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PMID:22449746
| Citation |
Honda, H, Nagaoka, S, Kawai, Y, Kemperman, R, Kok, J, Yamazaki, Y, Tateno, Y, Kitazawa, H and Saito, T (2012) Purification and characterization of two phospho-β-galactosidases, LacG1 and LacG2, from Lactobacillus gasseri ATCC33323(T). J. Gen. Appl. Microbiol. 58:11-7 |
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| 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. |
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| Keywords |
Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/isolation & purification; Base Sequence; Catalytic Domain; Cluster Analysis; Culture Media/chemistry; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme Assays; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Glycoside Hydrolases/chemistry; Glycoside Hydrolases/classification; Glycoside Hydrolases/genetics; Glycoside Hydrolases/isolation & purification; Kinetics; Lactobacillus/enzymology; Lactobacillus/genetics; Lactose/chemistry; Nitrophenylgalactosides/chemical synthesis; Nitrophenylgalactosides/chemistry; Phylogeny; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Substrate Specificity |
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Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0033920: 6-phospho-beta-galactosidase activity |
ECO:0000314: direct assay evidence used in manual assertion |
F |
Table 3. |
complete | ||||
| GO:0033920: 6-phospho-beta-galactosidase activity |
ECO:0000314: direct assay evidence used in manual assertion |
F |
Table 3. |
complete | ||||
See also
References
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