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Activation of LacZ gene in Escherichia coli DH5? via ?-complementation mechanism for ?-galactosidase production and its biochemical characterizations.


ABSTRACT: Background: Plasmid propagation in recombination strains such as Escherichia coli DH5? 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 ?-complementation mechanism in order to activate ?-D-galactosidase gene in DH5? strain (non-lactose-fermenting host).

Results: Trans-conjugation with pGEM-T allows correction of LacZ gene deletion through Tn5, and successful trans-conjugants in DH5? host cells can be able to produce active enzyme, thus described as lactose fermenting strain. The intracellular ?-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 DH5?- 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.

SUBMITTER: Hamed AA 

PROVIDER: S-EPMC7710787 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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

Hamed Ahmed A AA   Khedr Mohamed M   Abdelraof Mohamed M  

Journal, genetic engineering & biotechnology 20201202 1


<h4>Background</h4>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).<h4>Results</h4>Trans-conjugation wi  ...[more]

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