Enzyme assay with fluoresceinamine labeled biopolymers
Template:BreadcrumbsSecondaryProduction
| Polysaccharide hydrolysis |
|---|
| Approach: enzyme assay, fluorescent substrate |
| Context: incubation, lab |
| Spatial scale: low (> 1000 m) |
| Temporal scale: low; hours to days integration |
| Units: mol monomer L-1 h-1 |
| Community captured: heterotrophs |
| Co-measurements: cell abundance |
Method Overview
This enzyme assay quantifies polysaccharide hydrolysis rates by tracking the release of fluorescently labeled monomers from biopolymer substrates. Fluoresceinamine is covalently coupled to polysaccharides, which are then added to seawater samples at saturating concentrations. As extracellular enzymes produced by heterotrophic microbes cleave glycosidic bonds in the polymer, fluorescent monomers are released into solution. Fluorescence is measured over a time course, and the linear rate of increase is converted into a hydrolysis rate [1].
The assay can be applied to bulk community samples, size-fractionated subsets, or at the single-cell level.
Scale of measurement
As a bottle-based incubation, this method provides a point measurement with low spatial resolution in both horizontal and vertical dimensions. The temporal resolution is also low, with incubations integrating enzymatic activity over hours to days.
Data generated
The assay yields potential polysaccharide hydrolysis rates at maximum enzymatic turnover capacity (Vmax).
Units & currency
Units are mol monomer L-1 h-1. The currency is carbon.
Sample size
Sample volumes are less than one liter (< L).
Repositories & databases
Limitations
Measured rates represent maximum potential hydrolysis (Vmax); substrates are added at saturating concentrations. Bottle effects apply.
Example Applications & Protocols
Classic examples
- Arnosti (1996) A new method for measuring polysaccharide hydrolysis rates in marine environments. [1]
Recent applications
- Martinez-Garcia et al. (2012) Capturing Single Cell Genomes of Active Polysaccharide Degraders: An Unexpected Contribution of Verrucomicrobia. [2]
Common calculations/conversions
- Conversion to carbon units requires the molecular weight and carbon content of the released monomer (e.g., glucose: 180 g mol-1, 40% C by mass).
References
- ↑ 1.0 1.1 Arnosti, C. (1996). A new method for measuring polysaccharide hydrolysis rates in marine environments. Organic Geochemistry, 25(1–2), 105–115. https://doi.org/10.1016/S0146-6380(96)00112-X
- ↑ Martinez-Garcia, M., Brazel, D. M., Swan, B. K., Arnosti, C., Chain, P. S. G., Reitenga, K. G., Xie, G., Poulton, N. J., Lluesma Gomez, M., Masland, D. E. D., Thompson, B., Bellows, W. K., Ziervogel, K., Lo, C. C., Ahmed, S., Gleasner, C. D., Detter, C. J., & Stepanauskas, R. (2012). Capturing Single Cell Genomes of Active Polysaccharide Degraders: An Unexpected Contribution of Verrucomicrobia. PLoS ONE, 7(4), e35314. https://doi.org/10.1371/journal.pone.0035314