Burkholderia pickettii中D-乙内酰脲酶基因(dha)的克隆、测序及表达  

Cloning, Sequencing and High Expression in Escherichia coli of D-hydantoinase Gene from Burkholderia pickettii

作　　者：许祯[1] 姜卫红[1] 焦瑞身[1] 杨蕴刘[1] 

机构地区：[1]中国科学院上海植物生理生态研究所,上海200032

出　　处：《生物工程学报》2002年第2期149-154,共6页Chinese Journal of Biotechnology

基　　金：浙江海正药业股份有限公司资助项目~~

摘　　要：对一株能转化D ,L 对羟基苯乙内酰脲为D 对羟基苯甘氨酸的菌株MMR0 0 3进行了细菌分类学鉴定 ,该菌为皮氏伯克霍尔德氏菌 (Burkholderiapickettii)。实验通过Southern杂交 ,部分文库构建和筛选 ,并经一系列亚克隆测序分析 ,获得一长度为 1374bp的完整开放阅读框 ,编码 45 8个氨基酸的D 乙内酰脲酶基因。用该基因序列构建的高表达质粒pXZPH2转化E .coliBL2 1(DE3) ,经IPTG诱导后 ,检测到D 乙内酰脲酶活性。该基因编码的氨基酸序列经Blast同源比较分析与放射形土壤杆菌NRRLB112 91所产相应酶有 85 %的同源性。以D ,L 对羟基苯乙内酰脲为底物测得的表达酶的活力为 0 6 6u mL ,比相同条件下所测出发菌株MMR0 0 3的酶活提高了A strain, MMR003, used for D\|p\|HPG production in industry was classified as Burkholderia pickettii by morphological observation and biochemical characterization. The gene encoding the D\|hydantoinase enzyme was cloned, sequenced and expressed in Escherichia coli. The nucleotide sequence of the 5.0kb insert of subclone pXZ\|total was determined. One open reading frame of 1374bp was found and predicted to encode a polypeptide consisting of 458 amino acids in size of 50kD. The amino acid sequence alignment of D\|hydantoinase from Burkholderia pickettii shows the 85% homologous with the corresponding enzyme from Agrobacterium radiobacter NRRL B11291. The D\|hydantoinase gene ( dha )harboured in the plasmid pXZPH2 in E.coli BL21(DE3) was highly expressed by IPTG induction. The D\|hydantoinase activity for D, L\|p\|hydroxyphenylhydantion is 0.66u/mL broth, which is 2\|fold increase compared to the parent strain Burkholderia pickettii.

关 键 词：皮氏伯克霍尔德氏菌 D-乙内酰脲酶 基因克隆 表达 序列分析 

分 类 号：Q785[生物学—分子生物学]