title: Overexpression and characterization of a novel transgalactosylic and hydrolytic β-galactosidase from a human isolate Bifidobacterium breve B24.
abstract: After the complete gene of a β-galactosidase from human isolate Bifidobacterium breve B24 was isolated by PCR and overexpressed in E. coli, the recombinant β-galactosidase was purified to homogeneity and characterized for the glycoside transferase (GT) and glycoside hydrolase (GH) activities on lactose. One complete ORF encoding 691 amino acids (2,076 bp) was the structural gene, LacA (galA) of the β-gal gene. The recombinant enzyme shown by activity staining and gel-filtration chromatography was composed of a homodimer of 75 kDa with a total molecular mass of 150 kDa. The K(m) value for lactose (95.58 mM) was 52.5-fold higher than the corresponding K(m) values for the synthetic substrate ONPG (1.82 mM). This enzyme with the optimum of pH 7.0 and 45°C could synthesize approximately 42.00% of GOS from 1M of lactose. About 97.00% of lactose in milk was also quickly hydrolyzed by this enzyme (50 units) at 45°C for 5h to produce 46.30% of glucose, 46.60% of galactose and 7.10% of GOS. The results suggest that this recombinant β-galactosidase derived from a human isolate B. breve B24 may be suitable for both the hydrolysis and synthesis of galacto-oligosaccharides (GOS) in milk and lactose processing.
authors: Department of Food Science, McGill University, Montreal, QC, Canada H3A 2B4.
authors: Sung Hun
authors: SH
authors: Yi
authors: Inteaz
authors: I
authors: Alli
authors: Kwan Hwa
authors: KH
authors: Park
authors: Byonghoon
authors: B
authors: Lee
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D001644:Bifidobacterium / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005525:Food-Processing Industry; D005798:Genes, Bacterial / Q000502:physiology; D006801:Humans; D007785:Lactose / Q000737:chemistry / Q000378:metabolism; D009598:Nitrophenylgalactosides / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; C055012:2-nitrophenylgalactoside; D009598:Nitrophenylgalactosides; D001616:beta-Galactosidase; D007785:Lactose