Impact of bioavailability restrictions on microbially induced stable isotope fractionation. 2. Experimental evidence

Stable isotope fractionation analysis (SIFA) of contaminants is an emerging technique to characterize in situ microbial activity. The kinetic isotope effect in microbial degradation reactions, or enzyme catalysis, is caused by the preferential cleavage of bonds containing light rather than heavy isotopes. This leads to a relative enrichment of the heavier isotopes in the residual substrate pool. However, a number of nonisotopically sensitive steps preceding the isotopically sensitive bond cleavage may affect the reaction kinetics of a degradation process, thus reducing the observed (i.e., the macroscopically detectable) isotope fractionation. Low bioavailability of contaminants poses kinetic limitations on the biodegradation process and can significantly reduce the observed kinetic isotope fractionation. Here we present experimental evidence for the influence of bioavailability-limited pollutant biodegradation on observed stable isotope fractionation. Batch laboratory experiments were performed to quantify the toluene hydrogen isotope fractionation of Pseudomonas putida mt-2 (pWWO) subjected to different small concentrations of toluene with and without deuterium label, which corresponded to realistic environmental mass transfer scenarios. Detected isotope fractionations depended significantly on the toluene concentration, hence confirming the influence of substrate mass transfer limitation on observed isotope fractionation, hypothesized by Thullner et al. (Environ. Sci. Technol. 2008, 42,6544-6551). Our results indicate that the bioavailability of a substrate should be considered during quantitative analysis of microbial degradation based on SIFA.     

Quantification of sequential chlorinated ethene degradation by use of a reactive transport model incorporating isotope fractionation

Compound-specific isotope analysis (CSIA) enables quantification of biodegradation by use of the Rayleigh equation. The Rayleigh equation fails, however, to describe the sequential degradation of chlorinated aliphatic hydrocarbons (CAHs) involving various intermediates that are controlled by simultaneous degradation and production. This paper shows how isotope fractionation during sequential degradation can be simulated in a 1D reactive transport code (PHREEQC-2). 12C and 13C isotopes of each CAH were simulated as separate species, and the ratio of the rate constants of the heavy to light isotope equaled the kinetic isotope fractionation factor for each degradation step. The developed multistep isotope fractionation reactive transport model (IF-RTM) adequately simulated reductive dechlorination of tetrachloroethene (PCE) to ethene in a microcosm experiment. Transport scenarios were performed to evaluate the effect of sorption and of different degradation rate constant ratios among CAH species on the downgradient isotope evolution. The power of the model to quantify degradation is illustrated for situations where mixed sources degrade and for situations where daughter products are removed by oxidative processes. Finally, the model was used to interpret the occurrence of reductive dechlorination at a field site. The developed methodology can easily be incorporated in 3D solute transport models to enable quantification of sequential CAH degradation in the field by CSIA.     

Quantifying the degradation and dilution contribution to natural attenuation of contaminants by means of an open system rayleigh equation

Quantifying the share of destructive and nondestructive processes to natural attenuation (NA) of groundwater pollution plumes is of high importance to the evaluation and acceptance of NA as remediation strategy. Dilution as consequence of hydrodynamic dispersion may contribute considerably to NA, however, without reducing the mass of pollution. Unfortunately, tracers to quantify dilution are usually lacking. Degradation though of low-molecular-weight organic chemicals such as BTEX, chlorinated ethenes, and MTBE is uniquely associated with increases in isotope ratios for steady-state plumes. Compound-specific isotope analysis (CSIA) data are commonly interpreted by means of the Rayleigh equation, originally developed for closed systems, to calculate the extent of degradation under open system field conditions. For that reason, the validity of this approach has been questioned. The Rayleigh equation was accordingly modified to account for dilution, and showed that dilution contributed several to many times more to NA than biodegradation at a groundwater benzene plume. Derived equations also (i) underlined that field-derived isotopic enrichment factors underestimate actual values operative as a consequence of dilution, and (ii) provided a check on the lower limit of isotopic fractionation, thereby resulting in more reliable predictions on the extent of degradation.     

