纤维素降解复合菌系中纤维结合蛋白的分离及鉴定

Isolation and identification of cellulose binding protein of a cellulolytic microbial consortium

采用亲和消化法分离纯化了纤维素降解复合菌系中的纤维素结合蛋白,纯化后的纤维结合蛋白液经SDS-PAGE（聚丙烯酰氨凝胶电泳）后得到13条条带（命名为CBP1- 13）,通过对其进行酶谱分析,发现低分子量的蛋白主要表现CMC（羧甲基纤维素钠）酶活性,而高分子量（66- 200 kDa）的蛋白兼有CMC酶和木聚糖酶的活性,其中纤维结合蛋白CBP6表现出的木聚糖酶和CMC酶活性最强.这些条带经质谱鉴定后被确定由8种蛋白组成,除CBP4为Paenibacillus sp.分泌的木聚糖酶5外,其余的蛋白均由C.clariflavum DSM 19732所产生,其中CBP1-3为β-1,4木聚糖酶,CBP5,6为碳水化合物结合蛋白,CBP7-9,11,12为锚定蛋白,CBP10和CBP13为未知蛋白.通过结构域预测,发现除纤维结合蛋白CBP13外所有的蛋白均具有一种催化结构域,分别隶属于第8、9、10及48家族的糖苷水解酶,而部分蛋白（CBP1,2,4-6）还具有碳水化合物结构域或者锚定蛋白结构域（CBP5- 12）.

The cellulose binding proteins of a cellulolytic microbial consortium were isolated and purified by affinity digestion, where 13 bands （named CBP 1 -13 ） were obtained after SDS-PAGE from these purified proteins. Zymogram analysis found that the proteins with higher molecular weight （66 - 200 kDa） exhibited both xylanase and CMCase activities, while those with lower molecular weight exhibited CMCase activity only. Among these proteins, the ceUulose binding protein of CBP6 showed the highest xylana and CMCase activity. In addition, these bands were found to be consisted with 8 kinds of proteins by the identification of mass spectrum. The results showed that except protein CBP4 was secreted by PaenibaciUus sp. and confirmed as xlylanase 5, the other proteins were secreted by C. clariflavum DSM 19732. Among these proteins, CBP1 N 3 were identified as/3- 1, 4 xylanase, CBP5, 6 were putative carbohydrate binding proteins, CBP7- 9, 11, 12 were dockerin-like proteins and CBP 10, 13 were unnamed proteins. According to the domain prediction of these proteins, each of the proteins （except CBP 13 ） own a kind of catalysis domain and belongs to the glucoside hydrolase family of 8, 9, 10 and 48, respectively. In addition, parts of the proteins （ CBP1, 2, 4-6） owned carbohydrate binding domains or dockerin-like protein domains （ CBP5- 12）.