【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。更多还原

【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.更多还原