Extending the Rayleigh equation to allow competing isotope fractionating pathways to improve quantification of biodegradation

The Rayleigh equation relates the change in isotope ratio of an element in a substrate to the extent of substrate consumption via a single kinetic isotopic fractionation factor (alpha). Substrate consumption is, however, commonly distributed over several metabolic pathways each potentially having a different alpha. Therefore, extended Rayleigh-type equations were derived to account for multiple competing degradation pathways. The value of alpha as expressed in the environment appears a function of the alpha values and rate constants of the various involved degradation pathways. Remarkably, the environmental or apparent alpha value changes and shows non-Rayleigh behavior over a large and relevant concentration interval if Monod kinetics applies and the half-saturation constants of the competing pathways differ. Derived equations were applied to previously published data and enabled (i) quantification of the share that two competing degradation pathways had on aerobic 1,2-dichloroethane (1,2-DCA) biodegradation in laboratory batch experiments and (ii) calculation of the extent of methyl tert-butyl ether (MTBE) biodegradation shared over aerobic and anaerobic degradation at a field site by means of an improved solution to two-dimensional (carbon and hydrogen) compound-specific isotope analysis (CSIA).     

Chlorine and carbon isotopes fractionation during volatilization and diffusive transport of trichloroethene in the unsaturated zone

To apply compound-specific isotope methods to the evaluation of the origin and fate of organic contaminants in the unsaturated subsurface, the effect of physicochemical processes on isotope ratios needs to be known. The main objective of this study is to quantify chlorine and carbon isotope fractionation during NAPL-vapor equilibration, air-water partitioning, and diffusion of trichloroethene (TCE) and combinations of these effects during vaporization in porous media. Isotope fractionation is larger during NAPL-vapor equilibration than air-water partitioning. During NAPL-vapor equilibration, carbon, and chlorine isotope ratios evolve in opposite directions although both elements are present in the same bond, with a normal isotope effect for chlorine (ε(Cl) = -0.39 ± 0.03‰) and an inverse effect for carbon (ε(C) = +0.75 ± 0.04‰). During diffusion-controlled vaporization in a sand column, no significant carbon isotope fractionation is observed (ε(C) = +0.10 ± 0.05‰), whereas fairly strong chlorine isotope fractionation occurs (ε(Cl) = -1.39 ± 0.06‰) considering the molecular weight of TCE. In case of carbon, the inverse isotope fractionation associated with NAPL-vapor equilibration and normal diffusion isotope fractionation cancel, whereas for chlorine both processes are accompanied by normal isotope fractionation and hence they cumulate. A source of contamination that aged might thus show a shift toward heavier chlorine isotope ratios.     

Carbon and chlorine isotope ratios of chlorinated ethenes migrating through a thick unsaturated zone of a sandy aquifer

Compound-specific isotope analysis (CSIA) can potentially be used to relate vapor phase contamination by volatile organic compounds (VOCs) to their subsurface sources. This field and modeling study investigated how isotope ratios evolve during migration of gaseous chlorinated ethenes across a 18 m thick unsaturated zone of a sandy coastal plain aquifer. At the site, high concentrations of tetrachloroethene (PCE up to 380 μg/L), trichloroethene (TCE up to 31,600 μg/L), and cis-1,2-dichloroethene (cDCE up to 680 μg/L) were detected in groundwater. Chlorinated ethene concentrations were highest at the water table and steadily decreased upward toward the land surface and downward below the water table. Although isotopologues have different diffusion coefficients, constant carbon and chlorine isotope ratios were observed throughout the unsaturated zone, which corresponded to the isotope ratios measured at the water table. In the saturated zone, TCE became increasingly depleted along a concentration gradient, possibly due to isotope fractionation associated with aqueous phase diffusion. These results indicate that carbon and chlorine isotopes can be used to link vapor phase contamination to their source even if extensive migration of the vapors occurs. However, the numerical model revealed that constant isotope ratios are only expected for systems close to steady state.     

Transverse hydrodynamic dispersion effects on isotope signals in groundwater chlorinated solvents' plumes

The effects of transverse hydrodynamic dispersion on altering transformation-induced compound-specific isotope analysis (CSIA) signals within groundwater pollution plumes have been assessed with reactive transport modeling accommodating diffusion-induced isotope fractionation (DIF) and implementing different parameterizations of local transverse dispersion. The model reproduced previously published field data showing a negative carbon isotope pattern (-2 ‰) at the fringes of a nondegrading PCE plume. We extended the study to reactive transport scenarios considering vinyl chloride as a model compound and assessing, through a detailed sensitivity analysis, the coupled effects of transverse hydrodynamic dispersion (with and without DIF) and aerobic fringe degradation on the evolution of carbon and chloride isotope ratios. Transformation-induced positive isotope signals were increasingly attenuated with distance from the source and higher degradation rate. The effect of DIF on the overall isotope signal attenuation was greatest near the source and for low values of groundwater flow velocity, transverse dispersion coefficient, molecular weight, rate constant, and isotope fractionation factor, α, of the degradation reaction. Models disregarding DIF underestimate the actual α. The approximately twice larger DIF effect for chlorine than for carbon together with the low α for oxidation resulted in strong chlorine CSIA depletions for VC at the plume fringe.     

