β-Galactosidase from <i>Lactobacillus helveticus</i> DSM 20075: Biochemical Characterization and Recombinant Expression for Applications in Dairy Industry
oleh: Suwapat Kittibunchakul, Mai-Lan Pham, Anh-Minh Tran, Thu-Ha Nguyen
| Format: | Article |
|---|---|
| Diterbitkan: | MDPI AG 2019-02-01 |
Deskripsi
β-Galactosidase encoding genes <i>lacLM</i> from <i>Lactobacillus helveticus</i> DSM 20075 were cloned and successfully overexpressed in <i>Escherichia coli</i> and <i>Lactobacillus plantarum</i> 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 <i>E. coli</i>, which is more than 1000-fold or 28-fold higher than the production of native β-galactosidase from <i>L. helveticus</i> DSM 20075 when grown on glucose or lactose, respectively. The overexpression in <i>L. plantarum</i> 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 <i>E. coli</i>. The recombinant β-galactosidase from <i>L. helveticus</i> overexpressed in <i>E. coli</i> was purified to apparent homogeneity and subsequently characterized. The <i>K</i><sub>m</sub> and <i>v</i><sub>max</sub> values for lactose and <i>o</i>-nitrophenyl-β-<span style="font-variant: small-caps;">d</span>-galactopyranoside (<i>o</i>NPG) 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 <i>o</i>-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of <i>o</i>NPG with <i>K</i><sub>i,s</sub> = 3.6 ± 0.8 mM. The optimum pH for hydrolysis of both substrates, lactose and <i>o</i>NPG, 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 <i>L. plantarum</i> and purified recombinant enzyme from <i>E. coli</i> revealed high transgalactosylation activity of β-galactosidases from <i>L. helveticus</i>; hence, this enzyme is an interesting candidate for applications in lactose conversion and GOS formation processes.