Biochemical Characterization of a β-Galactosidase with a Low Temperature Optimum Obtained from an Antarctic Arthrobacter Isolate
Open Access
- 15 September 2003
- journal article
- research article
- Published by American Society for Microbiology in Journal of Bacteriology
- Vol. 185 (18), 5473-5482
- https://doi.org/10.1128/jb.185.18.5473-5482.2003
Abstract
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.Keywords
This publication has 46 references indexed in Scilit:
- Did psychrophilic enzymes really win the challenge?Extremophiles, 2001
- Molecular and Biochemical Analysis of Two β-Galactosidases fromBifidobacterium infantisHL96Applied and Environmental Microbiology, 2001
- Distributions of structural features contributing to thermostability in mesophilic and thermophilic α/β barrel glycosyl hydrolasesBiochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 2000
- Stabilities of uncomplemented and complemented M15 β-galactosidase (Escherichiacoli) and the relationship to α-complementationBiochemistry and Cell Biology, 1999
- Folding and association of β-galactosidaseJournal of Molecular Biology, 1998
- His-357 of β-Galactosidase (Escherichia coli) Interacts with the C3 Hydroxyl in the Transition State and Helps To Mediate CatalysisBiochemistry, 1998
- Quaternary structure, Mg2+ interactions, and some kinetic properties of the (β-galactosidase fromThermoanaerobacterium thermosulfurigenes EM1Protein Journal, 1996
- The β-Galactosidase ( ) Reaction Is Partly Facilitated by Interactions of His-540 with the C6 Hydroxyl of GalactosePublished by Elsevier BV ,1996
- Cloning and analysis of the β-galactosidase-encoding gene from Clostridium thermosulfurogenes EM1Gene, 1991
- pH Dependence of the Activity of β‐Galactosidase from Escherichia coliEuropean Journal of Biochemistry, 1971