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Applied and Environmental Microbiology, December 1999, p. 5443-5450, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Biochemical and Phylogenetic Analyses of a Cold-Active beta -Galactosidase from the Lactic Acid Bacterium Carnobacterium piscicola BA

Jonna M. Coombs and Jean E. Brenchley*

Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802

Received 28 June 1999/Accepted 19 September 1999

We are investigating glycosyl hydrolases from new psychrophilic isolates to examine the adaptations of enzymes to low temperatures. A beta -galactosidase from isolate BA, which we have classified as a strain of the lactic acid bacterium Carnobacterium piscicola, was capable of hydrolyzing the chromogen 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside (X-Gal) at 4°C and possessed higher activity in crude cell lysates at 25 than at 37°C. Sequence analysis of a cloned DNA fragment encoding this activity revealed a gene cluster containing three glycosyl hydrolases with homology to an alpha -galactosidase and two beta -galactosidases. The larger of the two beta -galactosidase genes, bgaB, encoded the 76.8-kDa cold-active enzyme. This gene was homologous to family 42 glycosyl hydrolases, a group which contains several thermophilic enzymes but none from lactic acid bacteria. The bgaB gene from isolate BA was subcloned in Escherichia coli, and its enzyme, BgaB, was purified. The purified enzyme was highly unstable and required 10% glycerol to maintain activity. Its optimal temperature for activity was 30°C, and it was inactivated at 40°C in 10 min. The Km of freshly purified enzyme at 30°C was 1.7 mM, and the Vmax was 450 µmol · min-1 · mg-1 with o-nitrophenyl beta -D-galactopyranoside. This cold-active enzyme is interesting because it is homologous to a thermophilic enzyme from Bacillus stearothermophilus, and comparisons could provide information about structural features important for activity at low temperatures.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802. Phone: (814) 863-7794. Fax: (814) 865-3330. E-mail: JEB7{at}psu.edu.

Applied and Environmental Microbiology, December 1999, p. 5443-5450, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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