文献类型：期刊文献

中文题名：大肠杆菌酸性磷酸酶基因克隆表达及应用

英文题名：Cloning,Expression and Application of Acid Phosphatase from Escherichia coli K-12

作者：黄莹[1];丁庆豹[1];欧伶[1];魏晓琨[2];许彦梅[2];张春艳[2];王玥[2]

机构：[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

年份：2012

卷号：28

期号：5

起止页码：110

中文期刊名：生物技术通报

外文期刊名：Biotechnology Bulletin

收录：CSTPCD;;北大核心:【北大核心2011】；CSCD:【CSCD_E2011_2012】；

语种：中文

中文关键词：大肠杆菌;酸性磷酸酶;IMP

外文关键词：Escherichia coli Acid phosphatase IMP

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(+)载体上,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。
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.