β-Glucoside metabolism in Oenococcus oeni: Cloning and characterization of the phospho-β-glucosidase CelD

Abstract

. Uptake and phosphorylation of β-glucosides via the PEP-PTS, allowing the cell to utilize alternative carbon sources and break down glycosylated aroma compounds found in wine. These compounds are not volatile in their glycosylated form, however are rendered perceivable once deglycosylated. ▶ celD characterized as a glycosyl hydrolase family 1 phospho-β-glucosidase. ▶ celD in conjunction with the PEP-PTS most likely responsible for β-glucosidase metabolism in whole cell Oenococcus oeni , an oenologically important lactic acid bacterium. ▶ The hydrolysis of β-glucosides in wine affects the aroma profile of a wine by liberating non volatile aroma compounds in their glycosylated form, but are rendered perceivable once deglycosylated. ▶ CelD has an optimum pH between 4.0 and 5.0 and is most active at 40 °C. In previous work, we reported characterization of a phospho-β-glucosidase gene bglD in a β-glucoside metabolizing operon in the oenologically important lactic acid bacterium Oenococcus oeni . Here we report a second phospho-β-glucosidase gene celD which has been cloned and expressed in Escherichia coli . This gene is found in a putative operon 6043 bp long encoding six genes designated celA to celF . Comparative sequence analyses of lactic acid bacteria suggest that the open reading frames of celA , B and F from the sequenced O. oeni PSU-1 encode phosphoenolpyruvate dependent phosphotransferase system (PEP-PTS) components IIB, IIA and IIC, respectively, which regulate the uptake and phosphorylation of β-glucosides across the cytoplasmic membrane. celE is speculated to have a regulatory function. celD was cloned and expressed in E. coli followed by purification of the gene product. The purified protein His-tagged CelD (485 residues, Mw = 55.8 kDa) has high homology to known phospho-β-glucosidases and has high activity towards the phosphorylated β-glucoside para-nitrophenol-β- d -glucopyranoside-6-phophate (pNPβG6P). CelD has an optimum pH between 4.0 and 5.0 and is most active at 40 °C. The gene celC was cloned, heterologously expressed and purified (481 residues, Mw = 55.7 kDa) but showed no significant activity towards pNPβG6P despite high sequence homology to celD and characterized phospho-β-glucosidases. Neither CelC nor CelD are active against non-phosphorylated β-glucosides.