Abstract
BackgroundPlasmid 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). ResultsTrans-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. ConclusionTrans-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.
Highlights
Plasmid propagation in recombination strains such as Escherichia coli DH5α is regarded as a beneficial instrument for stable amplification of the DNA materials
Bacterial trans-conjugation and screening for βgalactosidase activity One bacterial strain E. coli LK111 harbors genetically engineered pGEM-T (Promega PCR product cloning vector) with tra genes, and transposable element (Tn5) was used to perform bacterial trans-conjugation with E. coli DH5α producing an inactive form of β-galactosidase enzyme due to LacZ mutation
After bacterial transconjugation process was conducted, all isolates were tested for their ability to produce LacZ activity according to α-complementation mechanism
Summary
Plasmid propagation in recombination strains such as Escherichia coli DH5α is regarded as a beneficial instrument for stable amplification of the DNA materials. People with lactose intolerance are unable to make enough lactase enzymes which in turn causes the inability to consume dairy products [1–4]. The β-galactosidase enzyme has a broad utilization in food-processing industries such as hydrolysis of lactose. Many sources such as plants, animal cells, and microorganisms have been investigated for their ability to produce valuable metabolites [10–12]. One of them is the inhibition of βgalactosidase caused by the hydrolysis-formed product D-galactose which was regarded as a big barrier to its utilization in the industrial sector [2, 16]. It is of great economic interest to explore a new source to generate β-galactosidase with improved processing characteristics for their utilization in dairy industries
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