Enzymatic assay of marine bacterial phosphatases by capillary electrophoresis with laser-induced fluorescence detection.
Microbial ectoenzyme activities in aquatic environments are important determinants of polymer hydrolysis and indicators of the state of microbial carbon, nitrogen, and phosphorus nutrition. Marine ectoenzymes are found on the cell surface or in the periplasmic space of gram-negative heterotrophic bacteria. Phosphatases, which remove phosphate groups from substrates, are one example of an ectoenzyme. Enzyme assays based on-capillary electrophoresis (CE) take advantage of CE's high-efficiency separation, extremely low sample volume requirements, and its ability to electrophoretically mix and separate zones of enzymes, substrates, and products all in one experimental run. CE has better resolving power and, when utilized with laser-induced fluorescence (LIF) detection, it is more sensitive than chromatography. CE-LIF is a promising tool for determining different phosphatases within a single microbial strain as well as the functional diversity between strains. In this study, four bacterial strains were studied (Shewanella sp., TW7, BB2AT2, and Vibrio alginolyticus) with each yielding at least one phosphatase that was kinetically characterized. K(m) values were calculated and found to be in the range of 0.0725-3.35 microM, whereas V(max) values ranged from 1.02 x 10(-3) to 1.05 x 10(-2) microM/min. The large range of values demonstrates differences among the phosphatases, suggesting different roles for each phosphatase not only between the species but also within a single bacterial species. This can have the important implications for organic matter processing in the sea.
Chichester
Kimberly D
KD
Department of Chemistry, Wake Forest University, Winston-Salem, NC 27019, USA.
Sebastian
Marta
M
Ammerman
James W
JW
Colyer
Christa L
CL
D001426:Bacterial Proteins / Q000032:analysis*; D001681:Biological Assay; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D005453:Fluorescence; D007834:Lasers; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001426:Bacterial Proteins; D010744:Phosphoric Monoester Hydrolases