Degradation of dissolved organic nitrogen: Endopeptidase activity
| Endopeptidase activity |
|---|
| Approach: fluorescent substrate assay (MCA-oligopeptide) |
| Context: lab incubation |
| Spatial scale: point sample |
| Temporal scale: hours to days |
| Units: nmol L-1 h-1 |
| Community captured: heterotrophic bacteria |
| Co-measurements: temperature, DOC, bacterial abundance |
Method Overview
Endopeptidase activity is measured using fluorogenic oligopeptide substrates that are cleaved internally by endoprotease enzymes. The most commonly used substrate is the fluorogenic casein derivative or MCA-labelled oligopeptides (e.g., MCA-Ala-Gly-Leu). When endopeptidases cleave the internal peptide bond, the MCA fluorophore is released and measured fluorimetrically over a time course. The linear rate of fluorescence increase is converted to a hydrolysis rate using an MCA standard. Unlike exopeptidases (e.g., LAP), endopeptidases cleave internal bonds in polypeptides, producing smaller peptide fragments that can then be further processed by exopeptidases to yield free amino acids[1].
Scale of measurement
Incubations of hours to days at controlled temperature.
Data generated
Potential endopeptidase hydrolysis rate at Vmax (nmol MCA released L-1 h-1). Complementary to LAP measurements; the combination of exo- and endopeptidase activities characterizes the full proteolytic capacity of the microbial community.
Units & currency
Units are nmol L-1 h-1. The currency is carbon (products contribute to the dissolved organic pools).
Sample size
Typical samples are < 1 L in volume.
Repositories & databases
Limitations
As with LAP, measured rates represent Vmax at saturating substrate and may overestimate in situ rates if ambient substrate concentrations are sub-saturating. Synthetic MCA-peptide substrates may not capture the full diversity of endopeptidase substrates in natural DOM. Bottle effects apply.
Example Applications & Protocols
Classic examples
- Obayashi & Suzuki (2005) Proteolytic enzymes in coastal surface seawater: significant activity of endopeptidases and exopeptidases [1]
Recent applications
Common calculations/conversions
- Endopeptidase rate (nmol L-1 h-1) = Δ[MCA] / incubation time; calibrated against MCA standard.
References
- ↑ 1.0 1.1 Obayashi, Y., & Suzuki, S. (2005). Proteolytic enzymes in coastal surface seawater: significant activity of endopeptidases and exopeptidases. Limnology and Oceanography, 50(2), 722–726. https://doi.org/10.4319/lo.2005.50.2.0722