Beyond the Rayleigh equation: reactive transport modeling of isotope fractionation effects to improve quantification of biodegradation

The Rayleigh equation is commonly applied to evaluate the extent of degradation at contaminated sites for which compound-specific isotope analysis (CSIA) data are available. However, it was shown recently that (i) the Rayleigh equation systematically underestimates the extent of biodegradation in physically heterogeneous systems, while (ii) it overestimates biodegradation if sorption-based carbon isotope fractionation is relevant. This paper further explores these two isotope effects not captured by the Rayleigh equation by means of a numerical modeling approach. The reactive multicomponent transport simulations show that the systematic underestimation is considerably larger for fringe-controlled and Monod-type degradation reactions than for previously assumed redox-insensitive first-order degradation kinetics, while forthe nonsteady state front portion of plumes, the Rayleigh equation may falsely indicate the occurrence of and/or overestimate biodegradation. The latter anomaly results from carbon isotope fractionation during sorption. It occurs for both supply-controlled degradation at the plume fringe and slow, reaction-controlled degradation inside the plume core. The numerical model approach enables a more accurate interpretation of CSIA data and thereby improves the quantification of biodegradation processes.     

Fractionation of stable zinc isotopes in the zinc hyperaccumulator Arabidopsis halleri and nonaccumulator Arabidopsis petraea

Zn isotope fractionation may provide new insights into Zn uptake, transport and storage mechanisms in plants. It was investigated here in the Zn hyperaccumulator Arabidopsis halleri and the nonaccumulator A. petraea. Plant growth on hydroponic solution allowed us to measure the isotope fractionation between source Zn (with Zn(2+) as dominant form), shoot and root. Zn isotope mass balance yields mean isotope fractionation between plant and source Zn Δ(66)Zn(in-source) of -0.19 ± 0.20‰ in the nonaccumulator and of -0.05 ± 0.12‰ in the hyperaccumulator. The isotope fractionation between shoot Zn and bulk Zn incorporated (Δ(66)Zn(shoot-in)) differs between the nonaccumulator and the hyperaccumulator and is function of root-shoot translocation (as given by mass ratio between shoot Zn and bulk plant Zn). The large isotope fractionation associated with sequestration in the root (0.37‰) points to the binding of Zn(2+) with a high affinity ligand in the root cell. We conclude that Zn stable isotopes may help to estimate underground and aerial Zn storage in plants and be useful in studying extracellular and cellular mechanisms of sequestration in the root.     

Microbial respiration and diffusive transport of O2, 16O2, and 18O15O in unsaturated soils and geologic sediments

Molecular oxygen (O2) in unsaturated geologic sediments plays an important role in soil respiration, biodegradation of organic contaminants, metal oxidation, and global oxygen and carbon cycling, yet little is known about oxygen isotope fractionation during the consumption and transport of O2 in unsaturated zones. We used a laboratory kinetic cell technique to quantify isotope fractionation due to respiration and a numerical model to quantify both consumptive and diffusive fractionation of O2 isotopes at a field site comprised of unsaturated lacustrine sandy materials. The combined use of laboratory-based kinetic cell experiments and field-based isotope transport modeling provided an effective tool to characterize microbial respiration in unsaturated media. Based on results from the closed-system kinetic cells, O2 consumption and isotope fractionation were attributed to the alternative cyanide-resistant respiration pathway. At the field site, the modeled depth profiles for O2 and delta18O matched the measured in situ data and confirmed that the consumption of O2 was via the alternative respiration pathway. If the cyanide-resistant respiration pathway is indeed widespread in soils, its high oxygen isotope enrichment factor could help to explain the discrepancy between the predicted present-day Dole effect (+20.8/1000) and the observed Dole effect (+23.5/1000). Thus, further soil O2 isotope studies are needed to better characterize and model the fractionation of oxygen isotopes during subsurface respiration and the potential impact on the isotopic content of atmospheric O2.     

