D-氨基酸氧化酶与麦芽糖结合蛋白和透明颤菌血红蛋白的融合表达  

Fusion Expression of D-amino Acid Oxidase from Trignoposis variabilis with Maltose Binding Protein and Vitreoscilla Hemoglobin

作　　者：于慧敏[1] 马现锋[1] 罗晖[2] 文程[1] 沈忠耀[1] 

机构地区：[1]清华大学化工系生物化工研究所,北京100084 [2]北京科技大学生物科学与技术系,北京100083

出　　处：《生物工程学报》2008年第6期1004-1009,共6页Chinese Journal of Biotechnology

基　　金：全国优秀博士学位论文作者专项资金项目(No.200345)资助~~

摘　　要：D-氨基酸氧化酶(DAAO)是一种重要的工业酶。为了进一步提高DAAO在大肠杆菌中的可溶性和活性表达,分别构建了麦芽糖结合蛋白(MBP)和透明颤菌血红蛋白与三角酵母DAAO(TvDAAO)的N-端融合蛋白。其中,MBP融合蛋白MBP-TvDAAO在组成型(JM105/pMKC-DAAO)和诱导型菌株(JM105/pMKL-DAAO)中表达时,目标蛋白的可溶性表达量分别达到全细胞蛋白表达量的28%以上和17%左右,比无MBP融合的对照菌株BL21(DE3)/pET-DAAO分别提高3.7和1.8倍;但其酶活水平显著下降。VHb融合蛋白VHb-TvDAAO在重组菌BL21(DE3)/pET-VDAAO中摇瓶诱导表达时,DAAO酶活达到了3.24u/mL,比对照菌株BL21(DE3)/pET-DAAO提高了约90%。D-amino acid oxidase （DAAO） is one of important industrial enzymes. To increase the solubility and activity of the TvDAAO from Trignoposis variabilis expressed in recombinant Escherichia coli （E. coli）, a maltose binding protein （MBP） and Vitreoscilla hemoglobin （VHb） was introduced to fuse with N-terminal of the TvDAAO, respectively. Fusion protein of MBP-TvDAAO was constitutively expressed in JM105/pMKC-DAAO and inductively expressed in JM105/pMKL-DAAO. With respect to the control strain of BL21 （DE3）/pET-DAAO without MBP fusion, the constitutive fusion expression obtained 28% of soluble protein with 3.7 folds of solubility improvement. As for the inductive fusion expression, corresponding results changed to 17% and 1.8 folds, respectively. However, the DAAO activity significantly decreased in the MBP-fusing expression. Fusion protein of VHb-TvDAAO was constructed and inductively expressed in BL21 （DE3）/pET-VDAAO. Its DAAO activity highly reached 3.24 u/mL in flask culture, about 90% increase in contrast to the control without VHb.

关 键 词：D-氨基酸氧化酶 大肠杆菌麦芽糖结合蛋白 透明颤菌血红蛋白 融合表达 

分 类 号：Q78[生物学—分子生物学] TQ465.1[化学工程—制药化工]