Enzyme assay with fluoresceinamine labeled biopolymers: Difference between revisions

Latest revision as of 11:30, 11 May 2026

Polysaccharide hydrolysis
Approach: enzyme assay
Context: incubation, lab
Spatial scale: low
Temporal scale: low; hours to days
Units: mol monomer L-1 h-1
Community captured: heterotrophs
Co-measurements: cell abundance

Method Overview

This enzyme assay measures polysaccharide hydrolysis by time course incubations with fluorescently labeled substrate (fluoresceinamine labeled biopolymers)^[1].

The assay is performed in discrete mode on bulk, size-fract, single cell samples.

Scale of measurement

Typically performed on replicate samples collected at a sampling station at one point in time. Sampling effort is relatively high impairing resolution in space or time. Time course incubations last hours to days, further restricting temporal resolution.

Data generated

These measurements can provide hydrolysis rates per unit time.

Units & currency

Units are mol monomer L-1 h-1. The currency is carbon.

Sample size

Typical samples are < 1 L in volume.

Limitations

Rate measurements to estimate Vmax, yielding potential rates. Bottle effects are likely to occur.

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]

References

↑ ^1.0 ^1.1 Arnosti (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 et al. (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

[Arnosti1996-1] 1.0 ^1.1 Arnosti (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-Garcia2012-2] Martinez-Garcia et al. (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

[1]

[2]

@@ Line 10: / Line 10: @@
 ! Polysaccharide hydrolysis
 |-
-| '''Approach:''' enzyme assay, fluorescent substrate
+| '''Approach:''' enzyme assay
 |-
 | '''Context:''' incubation, lab
 |-
-| '''Spatial scale:''' low (> 1000 m)
+| '''Spatial scale:''' low
 |-
-| '''Temporal scale:''' low; hours to days integration
+| '''Temporal scale:''' low; hours to days
 |-
-| '''Units:''' mol monomer L<sup>-1</sup> h<sup>-1</sup>
+| '''Units:''' mol monomer L-1 h-1
 |-
 | '''Community captured:''' heterotrophs
@@ Line 29: / Line 29: @@
 == 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 <ref name="Arnosti1996">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</ref>.
+This enzyme assay measures polysaccharide hydrolysis by time course incubations with fluorescently labeled substrate (fluoresceinamine labeled biopolymers)<ref name="Arnosti1996">Arnosti (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</ref>.
-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.
+The assay is performed in discrete mode on bulk, size-fract, single cell samples.
 === 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.
+Typically performed on replicate samples collected at a sampling station at one point in time. Sampling effort is relatively high impairing resolution in space or time. Time course incubations last hours to days, further restricting temporal resolution.
 === Data generated ===
-The assay yields potential polysaccharide hydrolysis rates representing the maximum enzymatic turnover capacity (V<sub>max</sub>) 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.
+These measurements can provide hydrolysis rates per unit time.
 === Units & currency ===
-Units are mol monomer L<sup>-1</sup> h<sup>-1</sup>. The currency is carbon, as polysaccharides are carbon-rich substrates and hydrolysis rates feed directly into estimates of heterotrophic carbon processing.
+Units are mol monomer L-1 h-1. The currency is carbon.
 === Sample size ===
-Sample volumes are less than one liter (< L), consistent with standard shipboard or laboratory incubation bottle sizes.
+Typical samples are < 1 L in volume.
-=== Repositories & databases ===
 == Limitations ==
-Measured rates represent maximum potential hydrolysis (V<sub>max</sub>) 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.
+Rate measurements to estimate Vmax, yielding potential rates. Bottle effects are likely to occur.
 == Example Applications & Protocols ==
 === Classic examples ===
-* Arnosti (1996) ''A new method for measuring polysaccharide hydrolysis rates in marine environments.'' <ref name="Arnosti1996" />
+* Arnosti (1996) ''A new method for measuring polysaccharide hydrolysis rates in marine environments'' <ref name="Arnosti1996" />
 === Recent applications ===
-* Martinez-Garcia et al. (2012) ''Capturing Single Cell Genomes of Active Polysaccharide Degraders: An Unexpected Contribution of Verrucomicrobia.'' <ref name="MartinezGarcia2012">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</ref>
+* Martinez-Garcia et al. (2012) ''Capturing Single Cell Genomes of Active Polysaccharide Degraders: An Unexpected Contribution of Verrucomicrobia'' <ref name="Martinez-Garcia2012">Martinez-Garcia et al. (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</ref>
-=== Common calculations/conversions ===
-* Conversion to carbon units requires the molecular weight and carbon content of the released monomer (e.g., glucose: 180 g mol<sup>-1</sup>, 40% C by mass).
 == References ==
 [[Category:Main Pages|Model types]]