您的位置:首页  > 论文页面

绿色木霉产双功能酶的基因克隆、序列分析及表达

发表时间:2009-08-31  浏览量:1500  下载量:693
全部作者: 刘萍,,夏文水,刘靖,高勤学
作者单位: 江南大学食品学院,食品科学与技术国家重点实验室;江苏畜牧兽医职业技术学院
摘 要: 以绿色木霉(Trichoderma viride)RNA为模板,采用反转录-聚合酶链反应(reverse transcription-polymerase chain reaction, RT-PCR)及Smart-RACE扩增的方法获得具有壳聚糖和纤维素降解活性的壳聚糖-纤维素双功能酶(chitosanase-cellulase bifuntional enzyme, CCBE)的全长cDNA序列共2 018 bp. 该酶cDNA序列与DNA序列完全一致,保留有2个内含子。序列比较及同源性分析表明:CCBE基因与糖苷酶7族的CBH Ⅰ基因具有很高的同源性,但与46族、75族、80族的壳聚糖酶无任何序列相似性,与5族、8族细菌类双功能酶也无任何同源性。用不带自身信号肽CCBE基因的cDNA片段(保留有内含子)构建了重组表达载体pPIC-CCBE,重组质粒 Sal Ⅰ线性化后用电转化法导入毕赤酵母(Pichia pastoris)菌株GS115 中,获得高效分泌表达CCBE的毕赤酵母工程菌株。用甲醇诱导培养基进行发酵,表达得到55 ku的目的蛋白。酶活力测定表明:该表达产物具有高的壳聚糖酶活力却无纤维素酶活力,说明CCBE的两种酶功能来自于同一基因中,但具有不同催化结构域,内含子的选择性剪接同CCBE的双功能特性之间具有关联性。
关 键 词: 生物化学与分子生物学;双功能酶;基因克隆;绿色木霉;毕赤酵母
Title: Cloning, sequence analysis and expression of chitosanase-cellulase bifunctional enzyme from Trichoderma viride
Author: LIU Ping, XIA Wenshui, LIU Jing, GAO Qinxue
Organization: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University;Jiangsu Animal Husbandry and Veterinary College
Abstract: The full-length cDNA sequence of chitosanase-cellulase bifunctional enzyme(CCBE) was cloned from Trichoderma viride RNA using reverse transcription-polymerase chain reaction (RT-PCR) and Smart-RACE methods. This cDNA of 2 018 bp was identical with its DNA sequence, which contained two introns. Sequence analysis indicated that CCBE gene had high homology to those of fungal CBHⅠ belonging to family 7 of glycosyl hydrolase, while none similarity with chitosanases of GH-46, GH-75, GH-80, or those bacterial CCBEs of GH-5 and GH-8.The cDNA sequence (intron-retaining) encoding the mature peptide of CCBE was inserted into the Pichia pastoris vector pPIC9K, downstream of α-factor signal peptide sequence. The resulted recombinant plasmid pPIC-CCBE was linearized by SalⅠ digestion and introduced into the P. pastoris GS115 by electro-transformation method. After screening, the recombinant P. pastoris strain was obtained and induced in 25 mL methylotropic culture medium. Expression of the CCBE gene in P. pastoris cells resulted in high chitosanase activity but no cellulase activity of CCBE and the appearance of a recombinant protein of 55 ku detected on SDS-PAGE indicated that the two enzyme activities of CCBE were determined by one single gene and had two distinct catalytic domains. Otherwise, the above results suggested that the fungi producing CCBE gene underwent alternative splicing pattern, and the introns were probably involved in its transcription and post-translation, which would be in relation to its bifunctional activities and isoforms.
Key words: biochemistry and molecular biology; chitosanase-cellulase bifunctional enzyme; gene cloning; Trichoderma viride; Pichia patoris
发表期数: 2009年8月第16期
引用格式: 刘萍,,夏文水,等. 绿色木霉产双功能酶的基因克隆、序列分析及表达[J]. 中国科技论文在线精品论文,2009,2(16):1736-1742.
 
0 评论数 0
暂无评论
友情链接