Biochemical and phylogenetic analyses of a cold-active beta-galactosidase from the lactic acid bacterium Carnobacterium piscicola BA

Abstract

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 degrees C and possessed higher activity in crude cell lysates at 25 than at 37 degrees 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 degrees C, and it was inactivated at 40 degrees C in 10 min. The K(m) of freshly purified enzyme at 30 degrees C was 1.7 mM, and the V(max) was 450 micromol. 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.

FIG. 1

Phylogenetic analysis of the sequence from the 16S rRNA gene from isolate BA. 16S rRNA sequences from all other organisms are from the Ribosomal Database Project. Sequences from GenBank were also analyzed for similarity to isolate BA 16S rRNA, but their inclusion did not change the position of isolate BA within the Carnobacterium clade. Phylogenetic relationships are based on MegAlign alignments, optimized in ESEE and analyzed with the program Phylip. Sequence differences between C. piscicola BA and its closest relatives are too small (2 to 3 bp) to show on this scale. B., Bacillus; C., Carnobacterium; L., Lactobacillus.

FIG. 2

Phylogenetic analysis of the bgaB gene, based on its deduced amino acid sequence. Sequences for β-galactosidases from GenBank were aligned with MegAlign and ESEE. Phylogenetic trees were constructed using the program PAUP. Haloferax alicantei was used as the outgroup for this analysis. Bootstrap values were calculated based on 100 replicates. Sequence sources were as follows; Haloferax alicantei (22), Thermus sp. A4 (GenBank accession no. BAA28362), Thermus sp. T2 (38), Thermotoga neapolitana (GenBank accession no. AAC24217), Bifidobacterium breve (GenBank accession no. E05040), Caldicellulosiruptor sp. 14B (GenBank accession no. CAA10365), Clostridium perfringens pbg (GenBank accession no. BAA08485), Bacillus circulans bgaB (GenBank accession no. AAA22260), Bacillus circulans bgaA (GenBank accession no. AAA22258), B. stearothermophilus bgaB (20).

FIG. 3

Conserved sequences found for glycosyl hydrolases. The alignment of the proposed acid-base site is compared to the similar sequences of LacG from L. lactis (10) and with the E. coli LacZ enzyme (23). The sequence of a conserved region which may contain the putative nucleophilic site does not demonstrate homology to either LacZ or the family 1 β-galactosidases and is compared to the B7-12 enzyme from Arthrobacter psychrolactophilus (14).

FIG. 4

Thermal dependency of activity of purified enzyme BgaB. Enzyme solutions were stored in Z buffer plus 10% glycerol. Activity was assayed for 2 to 10 min in a reaction mixture containing Z buffer without glycerol and with ONPG. The 100% specific activity value is 393 U/mg.

FIG. 5

Stability of purified BgaB enzyme activity at different temperatures. (A) Enzyme stored in Z buffer containing 10% glycerol was incubated at 30 (●), 35 (▴), or 40°C (■), and aliquots were removed at various times and assayed at 20°C. The 100% specific activity was 179.5 U/mg. (B) Enzyme was incubated at 20°C in Z buffer in the absence of glycerol. Aliquots of enzyme were removed at different times and assayed for activity at 20°C.