| 大肠杆菌酸性磷酸酶基因克隆表达及应用 |
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| 引用本文： | 黄莹,丁庆豹,欧伶,魏晓琨,许彦梅,张春艳,王玥.大肠杆菌酸性磷酸酶基因克隆表达及应用[J].生物技术通报,2012(5):110-115. |
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| 作者姓名： | 黄莹 丁庆豹 欧伶 魏晓琨 许彦梅 张春艳 王玥 |
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| 作者单位： | 1. 华东理工大学生物反应器工程国家重点实验室,上海,200237
2. 上海斯贝生物科技有限公司,上海,200237 |
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| 摘 要： | 将大肠杆菌K-12的酸性磷酸酶(AphA)完整基因和去信号肽基因分别克隆到pET-28a(+)栽体上,并转化入大肠杆菌BL21( DE3)中.经诱导检测,重组菌均能表达出高活性的可溶性酶蛋白,去信号肽表达更稳定.对重组菌的活性研究表明,相对于野生菌,重组菌酶活力得到大幅度提高,同时,以pNPP、肌苷为底物进行磷酸转移催化反应,在pH4.0-6.0、反应温度37℃条件下,约有30％的肌苷可转化为IMP,但随着反应的进行所形成的IMP又被该酶降解,向反应液中加入EDTA即可明显抑制酶的水解活性,减缓IMP的降解速率.
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| 关 键 词： | 大肠杆菌 酸性磷酸酶 IMP |
Cloning, Expression and Application of Acid Phosphatase from Escherichia coli K-12 |
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| Huang Ying
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Ding Qingbao
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Ou Ling
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Wei Xiaokun
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Xu Yanmei
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Zhang Chunyan
,
Wang Yue.Cloning, Expression and Application of Acid Phosphatase from Escherichia coli K-12[J].Biotechnology Bulletin,2012(5):110-115. |
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| Authors: | Huang Ying Ding Qingbao Ou Ling Wei Xiaokun Xu Yanmei Zhang Chunyan Wang Yue |
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| Institution: | Yue2(1State Key Laboratory of Bioreactor Engineering,East China University of Technology,Shanghai 200237; 2Shanghai SCIBEST Technology Co.Ltd,Shanghai 200237) |
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| Abstract: | Gene(AphA)from E.coli K-12,which had full-length sequence or part-length sequence that initialization segment encoding signal peptide was cut off,was cloned into plasmid pET-28a(+)and expressed in E.coli BL21(DE3)individually.The two kinds of recombinant strain can express soluble enzyme protein under the induction of IPTG,but the aimed protein amount in pET-28a-dAphA was stable than that in pET-28a-AphA through identification by SDS-PAGE and Zymogram detection.The recombinant strain both had higher phosphorylysis activity than original strain.Meantime,30% inosinic acid could be formed from pNPP and inosine at pH4.0-6.0 and 37℃,howe-ver,the product was soon degraded.EDTA can inhibit the hydrolytic activity of the enzyme,thus the product IMP can remain a period of time without degradation. |
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| Keywords: | Escherichia coli Acid phosphatase IMP |
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