Hydrogen and carbon isotope fractionation during anaerobic biodegradation of aromatic hydrocarbons--a field study

The aquifer of a former manufactured gas plant site, highly contaminated by dissolved monocyclic, heterocyclic, and polycyclic aromatic hydrocarbons, was studied to evaluate the applicability of carbon and hydrogen isotope fractionation to prove ongoing biodegradation of these compounds even in complex aquifer settings. The loss of toluene, o-xylene, p,m-xylene, and 2-methylnaphthalene was accompanied by a considerable carbon isotope fractionation. Additionally, a strong 2H enrichment in residual o-xylene was detected. All isotope fractionations observed could be related to established biochemical degradation mechanisms, each involving a C-H bond cleavage in the rate-determining step. In contrast, other compounds such as 1-methylnaphthalene, methylbenzofuran, and acenaphthene exhibited a uniform stable carbon isotope composition. However, a decrease in concentration for these compounds was observed in the flowpath of the aquifer. High threshold concentrations of acenaphthene downgradient indicate that this contaminant is, if at all, only marginally biodegraded. Detailed analyses of xylenes provided support that compound specific isotope analyses and subsequent application of the Rayleigh model may provide a valuable basis to distinguish between different biodegradation mechanisms as well as dissolution processes in heterogeneous aquifers.     

Evaluation of isotopic enrichment factors for the biodegradation of chlorinated ethenes using a parameter estimation model: toward an improved quantification of biodegradation

A model was developed to predict the concentrations of chlorinated ethenes and ethene during sequential reductive dechlorination of tetrachloroethene (PCE) from stable carbon isotope values using Rayleigh model principles and specified isotopic enrichment factors for each step of dechlorination. The model was tested using three separate datasets of concentration and isotope values measured during three experiments involving the degradation of PCE to vinyl chloride (VC), trichloroethene (TCE) to ethene, and cis-1,2-dichloroethene (cDCE) to ethene. The model was then coupled to a parameter estimation method to estimate values for the isotopic enrichment factors of TCE, cDCE, and VC when they are intermediates in the dechlorination to ethene. The enrichment factors estimated for TCE and cDCE when they were intermediates in biodegradation experiments were close to or within the published range of enrichment factors determined from experiments where TCE or cDCE were the initial substrates. In contrast, the enrichment factors determined by parameter estimation for experiments in which VC was an intermediate in biodegradation experiments were consistently more negative (by approximately 10 per thousandth) than the most negative published enrichment factor determined from experiments where VC was the initial substrate. This finding suggests that the range of enrichment factors for VC dechlorination may not be as narrow as previously suggested (-21.5 per thousandth to -26.6 per thousandth) and that fractionation during VC dechlorination when VC is an intermediate compound may be significantly larger than when VC is the initial substrate. These findings have important implications both for the current practice of extrapolating laboratory-derived isotopic enrichment factors to quantify biodegradation of chlorinated ethenes in the field and for understanding the details of enzymatic reductive dechlorination.     

34S/32S fractionation during sulfate reduction in groundwater treatment systems: reactive transport modeling

Isotope ratio measurements provide a tool for indicating the relative significance of biogeochemical reactions and for constraining estimates of the extent and rate of reactions in passive treatment systems. In this paper, the reactive transport model MIN3P is used to evaluate sulfur isotope fractionation in column experiments designed to simulate treatment of contaminated water by microbially mediated sulfate reduction occurring within organic carbon-based and iron and carbon-based permeable reactive barriers. A mass dependent fractionation model was used to determine reaction rates for 32S and 34S compounds during reduction, precipitation, and dissolution reactions and to track isotope-dependent mass transfer during SO4 removal. The δ34S values obtained from the MIN3P model were similar to those obtained from the Rayleigh equation, indicating that there was not a significant difference between the conceptual models. Differences between the MIN3P derived α value and the Rayleigh equation derived value were attributed to minor changes in the dissolution and precipitation rate of gypsum and mathematical differences in the fitting models. The results indicated that the prediction of δ34S was fairly insensitive to differences in the fractionation factor at the concentration ranges measured in the current study. However, more significant differences would be expected at low sulfate conditions.     

