宇佐美曲霉木聚糖酶(xynⅡ)基因在大肠杆菌中的表达  

Expression of Aspergillus usamii Xylanase (xyn Ⅱ) Gene in Escherichia coli

作　　者：周晨妍[1] 白剑宇[1] 邬敏辰[2] 王武[1] 

机构地区：[1]江南大学生物工程学院,无锡214064 [2]江南大学医学院,无锡214064

出　　处：《农业生物技术学报》2007年第3期514-518,共5页Journal of Agricultural Biotechnology

基　　金：江南大学校级科研项目(No.210000-52210569)资助

摘　　要：根据宇佐美曲霉(Aspergill ususamii)E001菌株木聚糖酶xynⅡcDNA序列(GenBank登录号为DQ114485),合成1对引物(含EcoRⅠ和SalⅠ酶切位点),以重组质粒pUCm-T-xynⅡ为模板,扩增得到编码xynⅡ成熟肽的cDNA片段(555bp)。将其与表达质粒pET-28a连接,构建重组表达质粒pET-28a-xynⅡ,并转化大肠杆菌(Escherichia.coli)BL21-CodonPlus(DE3)-RIL,获得重组工程菌BL21xynⅡ。经IPTG诱导表达,木聚糖酶的比酶活力最高可达35.6Umg。最后采用金属Ni2+螯和层析柱对所表达的木聚糖酶进行纯化,获得电泳纯的木聚糖酶。重组表达的木聚糖酶最适温度为45℃,最适pH4.6。A pair of primers （with the EcoR Ⅰ and Sal Ⅰ sites） were designed based on the xyn lit cDNA sequence （GenBank accession number DQ114485）.With the recombinant plasmid pUCm-T-xyn lit as a template,xyn lit cDNA fragment （555 bp） encoding mature peptide was amplified and inserted into the plasmid pET-28a.The resultant recombinant plasmid pET-28a-xyn Ⅱ was transformed into Escherichia coli BL21- CodonPlus （DE3）-RIL,and finally the recombinant strain BL21/xyn Ⅱ was obtained. A maximum activity of 35.6 U/mg was gotten from cellular extract of E. coli B21/xyn Ⅱ induced by IPTG.The recombinant protein was purified by immobilizing metal affinity chromatography.The optimum temperature and pH for recombinant enzyme were 45 ℃ and 4.6,respectively.

关 键 词：宇佐美曲霉 木聚糖酶 表达 酶学性质 

分 类 号：S188[农业科学—农业基础科学]