Identification and Characterization of a Novel β-Galactosidase from Victivallis vadensis ATCC BAA-548, an Anaerobic Fecal Bacterium

Uyangaa Temuujin; Won-Jae Chi; Jae-Sun Park; Yong-Keun Chang; Jae Yang Song; Soon-Kwang Hong

doi:10.1007/s12275-012-2478-6

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Identification and Characterization of a Novel β-Galactosidase from Victivallis vadensis ATCC BAA-548, an Anaerobic Fecal Bacterium: Uyangaa Temuujin ¹, Won-Jae Chi ¹, Jae-Sun Park ¹, Yong-Keun Chang ², Jae Yang Song ³, Soon-Kwang Hong ¹; Journal of Microbiology 2012;50(6):1034-1040.
DOI: https://doi.org/10.1007/s12275-012-2478-6
Published online: December 30, 2012

¹Division of Bioscience and Bioinformatics, Myongji University, Yongin 449-728, Republic of Korea, ²Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, Daejon 305-701, Republic of Korea, ³Energy R&D Center, SK Innovation Global Technology, Daejon 305-712, Republic of Korea

Corresponding author: Soon-Kwang Hong , Tel: +82-31-330-6198,

Received: 3 September 2012 • Accepted: 12 September 2012

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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 T7 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 Ni2+-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 His6-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 Km and Vmax 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.

Keywords: Victivallis vadensis;ZP_01923925;VadG925;β-galactosidase;lactose hydrolysis

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Identification and Characterization of a Novel β-Galactosidase from Victivallis vadensis ATCC BAA-548, an Anaerobic Fecal Bacterium

J. Microbiol. 2012;50(6):1034-1040. Published online December 30, 2012

DOI: https://doi.org/10.1007/s12275-012-2478-6

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