Soil Science Society of America Journal 64:215-221 (2000)
© 2000 Soil Science Society of America
DIVISION S-3-SOIL BIOLOGY & BIOCHEMISTRY
Arylamidase Activity of Soils
V. Acosta-Martíneza and
M.A. Tabatabaia
a Dep. of Agronomy, Iowa State Univ., Ames, IA 50011-1010 USA
malit{at}iastate.edu
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ABSTRACT
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The enzyme amino acid arylamidase [
-aminoacyl-peptide hydrolase (microsomal) EC 3.4.11.2] catalyzes the release of an N-terminal amino acid from peptides, amides, or arylamides. Because of the presence of such substrates in soils, it is likely that this enzyme is involved in N mineralization. We report on the detection of the activity of this enzyme in soils and describe a precise and accurate method for its assay. This method involves colorimetric determination of the ß-naphthylamine produced when soil is incubated with L-leucine ß-naphthylamide in 0.1 M THAM [tris(hydroxymethyl)aminomethane] buffer (pH 8.0) at 37°C for 1 h. The ß-naphthylamine that is produced is extracted with ethanol and converted into an azo compound by reacting with p-dimethylaminocinnamaldehyde, and the absorbance of the color is measured at 540 nm. This enzyme has its optimal activity at pH 8.0 and is inactivated at temperatures above 60°C. Preheating soil samples for 2 h at temperatures ranging from 20 to 120°C before assay showed that this enzyme is stable up to 40°C in field-moist soils and up to 60°C in air-dried samples. The Km values of arylamidase activity in seven surface soils ranged from 0.19 to 0.35 mM. The temperature response followed Arrhenius equation over the range from 20 to 50°C. The activation energy values ranged from 30.6 to 49.8 kJ mol-1 for field-moist soils and from 26.2 to 32.4 kJ mol-1 for their air-dried counterparts. The means of temperature coefficients (Q10) ranged from
. Several compounds inhibited the activity of this enzyme in soils.
Abbreviations: THAM, [tris(hydroxymethyl)aminomethane]
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INTRODUCTION
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THE ENZYME AMINO ACID ARYLAMIDASE [-aminoacyl-peptide hydrolase (microsomal) EC 3.4.11.2] catalyzes the hydrolysis of an N-terminal amino acid from peptides, amides, or arylamides. Arylamidase is widely distributed in the tissues and body fluids of all animals (Hiwada et al., 1980), plants, and microorganisms (Appel, 1974). The chemical nature of N in soils is such that a large proportion (15–25%) of organic N is often released as NH+4 by 6 M HCl hydrolysis. The information available suggests that a portion of the released NH+4 is derived from amino acid residues in linear amides, and arylamides of soil organic matter (Sowden, 1958). Several papers have been published on linear amidase in soils and on the enzyme involved in hydrolysis of amides and amino acids such as asparagine, aspartic acid, and glutamine (Frankenberger and Tabatabai, 1980, 1991a, 1991b; Senwo and Tabatabai, 1996), but the possibility of the presence of arylamidase in soils has not been explored. The activity of this enzyme in soils deserves investigation because present knowledge indicates that a variety of arylamides are present in soils (Stevenson, 1994). Arylamidase may play an important role as an initial limiting step in mineralization of organic N in soils. Thus, understanding the environmental controls on the activity of this enzyme in soil is important for better understanding the N-cycling process. This enzyme is capable of hydrolyzing the neutral amino acid ß-naphthylamides or p-nitroanilides according to the following reaction (using the amino acid L-leucine as an example):
The objectives of the present work were (i) to develop a simple and sensitive method for the assay of arylamidase in soils and to ascertain the factors affecting the observed activity, and (ii) to determine the kinetic parameters of the reaction catalyzed by this enzyme. The method developed involves colorimetric determination of the ß-naphthylamine produced by arylamidase activity when soil is incubated with 0.1 M THAM buffer (pH 8.0) and L-leucine ß-naphthylamide hydrochloride at 37°C for 1 h. The factors studied included pH, substrate concentration, amount of soil, time of incubation, temperature of incubation, air-drying of field-moist soils, preheating temperature, and selected inhibitors. The Km and Vmax values, energy of activation, and temperature coefficients were calculated.
