Biochemical Characterization of a β-Galactosidase with a Low Temperature Optimum Obtained from an Antarctic Arthrobacter Isolate
| Autor: | Kevin R. Gutshall, Jennifer Loveland-Curtze, James A. Coker, Peter P. Sheridan, Ann J. Auman, Jean E. Brenchley |
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| Rok vydání: | 2003 |
| Předmět: |
Hot Temperature
Molecular Sequence Data Lysine Antarctic Regions Lactose Diamino acid medicine.disease_cause Biochemistry Microbiology Substrate Specificity chemistry.chemical_compound Sequence Analysis Protein Arthrobacter Enzyme Stability Escherichia coli medicine Beta-galactosidase Cloning Molecular Enzyme Inhibitors Molecular Biology Phylogeny Histidine chemistry.chemical_classification biology Temperature Hydrogen-Ion Concentration Nitrophenylgalactosides beta-Galactosidase biology.organism_classification Enzymes and Proteins Molecular biology Kinetics Enzyme chemistry Metals biology.protein Homotetramer |
| Zdroj: | Journal of Bacteriology. 185:5473-5482 |
| ISSN: | 1098-5530 0021-9193 |
| DOI: | 10.1128/jb.185.18.5473-5482.2003 |
| Popis: | A psychrophilic gram-positive isolate was obtained from Antarctic Dry Valley soil. It utilized lactose, had a rod-coccus cycle, and contained lysine as the diamino acid in its cell wall. Consistent with these physiological traits, the 16S ribosomal DNA sequence showed that it was phylogenetically related to other Arthrobacter species. A gene ( bgaS ) encoding a family 2 β-galactosidase was cloned from this organism into an Escherichia coli host. Preliminary results showed that the enzyme was cold active (optimal activity at 18°C and 50% activity remaining at 0°C) and heat labile (inactivated within 10 min at 37°C). To enable rapid purification, vectors were constructed adding histidine residues to the BgaS enzyme and its E. coli LacZ counterpart, which was purified for comparison. The His tag additions reduced the specific activities of both β-galactosidases but did not alter the other characteristics of the enzymes. Kinetic studies using o -nitrophenyl-β- d -galactopyranoside showed that BgaS with and without a His tag had greater catalytic activity at and below 20°C than the comparable LacZ β-galactosidases. The BgaS heat lability was investigated by ultracentrifugation, where the active enzyme was a homotetramer at 4°C but dissociated into inactive monomers at 25°C. Comparisons of family 2 β-galactosidase amino acid compositions and modeling studies with the LacZ structure did not mimic suggested trends for conferring enzyme flexibility at low temperatures, consistent with the changes affecting thermal adaptation being localized and subtle. Mutation studies of the BgaS enzyme should aid our understanding of such specific, localized changes affecting enzyme thermal properties. |
| Databáze: | OpenAIRE |
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