Degradation of dissolved organic nitrogen: Leucine-aminopeptidase activity

• Page authors: PRIMO
• Responsible curator: Hagen Buck-Wiese

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Leucine-aminopeptidase activity
Approach: fluorescent substrate assay (MCA-Leu)
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

Leucine-aminopeptidase (LAP) activity is measured using the fluorogenic substrate L-leucine-7-amido-4-methylcoumarin (MCA-Leu). When the substrate is cleaved by exopeptidase enzymes at the cell surface or in solution, it releases the fluorescent product 7-amino-4-methylcoumarin (MCA), which is measured fluorimetrically (excitation ~380 nm, emission ~440 nm) over a time course. The linear rate of fluorescence increase, calibrated against an MCA standard curve, yields the LAP hydrolysis rate at saturating substrate concentration (V_max). LAP is a broadly distributed bacterial ectoenzyme that degrades peptide bonds in dissolved organic nitrogen compounds, releasing amino acids that can be directly assimilated.

Scale of measurement

Point sample using incubations of hours to days in the dark at in situ or controlled temperature.

Data generated

Potential LAP hydrolysis rate at V_max (nmol MCA released L^-1 h^-1). This reflects the maximum enzymatic capacity for leucyl-peptide bond cleavage in the community and is used as a proxy for the community's capacity to degrade proteinaceous dissolved organic nitrogen.

Units & currency

Units are nmol L^-1 h^-1. The currency is carbon (nitrogen hydrolysis products contribute to the dissolved organic carbon and nitrogen pools).

Sample size

Typical samples are < 1 L in volume.

Limitations

Rates measured represent V_max at saturating substrate, which may substantially overestimate in situ rates if ambient peptide concentrations are sub-saturating. Bottle effects can alter community composition during incubation. The synthetic MCA-Leu substrate is a proxy for natural peptides and may not be representative of the full range of substrates cleaved by LAP in situ.

Example Applications & Protocols

Classic examples

• Hoppe (1983) Significance of exoenzymatic activities in the ecology of brackish water: measurements by means of methylumbelliferyl-substrates

Recent applications

• Thomson et al. (2020) Relative importance of phosphodiesterase vs. phosphomonoesterase activities for dissolved organic phosphorus hydrolysis in epi- and mesopelagic waters

Common calculations/conversions

• LAP rate (nmol N L^-1 h^-1) = Δ[MCA] / incubation time; calibrated from MCA standard curve.