利用质谱分析法从酶谱鉴定嗜热真菌木聚糖酶  

Identification of a Thermophilic Fungi Protein with Xylanase Activity by MALDI-TOF and Zymogram Method

作　　者：吴亚宁[1,2] 刘刚[1] 余少文[1] 李水明[2] 王娟[1] 

机构地区：[1]深圳大学生命科学学院,深圳市微生物基因工程重点实验室,广东深圳518060 [2]深圳大学生命科学学院,深圳市海洋生物资源与生态环境重点实验室,广东深圳518060

出　　处：《食品研究与开发》2013年第17期116-120,共5页Food Research and Development

摘　　要：通过对前期筛选到的一株嗜热真菌Talaromyces thermophilus WYN9进行产木聚糖酶的诱导和非诱导培养。诱导培养的上清液中可以检测到木聚糖酶酶活。在非变性胶和变性胶SDS-PAGE中,诱导和非诱导的酶谱条带差异明显。以非诱导酶谱为对照,将诱导酶谱中与其有显著差异的条带切下,回收蛋白,测定酶活。将有木聚糖酶酶活的蛋白通过SDS-PAGE进一步分离纯化,切胶后使用胶内酶切法进行预处理,使用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF)进行分析。结果表明该蛋白与拟青霉属Paecilomyces sp.J18的内切木聚糖酶高度同源。根据质谱分析结果设计兼并引物,通过交错式热不对称PCR(thermal asymmetric interlaced PCR,TAIL-PCR)扩增,获得了TtXynA基因全长。根据Kimura双参数模型用临近相邻法构建了TtXynA及其同源蛋白的进化树。A thermophilic fungus with xylanase activity, Talaromyces thermophilus WYN9, isolated in our previous study was cultured by the induced and non-induced mode. The xylanase activity was detected in liquid supernatant of induced culture model. The significant differences were observed between induced and non- induced zymogram bands both in non-denaturing gel and denaturing gel SDS-PAGE. The different bands were cut and the xylanase activities were detected. The recovered protein with xylanase activity was analyzed by MALDI-TOF. The result showed that the identified protein has high homology with an endo-xylanse from Paecilomyces sp. J18. Degenerate primers were designed according to the analysis results of mass spectrum and the full-length of TtXynA was obtained by TAIL-PCR amplification. NJ tree of TtXynA homologous proteins was constructed based on Kimura two-parameter distance.

关 键 词：质谱 酶谱 11家族木聚糖酶 嗜热踝节菌 进化分析 

分 类 号：O657.63[理学—分析化学] TS201.25[理学—化学]