长梗木霉外切纤维素酶CBH Ⅱ基因的克隆及表达  

Cloning and Expression of Cellobiohydrolase Ⅱ Gene from Trichoderma Longibrachiatum

作　　者：王娟[1] 刘海英[1] 朱亚然[1] 舒正玉[1] 吴松刚[1] 黄建忠[1] 

机构地区：[1]福建师范大学工业微生物教育部工程研究中心生命科学学院福建省现代发酵技术工程研究中心,福建福州350108

出　　处：《药物生物技术》2009年第2期95-98,共4页Pharmaceutical Biotechnology

基　　金：福建省科技平台(2005Q007)

摘　　要：分别以长梗木霉(Trichoderma longibrachiatum)XST1的基因组DNA和cDNA为模板PCR扩增其外切纤维素酶Ⅱ(cellobiohydrolaseⅡ,CBHⅡ)的全长基因cbhⅡ。序列分析表明:cbhⅡ基因全长1583bp,含3个内含子,分别为94～144bp,529～584bp和832～894bp。该基因编码的外切纤维素酶Ⅱ蛋白全长470个氨基酸(其中前24个氨基酸为信号肽)。将全长的cbhⅡ基因连接到pPIC3.5K上,转化毕赤酵母GS115(Pichia pastoris GS115)。重组转化子96h诱导培养,发酵上清液水解pNPC的酶活为18.1U/L。SDS-PAGE检测结果表明:浓缩的重组转化子发酵液较对照菌株的发酵液中,有一明显加强的蛋白质条带,Mr约为67k。The cellobiohydrolase Ⅱ gene （cbh Ⅱ ） of Trichoderrna longibrachiatum XST1 was amplified by PCR with genomic DNA and cDNA as template respectively. Nucleotide sequencing revealed that the cbh Ⅱ has an ORF of 1583 bp, containing three introns （94 - 144, 529 - 584 and 832 - 894 bases）. It encodes a 470-amino acid protein with a calculated molecular mass of approximately 50 kD. The cbh Ⅱ cDNA gene was inserted into the plasmid pPIC3.5K, linearized by Bgl Ⅱ restriction enzyme and then transformed into Pichia pastoris GS115 by electroporation. After being induced with 1% methanol, the recombinant reached its highest cellobiohydrolase activity of 18. 1 U/L with pNPC as substrate at 96 hours. The SDS-PAGE analysis showed that the relative molecular mass of the recombinant cellobiohydrolase Ⅱ was 67 kD. The result demonstrated taht the recombinant yeast can successfully secrete the cellobiohydrolase Ⅱ in an active form, laying a good foundation for its further research and industry production.

关 键 词：长梗木霉 外切纤维素酶 毕赤酵母 表达 

分 类 号：Q78[生物学—分子生物学]