New alkali tolerant ß-galactosidase from Paracoccus marcusii KGP - A promising biocatalyst for the synthesis of oligosaccharides derived from lactulose (OsLu), the new generation prebiotics.

ABSTRACT

The enzyme ß-galactosidase can synthesise novel prebiotics such as oligosaccharides derived from lactulose (OsLu) which can be added as a supplement in infant food formula. In this study, the intracellular ß-galactosidase produced by the alkaliphilic bacterium Paracoccus marcusii was extracted and purified to homogeneity using hydrophobic and metal affinity chromatography. The purification resulted in 18 U/mg specific activity, with a yield of 8.86% and an 18-fold increase in purity. The purified enzyme was a monomer with an 86 kDa molecular weight as determined by SDS PAGE and Q-TOF-LC/MS. ß-Galactosidase was highly active at 50 °C and pH 6-8. The enzyme displayed an alkali tolerant nature by maintaining more than 90% of its initial activity over a pH range of 5-9 after 3 h of incubation. Furthermore, the enzyme activity was enhanced by 37% in the presence of 5 M NaCl and 3 M KCl, indicating its halophilic nature. The effects of metal ions, solvents, and other chemicals on enzyme activity were also studied. The kinetic parameters KM and Vmax of ß-galactosidase were 1 mM and 8.56 µmoles/ml/min and 72.72 mM and 11.81 µmoles/ml/min on using oNPG and lactose as substrates. P. marcusii ß-galactosidase efficiently catalysed the transgalactosylation reaction and synthesised 57 g/L OsLu from 300 g/L lactulose at 40 °C. Thus, in this study we identified a new ß-galactosidase from P. marcusii that can be used for the industrial production of prebiotic oligosaccharides.