Enzyme assay with fluoresceinamine labeled biopolymers - Revision history

Hagi BucknWise at 18:30, 11 May 2026

2026-05-11T18:30:25Z

← Older revision		Revision as of 11:30, 11 May 2026
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	! Polysaccharide hydrolysis		! Polysaccharide hydrolysis
	\|-		\|-
	\| '''Approach:''' enzyme assay~~, fluorescent substrate~~		\| '''Approach:''' enzyme assay
	\|-		\|-
	\| '''Context:''' incubation, lab		\| '''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		\| '''Community captured:''' heterotrophs
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	== Method Overview ==		== 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~~.		The assay is performed in discrete mode on bulk, size-fract, single cell samples.

	=== Scale of measurement ===		=== 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.		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 ===		=== Data generated ===

	~~The assay yields potential polysaccharide~~ hydrolysis rates ~~at maximum enzymatic turnover capacity (V<sub>max</sub>)~~.		These measurements can provide hydrolysis rates per unit time.

	=== Units & currency ===		=== Units & currency ===

	Units are mol monomer L~~<sup>~~-1~~</sup>~~ h~~<sup>~~-1~~</sup>~~. The currency is carbon.		Units are mol monomer L-1 h-1. The currency is carbon.

	=== Sample size ===		=== Sample size ===

	~~Sample volumes~~ are ~~less than one liter (~~< L).		Typical samples are < 1 L in volume.

	~~=== Repositories & databases ===~~

	== Limitations ==		== Limitations ==

	~~Measured~~ rates ~~represent maximum potential hydrolysis (V<sub>max</sub>); substrates are added at saturating concentrations~~. Bottle effects ~~apply~~.		Rate measurements to estimate Vmax, yielding potential rates. Bottle effects are likely to occur.

	== Example Applications & Protocols ==		== Example Applications & Protocols ==

	=== Classic examples ===		=== 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 ===		=== 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 ==		== References ==

	[[Category:Main Pages\|Model types]]		[[Category:Main Pages\|Model types]]

Hagi BucknWise at 17:28, 11 May 2026

2026-05-11T17:28:54Z

← Older revision		Revision as of 10:28, 11 May 2026
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	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 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>.

	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 can be applied to bulk community samples, size-fractionated subsets, or at the single-cell level.

	=== Scale of measurement ===		=== 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~~.		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 ===		=== 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.		The assay yields potential polysaccharide hydrolysis rates at maximum enzymatic turnover capacity (V<sub>max</sub>).

	=== Units & currency ===		=== 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<sup>-1</sup> h<sup>-1</sup>. The currency is carbon.

	=== Sample size ===		=== Sample size ===

	Sample volumes are less than one liter (< L)~~, consistent with standard shipboard or laboratory incubation bottle sizes~~.		Sample volumes are less than one liter (< L).

	=== Repositories & databases ===		=== Repositories & databases ===
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	== Limitations ==		== 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~~.		Measured rates represent maximum potential hydrolysis (V<sub>max</sub>); substrates are added at saturating concentrations. Bottle effects apply.

	== Example Applications & Protocols ==		== Example Applications & Protocols ==

Hagi BucknWise at 17:12, 11 May 2026

2026-05-11T17:12:02Z

← Older revision		Revision as of 10:12, 11 May 2026
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	* [[Page authors\|Page authors]]: [[~~User:Kate Evans\|Kate Evans~~]]		{{BreadcrumbsSecondaryProduction}}
	* [[Responsible curator\|Responsible curator]]: [[User:~~Kate Evans~~\|~~Kate Evans~~]]
			* [[Page authors\|Page authors]]: [[PRIMO]]
			* [[Responsible curator\|Responsible curator]]: [[User:Hagi BucknWise\|Hagen Buck-Wiese]]
	----		----

	__TOC__		__TOC__
	<div class="model-box">		<div class="model-box">
	{\| class="model-ib"		{\| class="model-ib" style="float:right; margin-left:1em; margin-bottom:1em;"
	! ~~What is being measured in 1 - 3 words~~		! Polysaccharide hydrolysis
	\|-		\|-
	\| '''Approach:''' ~~tracer quantification~~, ~~remote sensing, sequencing, etc.~~		\| '''Approach:''' enzyme assay, fluorescent substrate
	\|-		\|-
	\| '''Context:''' ~~''in situ''~~, ~~incubation~~		\| '''Context:''' incubation, lab
	\|-		\|-
	\| '''Spatial scale:''' ~~mL, km<sup>2</sup~~>		\| '''Spatial scale:''' low (> 1000 m)
	\|-		\|-
	\| '''Temporal scale:''' ~~seconds,~~ days~~, seasons~~		\| '''Temporal scale:''' low; hours to days integration
	\|-		\|-
	\| '''Units:'''		\| '''Units:''' mol monomer L<sup>-1</sup> h<sup>-1</sup>
	\|-		\|-
	\| '''Community captured:''' ~~all, 0.7 - 3 µm, target species~~		\| '''Community captured:''' heterotrophs
	\|-		\|-
	\| '''Co-measurements:''' ~~Other measurements required for interpretation of results e.g., temperature, salinity, wind speed history~~		\| '''Co-measurements:''' cell abundance
	\|}		\|}
	</div>		</div>
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	== Method Overview ==		== Method Overview ==

	=~~= Output ==~~		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>.

