| 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 |
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| 引用本文： | 于慧敏,马现锋,罗晖,文程,沈忠耀.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[J].微生物学报,2008,24(6):1004-1009. |
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| 作者姓名： | 于慧敏 马现锋 罗晖 文程 沈忠耀 |
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| 作者单位： | 清华大学化工系 生物化工研究所, 北京 100084;清华大学化工系 生物化工研究所, 北京 100084;北京科技大学 生物科学与技术系, 北京 100083;清华大学化工系 生物化工研究所, 北京 100084;清华大学化工系 生物化工研究所, 北京 100084 |
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| 基金项目： | 全国优秀博士学位论文作者专项资金项目(No. 200345)资助。 |
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| 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3.24 u/mL, 比对照菌株BL21(DE3)/pET-DAAO提高了约90%。
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| 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 |
Fusion Expression of D-amino Acid Oxidase from Trignoposis variabilis with Maltose Binding Protein and Vitreoscilla Hemoglobin |
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| Huimin Yu,Xianfeng M,Hui Luo,Cheng Wen and Zhongyao Shen.Fusion Expression of D-amino Acid Oxidase from Trignoposis variabilis with Maltose Binding Protein and Vitreoscilla Hemoglobin[J].Acta Microbiologica Sinica,2008,24(6):1004-1009. |
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| Authors: | Huimin Yu Xianfeng M Hui Luo Cheng Wen and Zhongyao Shen |
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| Institution: | Department of Chemical Engineering, Tsinghua University, Beijing 100084, China;Department of Chemical Engineering, Tsinghua University, Beijing 100084, China;Department of Biological Biological Science and Technology, University of Science and Technology Beijing, Beijing 100083, China;Department of Chemical Engineering, Tsinghua University, Beijing 100084, China;Department of Chemical Engineering, Tsinghua University, Beijing 100084, China |
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| Abstract: | 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. |
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| Keywords: | D-amino acid oxidase (DAAO) maltose binding protein (MBP) Vitreoscilla Hemoglobin (VHb) fusion expression |
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