Identification and characterization of a novel beta-galactosidase from Victivallis vadensis ATCC BAA-548, an anaerobic fecal bacterium

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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 T-7 promoter of pET28a(+). The expressed protein with a 6xHis 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 6xHis 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-beta-d-galactopyranoside, but not p-nitrophenyl-alpha-d-galactopyranoside. The optimum pH and temperature for this beta-galactosidase activity were pH 7.0 and 40A degrees C, respectively. The K (m) and V (max) of His6-VadG925 towards p-nitrophenyl-beta-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 beta-galactosidase that can hydrolyze lactose, which is unusual because of its low homology to validated beta-galactosidases.
Publisher
MICROBIOLOGICAL SOCIETY KOREA
Issue Date
2012-12
Language
English
Article Type
Article
Keywords

ENZYMATIC-SYNTHESIS; ESCHERICHIA-COLI; GENOME SEQUENCE; ALPHA-AGARASE; PURIFICATION; LACTOSE; GENE; HYDROLYSIS; EXPRESSION; OLIGOSACCHARIDES

Citation

JOURNAL OF MICROBIOLOGY, v.50, no.6, pp.1034 - 1040

ISSN
1225-8873
DOI
10.1007/s12275-012-2478-6
URI
http://hdl.handle.net/10203/102209
Appears in Collection
CBE-Journal Papers(저널논문)
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