wheat straw powder. Following protein precipitation with （NH4）2SO4, 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 （X Ⅰ, X Ⅱ 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 for X Ⅰ, 4.7 for X Ⅱ and 4.0 for XⅢ） and carbohydrate content （0.25% for X Ⅰ, 0.63% for XⅡ and 3.4% for XⅢ）. X Ⅰ 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＋ and Co^2＋. Its Km and Vmax were 0.75mg/mL and 5,000mmoL/min·mg, respectively.
出处
《菌物学报》
CAS
CSCD
北大核心
2010年第1期83-90,共8页
Mycosystema
关键词
白腐菌
同工酶
分离纯化
酶学性质
white-rot fungus, isoenzyme, xylanases, separation and purification, enzymatic properties
