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Heterogeneous Properties of Individual Molecules of β-Galactosidase from the Thermophilic Bacteria Geobacillus stearothermophilus

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Abstract

Single enzyme molecule assays were performed on β-galactosidase from the thermophilic bacteria Geobacillus stearothermophilus using a capillary electrophoresis-based continuous flow assay and the substrate DDAO-β-d-galactoside. The enzyme was found to be heterogeneous with respect to catalytic rate, electrophoretic mobility and activation energy of catalysis. Catalytic rate was also found to vary over time for individual molecules at elevated temperature. Comparison with β-galactosidase from the mesophilic bacteria Escherichia coli showed that the variation in activity over time was less pronounced and the average activation energy of catalysis was lower for the Geobacillus stearothermophilus enzyme. Attempts to measure the properties of individual β-galactosidase molecules from the thermophilic bacteria Thermus thermophilus and the cold-adapted bacteria Pseudoalteromas haloplanktis using this assay were unsuccessful.

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Abbreviations

ATCC:

American Type Culture Collection

DDAO:

7-hydroxy-9H-(1,3-dichloro-9,9-dimethyacridin-2-one)

DDAO-gal:

9H-(1, 3-dichloro-9,9-dimethyacridin-2-one-7-yl)-β-d-galactopyranoside

CE:

Capillary electrophoresis

EOF:

Electroosmotic flow

N.A.:

Numerical aperture

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Acknowledgments

This study was supported by a grant from the Natural Sciences and Engineering Research Council.

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  1. Chemistry Department, University of Winnipeg, Winnipeg, MB, R3B 2E9, Canada

    Douglas B. Craig

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  1. Douglas B. Craig
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Correspondence to Douglas B. Craig.

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Craig, D.B. Heterogeneous Properties of Individual Molecules of β-Galactosidase from the Thermophilic Bacteria Geobacillus stearothermophilus . Protein J 29, 55–61 (2010). https://doi.org/10.1007/s10930-009-9220-1

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