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Activation of LacZ gene in Escherichia coli DH5α via α-complementation mechanism for β-galactosidase production and its biochemical characterizations
Journal of Genetic Engineering and Biotechnology Pub Date : 2020-12-02 , DOI: 10.1186/s43141-020-00096-w
Ahmed A. Hamed , Mohamed Khedr , Mohamed Abdelraof

Plasmid propagation in recombination strains such as Escherichia coli DH5α is regarded as a beneficial instrument for stable amplification of the DNA materials. Here, we show trans-conjugation of pGEM-T cloning vector (modified Promega PCR product cloning vector with tra genes, transposable element (Tn5)) and M13 sequence via α-complementation mechanism in order to activate β-d-galactosidase gene in DH5α strain (non-lactose-fermenting host). Trans-conjugation with pGEM-T allows correction of LacZ gene deletion through Tn5, and successful trans-conjugants in DH5α host cells can be able to produce active enzyme, thus described as lactose fermenting strain. The intracellular β-galactosidase was subjected to precipitation by ammonium sulfate and subsequently gel filtration, and the purified enzyme showed a molecular weight of approximately 72-kDa sodium dodecyl sulfate-polyacrylamid gel electrophoresis. The purified enzyme activity showed an optimal pH and temperature of 7.5 and 40 °C, respectively; it had a high stability within pH 6–8.5 and moderate thermal stability up to 50 °C. Trans-conjugant of E. coli DH5α- lacZ∆M15 was successfully implemented. UV mutagenesis of the potent trans-conjugant isolate provides an improvement of the enzyme productivity. The enzymatic competitive inhibition by d-galactose and hydrolysis of lactose at ambient temperatures could make this enzyme a promising candidate for use in the dairy industry.

中文翻译:

通过α-互补机制激活大肠杆菌DH5α中LacZ基因以产生β-半乳糖苷酶及其生化特性

质粒在重组菌株如大肠杆菌DH5α中的繁殖被认为是稳定扩增DNA材料的有益手段。在这里,我们展示了pGEM-T克隆载体(具有tra基因,转座因子(Tn5)的改良Promega PCR产物克隆载体)和M13序列通过α互补机制的转导缀合,以激活DH5α中的β-d-半乳糖苷酶基因。菌株(非乳糖发酵宿主)。与pGEM-T的转缀合可通过Tn5纠正LacZ基因的缺失,DH5α宿主细胞中成功的转缀合可产生活性酶,因此被称为乳糖发酵菌株。用硫酸铵使细胞内β-半乳糖苷酶沉淀,然后进行凝胶过滤,纯化的酶的分子量约为十二烷基硫酸钠-聚丙烯酰胺凝胶电泳。纯化的酶活性分别显示最佳pH和7.5的最适温度为40°C。在pH 6–8.5范围内具有很高的稳定性,在高达50°C的温度下具有中等的热稳定性。大肠杆菌DH5α-lacZΔM15的转结合成功实施。有效的反共轭分离物的紫外线诱变可提高酶的生产率。d-半乳糖对酶的竞争性抑制和乳糖在室温下的水解可能使该酶成为奶制品工业中很有希望的候选者。5,在高达50°C的温度下具有中等的热稳定性。大肠杆菌DH5α-lacZΔM15的转结合成功实施。有效的反共轭分离物的紫外线诱变可提高酶的生产率。d-半乳糖对酶的竞争性抑制和乳糖在室温下的水解可能使该酶成为奶制品工业中很有希望的候选者。5,在高达50°C的温度下具有中等的热稳定性。大肠杆菌DH5α-lacZΔM15的转结合成功实施。有效的反共轭分离物的紫外线诱变可提高酶的生产率。d-半乳糖对酶的竞争性抑制以及在室温下乳糖的水解可使该酶成为奶制品行业的有希望的候选者。
更新日期:2020-12-02
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