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KMID : 0880220120500061034
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Journal of Microbiology
2012 Volume.50 No. 6 p.1034 ~ p.1040
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Identification and characterization of a novel β-galactosidase from Victivallis vadensis ATCC BAA-548, an anaerobic fecal bacterium
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Uyangaa Temuujin
Chi Won-Jae
Park Jae-Sun
Hong Soon-Kwang
Chang Yong-Keun
Song Jae-Yang
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Abstract
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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.
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KEYWORD
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Victivallis vadensis, ZP_01923925, VadG925, β-galactosidase, lactose hydrolysis
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