Capaldo, A.Walker, M.Ford, C.Jiranek, V.2012-04-202012-04-202011Journal of Molecular Catalysis B: Enzymatic, 2011; 69(1-2):27-341381-11771873-3158http://hdl.handle.net/2440/70420In 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. © 2010 Elsevier B.V.en© 2010 Elsevier B.V. All rights reserved.Oenococcus oeniLactic acid bacteriaWinePEP-PTSMalolactic fermentationJournal article002011100110.1016/j.molcatb.2010.12.0060002879815000052-s2.0-7975153389828542Walker, M. [0000-0002-6934-3787]Ford, C. [0000-0003-1617-2977]Jiranek, V. [0000-0002-9775-8963]