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, making it suitable for examining which fractions of the heterotrophic community are responsible for polysaccharide degradation.
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; each incubation integrates enzymatic activity over hours to days, yielding a time-averaged rate rather than an instantaneous snapshot.
Data generated
The assay yields potential polysaccharide hydrolysis rates representing the maximum enzymatic turnover capacity (Vmax) of the sampled heterotrophic community. Rates reflect carbon flux from complex dissolved organic polymers to bioavailable monomers and are used to estimate the contribution of extracellular enzyme activity to the microbial carbon cycle.
Units & currency
Units are mol monomer L-1 h-1. The currency is carbon, as polysaccharides are carbon-rich substrates and hydrolysis rates feed directly into estimates of heterotrophic carbon processing.
Sample size
Sample volumes are less than one liter (< L), consistent with standard shipboard or laboratory incubation bottle sizes.
Repositories & databases
Limitations
Measured rates represent maximum potential hydrolysis (Vmax) because substrates are added at saturating concentrations. In situ rates may be substantially lower if environmental substrate concentrations are sub-saturating. As with all bottle incubations, enclosure introduces bottle effects that can alter microbial community composition and activity relative to ambient conditions.
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