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Open AccessArticle

β-Galactosidase from Lactobacillus helveticus DSM 20075: Biochemical Characterization and Recombinant Expression for Applications in Dairy Industry

1
Food Biotechnology Laboratory, BOKU University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria
2
Department of Biology, Faculty of Fundamental Sciences, Ho Chi Minh City University of Medicine and Pharmacy, 217 Hong Bang, Ho Chi Minh City, Vietnam
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(4), 947; https://doi.org/10.3390/ijms20040947
Received: 23 January 2019 / Revised: 15 February 2019 / Accepted: 19 February 2019 / Published: 22 February 2019
(This article belongs to the Special Issue Industrial Enzymes: Structure, Function and Applications)
β-Galactosidase encoding genes lacLM from Lactobacillus helveticus DSM 20075 were cloned and successfully overexpressed in Escherichia coli and Lactobacillus plantarum using different expression systems. The highest recombinant β-galactosidase activity of ∼26 kU per L of medium was obtained when using an expression system based on the T7 RNA polymerase promoter in E. coli, which is more than 1000-fold or 28-fold higher than the production of native β-galactosidase from L. helveticus DSM 20075 when grown on glucose or lactose, respectively. The overexpression in L. plantarum using lactobacillal food-grade gene expression system resulted in ∼2.3 kU per L of medium, which is approximately 10-fold lower compared to the expression in E. coli. The recombinant β-galactosidase from L. helveticus overexpressed in E. coli was purified to apparent homogeneity and subsequently characterized. The Km and vmax values for lactose and o-nitrophenyl-β-d-galactopyranoside (oNPG) were 15.7 ± 1.3 mM, 11.1 ± 0.2 µmol D-glucose released per min per mg protein, and 1.4 ± 0.3 mM, 476 ± 66 µmol o-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of oNPG with Ki,s = 3.6 ± 0.8 mM. The optimum pH for hydrolysis of both substrates, lactose and oNPG, is pH 6.5 and optimum temperatures for these reactions are 60 and 55 °C, respectively. The formation of galacto-oligosaccharides (GOS) in discontinuous mode using both crude recombinant enzyme from L. plantarum and purified recombinant enzyme from E. coli revealed high transgalactosylation activity of β-galactosidases from L. helveticus; hence, this enzyme is an interesting candidate for applications in lactose conversion and GOS formation processes. View Full-Text
Keywords: Lactobacillus helveticus; β-galactosidase; recombinant enzyme; expression systems Lactobacillus helveticus; β-galactosidase; recombinant enzyme; expression systems
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Kittibunchakul, S.; Pham, M.-L.; Tran, A.-M.; Nguyen, T.-H. β-Galactosidase from Lactobacillus helveticus DSM 20075: Biochemical Characterization and Recombinant Expression for Applications in Dairy Industry. Int. J. Mol. Sci. 2019, 20, 947.

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