产紫青霉β-葡萄糖醛酸苷酶基因的克隆与原核表达  

Cloning and prokaryotic expression of β-glucuronidase from Penicillium purpurogenum Li-3

作　　者：宋占科[1] 王小艳[2] 陈国强[1] 李春[1,2] 

机构地区：[1]北京理工大学生命科学与技术学院,北京100081 [2]石河子大学化学化工学院,新疆石河子832003

出　　处：《化工学报》2008年第12期3101-3106,共6页CIESC Journal

基　　金：国家自然科学基金项目(20776017);北京市自然科学基金项目(5072028);北京理工大学基础研究基金项目(20060642004)~~

摘　　要：产紫青霉(Penicillium purpurogenum Li-3)β-葡萄糖醛酸苷酶可定向转化甘草酸生成单葡萄糖醛酸基甘草次酸。根据β-葡萄糖醛酸苷酶氨基酸序列的同源保守区设计简并引物,PCR扩增克隆得到β-葡萄糖醛酸苷酶编码基因pgus(GenBank登录号:EU095019),该基因全长1815bp,编码604个氨基酸,理论单亚基分子量为67.77×103,含有4个潜在的N-糖基化位点。构建原核表达载体pET-28a(+)-pgus,转化大肠杆菌BL21(DE3),获得高效表达pgus基因的重组菌。经IPTG诱导表达、Ni2+-NTA亲和层析柱纯化的重组酶PGUS-E,纯度达到95%以上,得率为25.6mg·L-1,SDS-PAGE分析检测分子量约为70×103,比酶活达到6368U·mg-1。One new gene of β-glucuronidase （EC 3.2.1.31） which could biosynthesize glycyrrhetinic acid monoglucuronide （GAMG） from glycyrrhizin, was cloned from Penicillium purpurogenum Li-3 by degenerated PCR using primers designed from the conserved amino acid sequences. It is the first time that a β-glucuronidase gene （pgus） （GenBank Accession No. EU095019） was cloned from Penicillium species other than Penicillium canescens. Sequence analysis indicated that the gene of pgus has 1815 base pairs, encoding 604 amino acids with the putative potential molecular weight of 66.7 × 10^3 and 4 potential Nglycosylation sites. The prokaryotic expression system was constructed with pET-28a （＋）, as the vector. The fusion protein （PGUS-E） with activity was overexpressed in E. coli BL21 （DE3） after IPTG induction. The specific activity of enzyme, purified with Ni^2- -NTA column to homogeneity （accounting for 95% of soluble protein）, was up to 6368 U · mg^-1 . The production of soluble PGUS-E was about 25.6 mg · L^-1 culture medium. The molecular weight of sub-unit was estimated to be about 70 × 10a by SDS-PAGE.

关 键 词：甘草酸 Β-葡萄糖醛酸苷酶 克隆 产紫青霉 

分 类 号：Q78[生物学—分子生物学] TQ925[轻工技术与工程—发酵工程]