Archives of Microbiology

, 191:825 | Cite as

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

  • Aneta Monika Białkowska
  • Hubert Cieśliński
  • Karolina Maria Nowakowska
  • Józef Kur
  • Marianna Turkiewicz
Original Paper

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+).

Keywords

Cold-adapted enzymes β-Galactosidase Arthrobacter sp. Lactose hydrolysis Psychrotrophic microorganisms 

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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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Aneta Monika Białkowska
    • 1
  • Hubert Cieśliński
    • 2
  • Karolina Maria Nowakowska
    • 1
    • 2
  • Józef Kur
    • 2
  • Marianna Turkiewicz
    • 1
  1. 1.Institute of Technical Biochemistry (ITB)Technical University of ŁódźŁódźPoland
  2. 2.Department of MicrobiologyGdańsk University of TechnologyGdańskPoland

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