Adsorptive fractionation of humic acid at air-water interfaces

By using a simple bubble column, the adsorption behavior of a commercial soil-humic acid (CHA) at air-water interfaces was investigated. At pH 4.0, the concentrations of the CHA exhibited clear gradients in the bubble column, and increased significantly along the column height; smaller concentration gradients were also observed at pH 6.0. These concentration profiles demonstrate the surface activity of humic acid and pH-dependent affinity toward air-water interfaces. Taking advantage of the bubble column method, we interestingly found that the adsorptive fractionation of the CHA at air-water interfaces did occur. The components with higher molecular weight and stronger UV absorptivity showed greater affinity toward air-water interfaces, despite that the fractionation pattern was reduced to a certain extent as solution pH increased. The organic carbon-normalized pyrene partition coefficient Koc values deviated from the corresponding values of original bulk solutions at both pH 4.0 and 6.0, and increased along the height of the column. Our results demonstrate the usefulness of the simple bubble column, and suggest that the adsorptive fractionation of humic acid at air-water interfaces might have implications for some natural environments and engineered systems where air-water interfaces exist extensively.     

Using chromium stable isotope ratios to quantify Cr(VI) reduction: lack of sorption effects

Chromium stable isotope values can be effectively used to monitor reduction of Cr(VI) in natural waters. We investigate effects of sorption during transport of Cr(VI) which may also shift Cr isotopes values, complicating efforts to quantify reduction. This study shows that Cr stable isotope fractionation caused by sorption is negligible. Equilibrium fractionation of Cr stable isotopes between dissolved Cr(VI) and Cr(VI) adsorbed onto gamma-Al2O3 and goethite is less than 0.04 per thousand (53Cr/52Cr) under environmentally relevant pH conditions. Batch experiments at pH 4.0 and pH 6.0 were conducted in series to sequentially magnify small isotope fractionations. A simple transport model suggests that adsorption may cause amplification of a small isotope fractionation along extreme fringes of a plume, leading to shifts in 53Cr/52Cr values. We therefore suggest that isotope values at extreme fringes of Cr plumes be critically evaluated for sorption effects. A kinetic effect was observed in experiments with goethite at pH 4 where apparently lighter isotopes diffuse into goethite clumps at a faster rate before eventually reaching equilibrium. This observed kinetic effect may be important in a natural system that has not attained equilibrium and is in need of further study. Cr isotope fractionation caused by speciation of Cr(VI) between HCrO4- and CrO4(2-) was also examined, and we conclude that it is not measurable. In the absence of isotope fractionation caused by equilibrium speciation and sorption, most of the variation in delta53Cr values may be attributed to reduction, and reliable estimates of Cr reduction can be made.     

Effects of trace element concentration on enzyme controlled stable isotope fractionation during aerobic biodegradation of toluene

The effects of iron concentration on carbon and hydrogen isotopic fractionation during aerobic biodegradation of toluene by Pseudomonas putida mt-2 were investigated using a low iron medium and two different high iron media. Mean carbon enrichment factors (epsilonc) determined using a Rayleigh isotopic model were smaller in culture grown under high iron conditions (epsilonc = -1.7+/-0.1%) compared to low iron conditions (epsilonc = -2.5+/-0.3%). Mean hydrogen enrichment factors (epsilonH) were also significantly smaller for culture grown under high iron conditions (epsilonH = -77 +/-4%) versus low iron conditions (EpsilonH = -159+/-11%). A mechanistic model for enzyme kinetics was used to relate differences in the magnitude of isotopic fractionation for low iron versus high iron cultures to the efficiency of the enzymatic transformation. The increase of carbon and hydrogen enrichment factors at low iron concentrations suggests a slower enzyme-catalyzed substrate conversion step (k2) relative to the enzyme-substrate binding step (k-l) at low iron concentration. While the observed differences were subtle and, hence, do not significantly impact the ability to use stable isotope analysis in the field, these results demonstrated that resolvable differences in carbon and hydrogen isotopic fractionation were related to low and high iron conditions. This novel result highlights the need to further investigate the effects of other trace elements known to be key components of biodegradative enzymes.     

