里氏木霉木聚糖酶XYN II基因在毕赤酵母中的分泌表达  

Secretory Expression of Trichoderma reesei XYN II Gene in Pichia pastoris

作　　者：欧阳嘉[1] 王向明[2] 张清[2] 严明[2] 许琳[2] 

机构地区：[1]南京林业大学化学工程学院,江苏南京210037 [2]南京工业大学制药与生命科学学院,江苏南京210009

出　　处：《林产化学与工业》2007年第5期83-88,共6页Chemistry and Industry of Forest Products

基　　金：国家重点基础研究发展计划(2003CB716005;2007CB707801);国家高技术研究发展计划(2006AA020204)

摘　　要：采用RT—PCR方法克隆到里氏木霉RutC-30木聚糖酶（XYNⅡ）的cDNA序列。测序结果表明，XYNⅡ的cDNA基因开放阅读框长度为669bp，编码223个氨基酸，N端1～19个氨基酸为潜在的信号肽序列，删去潜在信号肽序列，将里氏木霉木聚糖酶的基因（xynⅡ）构建到巴斯德毕赤酵母分泌表达载体pPIC9K上，线性化后电击转化到巴斯德毕赤酵母中，经G418筛选和PCR鉴定后的转化子用1％的甲醇进行诱导，对重组木聚糖酶活检测显示该基因能在毕赤酵母中表达有生物活性的XYNⅡ并分泌到胞外。发酵液中的酶活在诱导培养60h达到1．45IU／mL，最适酶解温度为50℃，最适pH值为6．0。The gene of xylanase （XYNⅡ） was amplified by RT-PCR from the total RNA of Trichoderma reesei Rut C-30 and sequenced. The gene comprised an open reading frame that encoded a polypeptide of 223 amino acid residues, containing a signal peptide of 19 amino acid residues. A recombinant plasmid pPIC9K-xyn Ⅱ was constructed by inserting gene xyn Ⅱ into Pichia pastoris secretory vector pPIC9K. Linearized pPIC9K-xyn Ⅱ was transformed into P, pastoris GS115 with the method of electropo- ration, The recombinant strain identified by G418 selection and confirmed by PCR analysis was induced by 1.0 % methanol at 28 ℃ to express the recombinant xylanase, the highest enzyme activity of 1.45 IU/mL was detected for 60 h incubation. The optimal pH value and temperature of the enzyme activity is 6, 0 and 50 ℃ , respectively

关 键 词：里氏木霉 木聚糖酶 毕赤酵母 分泌表达 

分 类 号：TQ91[化学工程] Q786[生物学—分子生物学]