Functional expression of human liver cytosolic beta-glucosidase in Pichia pastoris. Insights into its role in the metabolism of dietary glucosides.
Human tissues such as liver, small intestine, spleen and kidney contain a cytosolic beta-glucosidase (CBG) that hydrolyses various beta-d-glycosides, but whose physiological function is not known. Here, we describe the first heterologous expression of human CBG, a system that facilitated a detailed assessment of the enzyme specificity towards dietary glycosides. A full-length CBG cDNA (cbg-1) was cloned from a human liver cDNA library and expressed in the methylotrophic yeast Pichia pastoris at a secretion yield of approximately 10 mg x L-1. The recombinant CBG (reCBG) was purified from the supernatant using a single chromatography step and was shown to be similar to the native enzyme isolated from human liver in terms of physical properties and specific activity towards 4-nitrophenyl-beta-D-glucoside. Furthermore, the reCBG displayed a broad specificity with respect to the glycone moiety of various aryl-glycosides (beta-D-fucosides, alpha-L-arabinosides, beta-D-glucosides, beta-D-galactosides, beta-L-xylosides, beta-D-arabinosides), similar to the native enzyme. For the first time, we show that the human enzyme has significant activity towards many common dietary xenobiotics including glycosides of phytoestrogens, flavonoids, simple phenolics and cyanogens with higher apparent affinities (K(m)) and specificities (k(cat)/K(m)) for dietary xenobiotics than for other aryl-glycosides. These data indicate that human CBG hydrolyses a broad range of dietary glucosides and may play a critical role in xenobiotic metabolism.
Berrin
Jean-Guy
JG
Nutrition, Health and Consumer Sciences Division, Institute of Food Research, Norwich, UK.
McLauchlan
W Russell
WR
Needs
Paul
P
Williamson
Gary
G
Puigserver
Antoine
A
Kroon
Paul A
PA
Juge
Nathalie
N
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology*; D005960:Glucosides / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000502:physiology*
D005960:Glucosides; D011994:Recombinant Proteins; D001617:beta-Glucosidase