A new beta-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 beta-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-beta-D-galactopyranoside)-containing agar plates led to the isolation of beta-galactosidase gene. The beta-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 beta-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 beta-galactosidase was optimally active at pH 6.0-8.0 and 25 degrees Celsius. P-nitrophenyl-beta-D-galactopyranoside (PNPG) is its preferred substrate (three times higher activity than for ONPG-o-nitrophenyl-beta-D-galactopyranoside). The Arthrobacter sp. 20B beta-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 Mn(2+)) and inactivated by pCMB (4-chloro-mercuribenzoic acid) and heavy metal ions (Pb(2+), Zn(2+), Cu(2+)).
Białkowska
Aneta Monika
AM
Institute of Technical Biochemistry (ITB), Technical University of Łódź, Stefanowskigo 4/10, 90-924 Łódź, Poland. aneta.bialkowska@p.lodz.pl
Cieśliński
Hubert
H
Nowakowska
Karolina Maria
KM
Kur
Józef
J
Turkiewicz
Marianna
M
D000595:Amino Acid Sequence; D000864:Antarctic Regions; D001173:Arthrobacter / Q000145:classification / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D003080:Cold Temperature; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D015698:Genomic Library; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009598:Nitrophenylgalactosides / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity / Q000235:genetics; D013696:Temperature; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; C055012:2-nitrophenylgalactoside; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; D001616:beta-Galactosidase