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

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Abstract

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+).

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Abbreviations

ORF:

Open reading frame

X-Gal:

5-Bromo-4-chloro-3-indolyl-β-d-galactopyranoside

IPTG:

Isopropyl-β-d-thiogalactopyranoside

PNPG:

p-Nitrophenyl-β-d-galactopyranoside

ONPG:

o-Nitrophenyl-β-d-galactopyranoside

pCMB:

4-Chloro-mercuribenzoic acid

PABTG:

p-Amino-benzyl-1-thio-β-d-galactopyranoside

PMSF:

Phenylmethylsulphonyl fluoride

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Author notes

  1. Karolina Maria Nowakowska

    Present address: Institute of Technical Biochemistry (ITB), Technical University of Łódź, Stefanowskigo 4/10, 90-924, Łódź, Poland

Authors and Affiliations

  1. Institute of Technical Biochemistry (ITB), Technical University of Łódź, Stefanowskigo 4/10, 90-924, Łódź, Poland

    Aneta Monika Białkowska & Marianna Turkiewicz

  2. Department of Microbiology, Gdańsk University of Technology, Narutowicza 11/12, 80-952, Gdańsk, Poland

    Hubert Cieśliński, Karolina Maria Nowakowska & Józef Kur

Authors
  1. Aneta Monika Białkowska
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  2. Hubert Cieśliński
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  3. Karolina Maria Nowakowska
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  4. Józef Kur
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  5. Marianna Turkiewicz
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Corresponding author

Correspondence to Aneta Monika Białkowska.

Additional information

Communicated by Erko Stackebrandt.

A. M. Białkowska and H. Cieśliński contributed equally to this work.

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Białkowska, A.M., Cieśliński, H., Nowakowska, K.M. et al. A new β-galactosidase with a low temperature optimum isolated from the Antarctic Arthrobacter sp. 20B: gene cloning, purification and characterization. Arch Microbiol 191, 825–835 (2009). https://doi.org/10.1007/s00203-009-0509-4

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