Study of transgalactosylation mechanisms of lactase-phlorizin hydrolase from pig small intestine

Abstract

[Introduction]: Lactulose is a synthetic sugar mainly used in medicine for the treatment of portal systemic encephalopathy and chronic constipation and it is one of the most widely used prebiotic in the pharmaceutical industry. On the other hand, the high physiological and anatomical similarity of pig and human digestive tracts makes the use of brush border membrane vesicles (BBMV) of the pig small intestine an ideal model for gathering information about transgalactosylation mechanisms of disaccharidases from pig small intestines and in consequence, the potential hydrolysis mechanism of new synthesized carbohydrates. The aim of this work has been to study the hydrolysis and transglycosylation of lactulose by lactase-phlorizin hydrolase from brush border membrane vesicles of pig small intestinal and compare the results with those obtained for lactose.

[Methods]: The synthesis was carried out using lactulose (250 g/L) in phosphate buffer saline (PBS) at pH 7.3 and lyophilized pig small intestinal BBMV (180 g/L) at 37 ºC in an orbital shaker at 900 rpm during 6h. The synthesis was monitored by Gas-Chromatography coupled to a flame ionization detector (GC-FID). To accomplish a comprehensive structural characterization of the main synthesized oligosaccharides, a selective fractionation of mono- di- and trisaccharides, was achieved by activated charcoal treatment using 8% v/v and 50% v/v of ethanol, respectively. Subsequently, the trisaccharide mixture was fractionated by semi preparative High-Performance Liquid Chromatography with refractive index detector (HPLC-RI). The structure of the purified trisaccharide fraction was elucidated by Nuclear Magnetic Resonance spectroscopy.

[Results]: The pig small intestinal BBMV presented low transgalactosylation activity when lactulose acted as donor and acceptor, giving rise to the formation of small amounts of oligosaccharides with different degree of polymerization, mainly di- and trisaccharides, with percentage of 4.5% and 3.1%, respectively. The analysis by Nuclear Magnetic Resonance spectroscopy delivered significant results, showing that major trisaccharide was β-D-Galactopyranosyl – (1-3) -β-D-Galactopyranosyl- (1-4) -β-D-Fructopyranoside (3’-galactosyl-lactulose), which was produced by the transfer of a galactose unit to the C-3 of the non-reducing moiety of lactulose. These findings are in line with those previously obtained using lactose as substrate, since major trisaccharide was produced by the transfer of a galactose unit to the C-3 of the non-reducing moiety of lactose.

[Discussion]: These results reaffirm that enzyme from pig small intestinal BBMV preferably synthesizes oligosaccharides linked by β-(1→3) bonds and could be crucial for future studies investigating the structure-small intestinal digestibility relationship of a great variety of available prebiotics, as well as for designing tailored fully non-digestible GOS.