title: Purification and characterization of two phospho-β-galactosidases, LacG1 and LacG2, from Lactobacillus gasseri ATCC33323(T).
abstract: Lactobacillus gasseri ATCC33323(T) expresses four enzymes showing phospho-β-galactosidase activity (LacG1, LacG2, Pbg1 and Pbg2). We previously reported the purification and characterization of two phospho-β-galactosidases (Pbg1 and Pbg2) from Lactobacillus gasseri JCM1031 cultured in lactose medium. Here we aimed to characterize LacG1 and LacG2, and classify the four enzymes into 'phospho-β-galactosidase' or 'phospho-β-glucosidase.' LacG1 and recombinant LacG2 (rLacG2), from Lb. gasseri ATCC33323(T), were purified to homogeneity using column chromatography. Kinetic experiments were performed using sugar substrates, o-nitrophenyl-β-D-galactopyranoside 6-phosphate (ONPGal-6P) and o-nitrophenyl-β-D-glucopyranoside 6-phosphate (ONPGlc-6P), synthesized in our laboratory. LacG1 and rLacG2 exhibited high k(cat)/K(m) values for ONPGal-6P as compared with Pbg1 and Pbg2. The V(max) values for ONPGal-6P were higher than phospho-β-galactosidases previously purified and characterized from several lactic acid bacteria. A phylogenetic tree analysis showed that LacG1 and LacG2 belong to the phospho-β-galactosidase cluster and Pbg1 and Pbg2 belong to the phospho-β-glucosidase cluster. Our data suggest two phospho-β-galactosidase, LacG1 and LacG2, are the primary enzymes for lactose utilization in Lb. gasseri ATCC33323(T). We propose a reclassification of Pbg1 and Pbg2 as phospho-β-glucosidase.
authors.affiliation: Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Honda
authors.forename: Seiji
authors.initials: S
authors.lastname: Nagaoka
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kawai
authors.forename: Robèr
authors.initials: R
authors.lastname: Kemperman
authors.forename: Jan
authors.initials: J
authors.lastname: Kok
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Yamazaki
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Tateno
authors.forename: Haruki
authors.initials: H
authors.lastname: Kitazawa
authors.forename: Tadao
authors.initials: T
authors.lastname: Saito
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D020134:Catalytic Domain; D016000:Cluster Analysis; D003470:Culture Media / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D057075:Enzyme Assays; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D007785:Lactose / Q000737:chemistry; D009598:Nitrophenylgalactosides / Q000138:chemical synthesis / Q000737:chemistry; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D011994:Recombinant Proteins; C055012:2-nitrophenylgalactoside; D009598:Nitrophenylgalactosides; D006026:Glycoside Hydrolases; C022361:6-phospho-beta-galactosidase; D007785:Lactose