title: Identification and characterization of a novel β-galactosidase from Victivallis vadensis ATCC BAA-548, an anaerobic fecal bacterium.
abstract: Victivallis vadensis ATCC BAA-548 is a Gram-negative, anaerobic bacterium that was isolated from a human fecal sample. From the genomic sequence of V. vadensis, one gene was found to encode agarase; however, its enzymatic properties have never been characterized. The gene encoding the putative agarase (NCBI reference number ZP_01923925) was cloned by PCR and expressed in E. coli Rosetta-gami by using the inducible T(7) promoter of pET28a(+). The expressed protein with a 6×His tag at the N-terminus was named His6-VadG925 and purified as a soluble protein by Ni(2+)-NTA agarose affinity column chromatography. The purification of the enzyme was 26.8-fold, with a yield of 73.2% and a specific activity of 1.02 U/mg of protein. The purified His6-VadG925 produced a single band with an approximate MW of 155 kDa, which is consistent with the calculated value (154,660 Da) including the 6×His tag. Although VadG925 and many of its homologs were annotated as agarases, it did not hydrolyze agarose. Instead, purified His(6)-VadG925 hydrolyzed an artificial chromogenic substrate, p-nitrophenyl-β-D-galactopyranoside, but not p-nitrophenyl-α-D-galactopyranoside. The optimum pH and temperature for this β-galactosidase activity were pH 7.0 and 40°C, respectively. The K(m) and V(max) of His6-VadG925 towards p-nitrophenyl-β-D-galactopyranoside were 1.69 mg/ml (0.0056 M) and 30.3 U/mg, respectively. His6-VadG925 efficiently hydrolyzed lactose into glucose and galactose, which was demonstrated by TLC and mass spectroscopy. These results clearly demonstrated that VadG925 is a novel β-galactosidase that can hydrolyze lactose, which is unusual because of its low homology to validated β-galactosidases.
authors.affiliation: Division of Bioscience and Bioinformatics, Myongji University, Yongin 449-728, Republic of Korea.
authors.forename: Uyangaa
authors.initials: U
authors.lastname: Temuujin
authors.forename: Won-Jae
authors.initials: WJ
authors.lastname: Chi
authors.forename: Jae-Sun
authors.initials: JS
authors.lastname: Park
authors.forename: Yong-Keun
authors.initials: YK
authors.lastname: Chang
authors.forename: Jae Yang
authors.initials: JY
authors.lastname: Song
authors.forename: Soon-Kwang
authors.initials: SK
authors.lastname: Hong
mesh_terms: D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D004789:Enzyme Activation; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D007785:Lactose / Q000737:chemistry; D013696:Temperature; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D001616:beta-Galactosidase; D007785:Lactose