Fluorescein diacetate hydrolysis

Enzyme activity (urease, amidase, dehydrogenase, pl-glucosidase, phosphatase, arylsulfatase, fluorescein diacetate hydrolysis) Laboratory incubation Indicates potential microbial activity and nutrient cycling reactions determined in nonstandard laboratory with specialized equipment highly spatially and temporally variable dependent upon organic inputs Dick et al. (1996) Parham et aL (2002)... 

Schnurer, J., and RosswaU, T. (1982). Fluorescein diacetate hydrolysis as a measure of total microbial activity in soil and litter. Appl. Environ. Microbiol. 43, 1256—1261. 

Swisher, R. G.C. Carroll. 1980. Fluorescein diacetate hydrolysis as an estimator of microbial biomass on coniferous needle surfaces. Microbial Ecol. 6 217-226. 

Fontvieille, D. A. Outaguerouine, A. Thevenot, D. R. Fluorescein diacetate hydrolysis as a measure of microbial activity in aquatic systems application to activated sludges. Environ. Technol. 1992, 13, 531-540. 

Leszczynska, M. Oleszkiewic, J. A. Application of the fluorescein diacetate hydrolysis as an acute toxicity test. Environ. Technol. 1996,17,79-85. 

Measurements other than respiration rate can also be used as indicators of soil microbial activity. These include measurements of the rate of multienzyme processes such as arginine ammonification rate (Alef and Kleiner 1995) fluorescein diacetate (FDA) hydrolysis rate (Alef 1995) and measurement of key endocellular enzymes such as dehydrogenase (Tabatabai 1994). 

Alef K (1995) Estimation of the hydrolysis of fluorescein diacetate. In Alef K, Nannipieri P (eds) Methods in Applied Soil Microbiology and Biochemistry. Academic Press, London, pp 232-238... 

Instruments of this type may also be used quite effectively to evaluate kinetics of time-dependent changes in foods, be they enzymatic or reactive changes of other types. The computerized data-acquisition capabilities of these instruments allow precise measurement of absorbance or fluorescence changes, often over very brief time periods ( milliseconds). This is particularly useful for analysis of fluorescence decay rates, and in measurement of enzymatic activity in situ. A number of enzyme substrates is available commercially which, although non-fluorescent initially, release fluorescent reaction products after hydrolysis by appropriate enzymes. This kinetic approach is a relatively underused capability of computerized microspectrophotometers, but one which has considerable capability for comparing activities in individual cells or cellular components. Fluorescein diacetate, for example, is a non-fluorescent compound which releases intensely fluorescent fluorescein on hydrolysis. This product is readily quantified in individual cells which have high levels of esterase [50]. Changes in surface or internal color of foods may also be evaluated over time by these methods. 

Breeuwer, P., Drocourt, J. L, Bunschoten, N., Zwietering, M. H., Rombouts, F. M. and Abee, T. Characterization of uptake and hydrolysis of fluorescein diacetate and carboxyfluorescein diacetate by intracellular esterases in Saccharomyces cerevisiae, which result in accumulation of fluores-... 

EPTC and Butvlate Fluorescein Diacetate Assay. Spectrophotometric determinations of the hydrolysis of fluorescein diacetate have been shown to be simple, rapid, and sensitive methods for determining microbial activity in soil (18). Essentially, the hydrolytic cleavage of diacetate from fluorescein is responsible for the reaction products including fluorescein, which may be detected spectrophotometrically at 490 nm. This method is somewhat nonspecific in that it is indicative of overall activity of several enzymes (protease, lipase, esterase) rather than of a specific class of enzymes. Enzyme activity may be influenced by subtle pH changes in the sample since abiotic hydrolysis of fluorescein diacetate may occur. Also, an associated lag phase in soil hydrolytic activity must be accounted for in each assay. 

A wide range of bacteria and fungi, and some protozoan, algal and mammalian cells have the ability to hydrolyse fluorescein diacetate. Recently Schnurer and Rosswall have used this substrate to develop an assay for esterase activity in soil and straw based on the spectrophotometric determination of extracted fluorescein. The amounts formed increased linearly with time (up to 3h). The rates of hydrolysis were directly proportional to the amounts of soil used, to the amounts of fungal mycelium (Fusarium culmorum) or bacterial biomass (Pseudomonas denitrifleans) in buffered (pH 7.6) pure culture suspensions, and, in the case of fungi, to the amounts added to autoclaved soil, which alone was inactive. Esterase activities correlated with respiratory (O2 uptake) activities for samples from different depths in the soil profile. 

A variety of techniques have been used to estimate microbial biomass in natural habitats. Visual measurements of cell volume are laborious, and subject to a variety of errors when converted to dry-weight equivalents (Jones Mollison 1948 Carroll et al. 1980). Hydrolysis of fluorescein diacetate has been shown to be correlated to microbial biomass, especially for epiphytic fungi (Swisher Carroll 1980). Other methods of sampling include dilution plating of tissue homogenates or surface wash fluids, incubation of leaves or other biological... 

Bruheim, P Eimhjellen, K. Effects of non-ionic surfactants on the uptake and hydrolysis of fluorescein diacetate by alkane-oxidizing bacteria. Can. J. Microbiol. 2000,46, 387-390. 

Heslop-Harrison, J. Heslop-Harrison, Y. Evaluation of pollen viability by enzymically induced fluorescence intracellular hydrolysis of fluorescein diacetate. Stain Technol. 1970,45,115-120. 

Alarcon-Gutierrez, E. Hoch, C. Ruaudel, F. Criquet, S. Non-enzymatic hydrolysis of fluorescein diacetate (FDA) in a Mediterranean oak (Quercus ilex L.) litter. Eur. J. Soil Sci. 2008,59,139-146. 

Hofmatm, J. Semetz, M. A kinetic study on the enzymatic hydrolysis of fluorescein diacetate and fluorescein di-P-D-galactopyranoside. Anal. Biochem. 1983,131,180-186. 

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