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.
出处
《药物生物技术》
CAS
CSCD
2009年第2期95-98,共4页
Pharmaceutical Biotechnology
基金
福建省科技平台(2005Q007)
关键词
长梗木霉
外切纤维素酶
毕赤酵母
表达
Trichoderma longibrachiatum, Cellobiohydrolase, Pichia pastoris, Expression
作者简介
王娟，1981年生，女，汉族，河北秦皇岛人，硕士研究生，主要研究方向为微生物基因工程，E-mail：Wangjuanpop@yahoo．com．cn．Tel：13774591719。
通讯作者：黄建忠；教授，E-mail：hjz@fjnu．edu．cn。
