Applied and Environmental Microbiology, April 2001, p. 1601-1606, Vol. 67, No. 4
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1601-1616.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
-Galactosidase
Expressed in Escherichia coli
Institute of Applied Biochemistry, University of Tsukuba, Ibaraki 305-0006,1 National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Ibaraki 305-8642,2 National Institute of Bioscience and Human Technology, MITI, Ibaraki 305-8566,3 Japan, and Department of Food Engineering and Biotechnology, Kyungwon University, Kyunggi-do 461-701, Korea4
Received 5 October 2000/Accepted 2 February 2001
The nucleotide sequence of the Thermus sp. strain T2
DNA coding for a thermostable
-galactosidase was determined. The
deduced amino acid sequence of the enzyme predicts a polypeptide of 474 amino acids (Mr, 53,514). The observed
homology between the deduced amino acid sequences of the enzyme and
-galactosidase from Thermus brockianus was over 70%.
Thermus sp. strain T2
-galactosidase was expressed in
its active form in Escherichia coli and purified. Native
polyacrylamide gel electrophoresis and gel filtration chromatography data suggest that the enzyme is octameric. The enzyme was most active
at 75°C for
p-nitrophenyl-
-D-galactopyranoside
hydrolysis, and it retained 50% of its initial activity after 1 h
of incubation at 70°C. The enzyme was extremely stable over a broad
range of pH (pH 6 to 13) after treatment at 40°C for 1 h. The
enzyme acted on the terminal
-galactosyl residue, not on the side
chain residue, of the galactomanno-oligosaccharides as well as those of
yeasts and Mortierella vinacea
-galactosidase I. The
enzyme has only one Cys residue in the molecule.
para-Chloromercuribenzoic acid completely inhibited the
enzyme but did not affect the mutant enzyme which contained Ala instead
of Cys, indicating that this Cys residue is not responsible for its
catalytic function.
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