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目的构建大肠杆菌色氨酸操纵子基因突变株,提高邻氨基苯甲酸合成酶和色氨酸合成酶的产量。方法利用依赖于DpnⅠ酶的PCR方法突变表达载体pET-22b(+)-Trp Operon上的关键位点,PCR扩增Trp OperonM基因,构建pET-22b(+)-Trp OperonM重组表达质粒,经酶切及测序鉴定正确后,转化大肠杆菌BL21(DE3),IPTG诱导表达。制备粗酶液,经比色法测定邻氨基苯甲酸合成酶和色氨酸合成酶的活性。结果 PCR扩增产物可见约7000bp的Trp OperonM条带;所构建的重组表达质粒经酶切及测序鉴定正确;邻氨基苯甲酸合成酶和色氨酸合成酶的活性比大肠杆菌BL21(DE3)空菌分别提高了4.5倍和5.2倍。结论已成功构建了大肠杆菌色氨酸操纵子基因突变株BL21(DE3)/pET-22b(+)-Trp OperonM,邻氨基苯甲酸合成酶和色氨酸合成酶的活性均有提高,为高产色氨酸基因工程菌的构建奠定了基础。
Objective To construct the tryptophan operon gene mutant of Escherichia coli and improve the yield of anthranilate synthase and tryptophan synthase. Methods The key sites of pET-22b (+) - Trp Operon were mutated by DpnⅠ-dependent PCR and the Trp OperonM gene was amplified by PCR. The recombinant plasmid pET-22b (+) - Trp OperonM was constructed. After sequencing and sequencing, the recombinant plasmid was transformed into E.coli BL21 (DE3) and induced by IPTG. The crude enzyme solution was prepared and the activities of anthranilate synthase and tryptophan synthase were determined by colorimetry. Results The PCR product was about 7000bp in Trp OperonM band. The constructed recombinant plasmid was confirmed by restriction enzyme digestion and sequencing. The activity of anthranilate synthase and tryptophan synthase was higher than that of E. coli BL21 (DE3) The bacteria increased by 4.5 times and 5.2 times respectively. Conclusion The E. coli tryptophan operon gene BL21 (DE3) / pET-22b (+) - Trp OperonM has been successfully constructed. The activities of anthranilate synthase and tryptophan synthase are all improved. Tryptophan gene engineering bacteria laid the foundation for the construction.