new β-galactosidase with a low temperature optimum isolated from the Antarctic Arthrobacter sp. 20B: gene cloning, purification and characterization

A psychrotrophic bacterium producing a cold-adapted β-galactosidase upon growth at low temperatures was classified as Arthrobacter sp. 20B. A genomic DNA library of strain 20B introduced into Escherichia coli TOP10F′ and screening on X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside)-containin...

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Bibliographic Details
Published in:	Archives of microbiology Vol. 191; no. 11; pp. 825 - 835
Main Authors:	Białkowska, Aneta Monika, Cieśliński, Hubert, Nowakowska, Karolina Maria, Kur, Józef, Turkiewicz, Marianna
Format:	Journal Article
Language:	English
Published:	Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01-11-2009 Springer-Verlag Springer
Subjects:	Amino Acid Sequence Antarctic Regions Arthrobacter - classification Arthrobacter - enzymology Arthrobacter - genetics Arthrobacter - isolation & purification Bacteriology beta-Galactosidase - chemistry beta-Galactosidase - genetics beta-Galactosidase - isolation & purification beta-Galactosidase - metabolism Biochemistry Biological and medical sciences Biomedical and Life Sciences Biotechnology Cell Biology Cloning, Molecular Cold Temperature DNA, Bacterial - genetics DNA, Bacterial - isolation & purification Ecology Electrophoresis, Polyacrylamide Gel Escherichia coli - genetics Fundamental and applied biological sciences. Psychology Genes, Bacterial Genomic Library Kinetics Life Sciences Microbial Ecology Microbiology Miscellaneous Molecular Sequence Data Molecular Weight Nitrophenylgalactosides - metabolism Original Paper RNA, Ribosomal, 16S - genetics RNA, Ribosomal, 16S - isolation & purification Sequence Alignment Sequence Analysis, DNA Substrate Specificity - genetics Temperature Antarctica Antarctic Regions Cold-adapted enzymes Psychrotrophic microorganisms sp Lactose hydrolysis β-Galactosidase Purification Enzyme Arthrobacter sp Glycosylases Hydrolysis Lactose Gene Glycosidases Bacteria Hydrolases Actinomycetes Psychrotrophy Microorganism Arthrobacter Low temperature
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Summary:	A psychrotrophic bacterium producing a cold-adapted β-galactosidase upon growth at low temperatures was classified as Arthrobacter sp. 20B. A genomic DNA library of strain 20B introduced into Escherichia coli TOP10F′ and screening on X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside)-containing agar plates led to the isolation of β-galactosidase gene. The β-galactosidase gene (bgaS) encoding a protein of 1,053 amino acids, with a calculated molecular mass of 113,695 kDa. Analysis of the amino acid sequence of BgaS protein, deduced from the bgaS ORF, suggested that it is a member of the glycosyl hydrolase family 2. A native cold-adapted β-galactosidase was purified to homogeneity and characterized. It is a homotetrameric enzyme, each subunit being approximately 116 kDa polypeptide as deduced from native and SDS-PAGE, respectively. The β-galactosidase was optimally active at pH 6.0-8.0 and 25°C. P-nitrophenyl-β-d-galactopyranoside (PNPG) is its preferred substrate (three times higher activity than for ONPG—o-nitrophenyl-β-d-galactopyranoside). The Arthrobacter sp. 20B β-galactosidase is activated by thiol compounds (53% rise in activity in the presence of 10 mM 2-mercaptoethanol), some metal ions (activity increased by 50% for Na⁺, K⁺ and by 11% for Mn²⁺) and inactivated by pCMB (4-chloro-mercuribenzoic acid) and heavy metal ions (Pb²⁺, Zn²⁺, Cu²⁺).
Bibliography:	http://dx.doi.org/10.1007/s00203-009-0509-4 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0302-8933 1432-072X
DOI:	10.1007/s00203-009-0509-4