Activation of LacZ gene in Escherichia coli DH5α via α-complementation mechanism for β-galactosidase production and its biochemical characterizations

Background

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).

Results

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.

Conclusion

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.

Loại tài liệu:

Article - Bài báo

Tác giả:

Hamed, Ahmed A.

Đề mục:

Journal of Genetic Engineering and Biotechnology

Nhà xuất bản:

Elsevier

Ngày xuất bản:

December 2020

Số trang/ tờ:

Định dạng:

pdf

Định danh tư liệu:

DOI: https://doi.org/10.1186/s43141-020-00096-w | ISSN 1687-157X

Nguồn gốc:

Journal of Genetic Engineering and Biotechnology, Volume 18, Issue 1, December 2020, 80

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