嗜热木聚糖酶基因在毕赤酵母中的表达及其酶学性质  

Expression of A Thermophilic Xylanase Gene in Pichia pastoris and Its Enzymatic Properties

作　　者：陈鲜[1] 郭颖[1] 胡胜伟[1] 刘君梁[1] 冯家勋[1] 

机构地区：[1]广西大学生命科学与技术学院,亚热带农业生物资源保护与利用国家重点实验室,南宁530004

出　　处：《基因组学与应用生物学》2016年第11期3060-3068,共9页Genomics and Applied Biology

基　　金：广西自然科学基金创新研究团队项目(2012GXNSFGA060005)资助

摘　　要：木聚糖酶Umxyn10A,属于GH10家族,包含一段跨膜区和GH10家族催化功能域,将跨膜区去掉后,剩余部分重命名为Umxyn10AQ。按毕赤酵母密码子偏好性将Umxyn10AQ序列进行密码子优化,与毕赤酵母表达载体p PIC9K相连。重组质粒p PIC9K-Umxyn10AQ经SalⅠ单酶切线性化后转至毕赤酵母(Pichia pastoris)GS115。经G418筛选得到重组毕赤酵母菌株GS115/Umxyn10AQ,每12 h添加1%甲醇诱导剂。DNS法测定重组木聚糖酶re Umxyn10AQ酶活,最高酶活达到15 U/m L,SDS-PAGE分析表明,re Umxyn10AQ相对分子质量为45.0 k D。重组木聚糖酶re Umxyn10AQ的最适p H为8.0,最适反应温度85℃,Co2+对该酶活有显著促进作用,提高了近20%,水解产物为木糖(14%)、木二糖(86%)。结果表明,木聚糖酶Umxyn10AQ在毕赤酵母中成功表达并且分泌到胞外,相较于大肠杆菌表达产物Umxyn10A,最适p H提高了1.5个单位、最适温度提高了10个单位,p H耐受性和温度耐受性都有所改善,并且主要水解产物仍为木二糖。Umxyn10A, a thermophilic xylanase, which belongs to GH10 family, contains a transmembrane domain and a GH 10 catalytic domain. The transmembrane domain was deleted, and the rest was renamed as Umxyn 10AQ. Then, the gene sequence of Umxynl0AQ was codon-optimized according to the Pichia pastoris codon preferences, and cloned into expression plasmid pPIC9K. The recombinant plasmid pPIC9K-Umxyn10AQ was linerized with Sal I and integrated into （P. pastoris） GS 115 by electroporation. The recombinant P. pastoris GS 115/Umxyn10AQ was screened by G418, and induced with 1% methanol every 12 hours. The recombinant xylanase reUmxyn 10AQ catalytic activity was detected by DNS, the highest activity was 15 U/mL, and the molecular weight of reUmxyn10AQ was 45.0 kD by SDS-PAGE anlaysis. The optimum pH of reUmxyn10AQ was 8.0, the optimum temper- ature was 85℃, and its activity was significantly sitimulated by Co^2＋, increased by 20%, the main hydrolysates were xylose （14%） and xylobiose （86%）. Results showed that, the xylanase Umxynl0AQ was functionally expressed in P. pastoris and secreted into the supernatant. Compared to the recombinant Umxyn10A from E. coli, the optimum pH increased by 1.5 units, optimum temperature increased by 10 units, and the pH and thermol stability of were improved, otherwise, its hydrolysate was still xylobiose mainly.

关 键 词：木聚糖酶 低聚木糖 毕赤酵母 酶学性质 

分 类 号：Q78[生物学—分子生物学] Q55