			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 ===		=== 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 ===		=== 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.

	=== Units & currency ===		=== 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.

	=== Sample size ===		=== Sample size ===

			Sample volumes are less than one liter (< L), consistent with standard shipboard or laboratory incubation bottle sizes.

	=== Repositories & databases ===		=== Repositories & databases ===

	~~e.g. [https://github.com/raw-lab/NFixDB nFixDB]~~		== Limitations ==

	~~== 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.

	== Example Applications & Protocols ==		== Example Applications & Protocols ==

	=== Classic examples ===		=== Classic examples ===
	* ~~Classic~~/~~original papers~~		* Arnosti (1996) ''A new method for measuring polysaccharide hydrolysis rates in marine environments.'' <ref name="Arnosti1996" />

	=== Recent applications ===		=== Recent applications ===
	* ~~New papers + protocol links~~ (e.g. ~~word doc used in lab~~) ~~if applicable~~		* 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>

	=== Common calculations/conversions ===		=== Common calculations/conversions ===
	* E.g. ~~conversion to standard units~~		* 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 ==		== References ==

	[[Category:Main Pages\|Model types]]		[[Category:Main Pages\|Model types]]

Kate Evans at 13:55, 3 February 2026

2026-02-03T13:55:42Z

← Older revision		Revision as of 06:55, 3 February 2026
Line 1:		Line 1:
	* [[Page authors\|Page authors]]: [[~~Melanie Staeubli]], [[Grommet the Cat]], [[PRIMO~~]]		* [[Page authors\|Page authors]]: [[User:Kate Evans\|Kate Evans]]
	* [[Responsible curator\|Responsible curator]]: [[User:Kate Evans\|Kate Evans]]		* [[Responsible curator\|Responsible curator]]: [[User:Kate Evans\|Kate Evans]]
	----		----

Kate Evans: Created page with "* Page authors: Melanie Staeubli, Grommet the Cat, PRIMO * Responsible curator: Kate Evans ---- TOC
{| class="model-ib" ! What is being measured in 1 - 3 words |- | '''Approach:''' tracer quantification, remote sensing, sequencing, etc. |- | '''Context:''' ''in situ'', incubation |- | '''Spatial scale:''' mL, km² |- | '''Temporal scale:''' seconds, days, seas..."

2026-02-03T13:53:36Z

Created page with "* Page authors: Melanie Staeubli, Grommet the Cat, PRIMO * Responsible curator: Kate Evans ---- __TOC__ <div class="model-box"> {| class="model-ib" ! What is being measured in 1 - 3 words |- | '''Approach:''' tracer quantification, remote sensing, sequencing, etc. |- | '''Context:''' ''in situ'', incubation |- | '''Spatial scale:''' mL, km<sup>2</sup> |- | '''Temporal scale:''' seconds, days, seas..."

New page

* [[Page authors|Page authors]]: [[Melanie Staeubli]], [[Grommet the Cat]], [[PRIMO]]
* [[Responsible curator|Responsible curator]]: [[User:Kate Evans|Kate Evans]]
----

__TOC__
<div class="model-box">
{| class="model-ib"
! What is being measured in 1 - 3 words
|-
| '''Approach:''' tracer quantification, remote sensing, sequencing, etc.
|-
| '''Context:''' ''in situ'', incubation
|-
| '''Spatial scale:''' mL, km<sup>2</sup>
|-
| '''Temporal scale:''' seconds, days, seasons
|-
| '''Units:'''
|-
| '''Community captured:''' all, 0.7 - 3 µm, target species
|-
| '''Co-measurements:''' Other measurements required for interpretation of results e.g., temperature, salinity, wind speed history
|}
</div>
<div style="clear:both"></div>

== Method Overview ==

== Output ==

=== Scale of measurement ===

=== Data generated ===

=== Units & currency ===

=== Sample size ===

=== Repositories & databases ===

e.g. [https://github.com/raw-lab/NFixDB nFixDB]

== Limitations ==

== Example Applications & Protocols ==

=== Classic examples ===
* Classic/original papers

=== Recent applications ===
* New papers + protocol links (e.g. word doc used in lab) if applicable

=== Common calculations/conversions ===
* E.g. conversion to standard units

== References ==

[[Category:Main Pages|Model types]]