Monitoring biodegradation of methyl tert-butyl ether (MTBE) using compound-specific carbon isotope analysis

Methyl tert-butyl ether (MTBE), the most common gasoline oxygenate, is frequently detected in surface water and groundwater. The aim of this study was to evaluate the potential of compound-specific isotope analysis to assess in situ biodegradation of MTBE in groundwater. For that purpose, the effect of relevant physical and biological processes on carbon isotope ratios of MTBE was evaluated in laboratory studies. Carbon isotope fractionation during organic phase/gas-phase partitioning (0.50 +/- 0.15@1000), aqueous phase/gas-phase partitioning (0.17 +/- 0.05@1000), and organic phase/aqueous-phase partitioning (0.18 +/- 0.24@1000) was small in comparison to carbon isotope fractionation measured during biodegradation of MTBE in microcosms based on aquifer sediments of the Borden site. In experiments with MTBE as the only substrate and a cometabolic experiment with 3-methypentane as primary substrate, MTBE became enriched in 13C by 5.1 to 6.9@1000 after 95 to 97% degradation. For both experiments, similar isotopic enrichment factors were obtained (-1.52 +/- 0.06 to -1.97 +/- 0.05@1000). Biodegradation of TBA, which accumulated transiently in the cometabolic microcosms, was also accompanied by carbon isotope fractionation, with an isotopic enrichment factor of -4.21 +/- 0.07@1000. This study suggests that carbon isotope analysis is a potential tool to trace in situ biodegradation of MTBE and TBA and thus to better understand the fate of these contaminants in the environment.     

Carbon isotope fractionation in the reductive dehalogenation of carbon tetrachloride at iron (hydr)oxide and iron sulfide minerals

Compound-specific isotope analysis (CSIA) is used increasingly in contaminant hydrology in the attempt to assess the nature as well as the extent of in situ transformation reactions. Potentially, variations of stable isotope ratios along a contaminant plume may be used to quantify in situ degradation. In the present study, the abiotic dehalogenation of CCl4 by Fe(II) present at the surface of different iron minerals has been characterized in terms of the reaction rates and carbon isotopic fractionation (delta13C) of carbon tetrachloride (CCl4) as well as the yields and isotopic signatures of chloroform (CHCl3), one of the main transformation products. The abiotic reductive dehalogenation of CCl4 was associated with substantial carbon isotopic enrichment effects. The observed enrichment factors, e, correlated neither with the surface-normalized reaction rate constants nor with the type of products formed but fell into two distinctly different ranges for the two principal groups of minerals studied. With iron (hydr)oxide minerals (goethite, hematite, lepidocrocite, and magnetite) and with siderite, the e-values for CCl4 dehalogenation were remarkably similar (-29 +/- 3 per thousand). Because this value matches well with the theoretical estimates for the cleavage of an aliphatic C-Cl bond, we suggest that dissociative electron transfer to CCl4 controls the reaction rates for this group of iron minerals. Conversely, CCl4 transformation by different preparations of the iron sulfide mackinawite was accompanied by a significantly lower carbon istotopic fractionation (e = -15.9 +/- 0.3 per thousand), possibly due to the presence of nonfractionating rate-determining steps or a significantly different transition state structure of the reaction. Isotopically sensitive branching of the reaction pathways (i.e., the effect of different product distributions on isotope fractionation of CCl4) did not play a significant role in our systems. The extensive data set presented in this study opens new perspectives toward an improved understanding of the factors that determine reaction mechanisms and isotopic fractionation of dehalogenation reactions by Fe(II) at iron containing minerals.     

Properties of wheat bran polyphenol oxidase

Polyphenol oxidase (PPO) obtained from wheat bran catalyzed the oxidation of 4-methyl catechol. Phenolic compounds found naturally in crude extract played role as an endogeneous substrate and activity of crude extract needed correction. Activity versus enzyme concentration gave a linear plot at high substrate concentration whereas a nonlinear plot was obtained at low substrate concentration which proved the presence of endogeneous substrate. Adsorption on celite and extraction with polyvinylpyrrolidone (PVPP) caused the removal of phenols. Adsorption of PPO on celite yielded a 4-fold increase in specific activity whereas extraction with PVPP yielded a 2.5-fold increase in specific activity compared to the crude extract. The kinetics of PPO catalyzed oxidation obeyed Michaelis-Menten model; Km and Vmax values were found as 218 mM and 99 microM/min, respectively. The enzyme was inhibited by ethyl alcohol, dithiothreitol (DTT) and isoproterenol and exhibited heat stability up to a temperature of 90 degrees C. The optimum pH of the enzyme was found to be 5.0.