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Material and methods
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Soils and their Properties
The soils (Table 1)
used were surface samples (0–15 cm) selected to provide a wide range of chemical and physical soil properties. In the properties reported in Table 1, pH was determined by a combination glass electrode (soil/water or soil/0.01 M CaCl2 ratio, 1:2.5), organic C by the Mebius method (Mebius, 1960), total N by the semimicro-Kjeldahl procedure (Bremner and Mulvaney, 1982), and particle-size distribution by the pipette method (Kilmer and Alexander, 1949).
Reagents
THAM buffer (0.1 M, pH 8.0)
Prepared by dissolving 2.44 g of tris(hydroxymethyl) aminomethane (THAM buffer, Fisher Scientific, Chicago) in about 50 mL of water, adjusting the pH by titration with 
0.05 M H2SO4, and diluting the solution to 200 mL with water.
L-leucine ß-naphthylamide solution (8.0 mM)
Prepared by dissolving 0.2342 g of hydrochloride salt of L-leucine ß-naphthylamide (Sigma Chemical, St. Louis) in water and adjusting the volume to 100 mL with water.
Ethanol
(95%).
Acidified ethanol (0.26 M HCl)
Prepared by adding 4.32 mL of concentrated HCl to ethanol and adjusting the volume to 200 mL with ethanol.
p-Dimethylaminocinnamaldehyde solution (0.6 mg mL-1)
(Sigma Chemical, St. Louis) Prepared by dissolving 0.12 g of p-dimethylaminocinnamaldehyde in ethanol and adjusting the volume to 200 mL with ethanol.
Standard ß-naphthylamine stock solution (125 µg mL-1)
Prepared by dissolving 12.5 mg of ß-naphthylamine (Sigma Chemical Co., St. Louis) in 75 mL deionized water containing 5 mL of ethanol in a 100-mL volumetric flask, and adjusting the volume with deionized water.
Standard ß-naphthylamine working solutions
Prepared by transferring 1, 2, 3, 4, 5, or 6 mL of the standard ß-naphthylamine stock solution (125 µg mL-1) into a 25-mL volumetric flask, and adjusting the volume with deionized water. These standard solutions contain 5, 10, 15, 20, 25, or 30 µg of ß-naphthylamine mL-1, respectively.
Assay of Arylamidase Activity
A 1-g soil sample (air-dried, <2mm) in a 25-mL Erlenmeyer flask was treated with 3 mL of 0.1 M THAM buffer (pH 8.0) and 1 mL of 8.0 mM L-leucine ß-naphthylamide hydrochloride. The flask was swirled for a few seconds to mix the contents and was stoppered and placed on a shaker in an incubator (37°C) for 1 h. After incubation, the reaction was stopped by adding 6 mL of ethanol (95%). The soil suspension was immediately mixed and transferred into a centrifuge tube and centrifuged for 1 min at 17000 x g. The supernatant was transferred to a test tube to prevent any further hydrolysis of the substrate, and a 1-mL aliquot of this supernatant was treated (in a second test tube) with 1 mL of ethanol, 2 mL of acidified ethanol, and 2 mL of the p-dimethylaminocinnamaldehyde reagent. The solution was mixed on a vortex mixer, after adding each of the reagents. The intensity of the resulting red azo compound was measured at 540 nm (Hiwada et al., 1977). The reaction involved is as follows:
In our assay method, when the color intensity of the red azo compound exceeded the concentration of the highest ß-naphthylamine standard, an aliquot of the red azo compound was diluted with ethanol until the reading was within the limits of the calibration graph. The calibration graph was prepared by treating 1 mL of each of the standard working solutions in a test tube with 1 mL of ethanol, 2 mL of the acidified ethanol, and 2 mL of reagent containing p-dimethylaminocinnamaldehyde.
Controls were included as described for the assay, but the 1 mL of the substrate was added after incubation. Subsets of samples were used to study the factors affecting the activity of this enzyme. These were selected to give ranges in the activity values and to avoid overlapping of the curves obtained. Unless otherwise indicated, all results reported are averages of duplicate assays on air-dried soils and are expressed on a moisture-free basis. Moisture was determined from the loss in weight after drying at 105°C for 36 h. At all data points reported in the figures, the differences between the duplicate values were smaller than the point size.
An alternative method is available for colorimetric determination of the ß-naphthylamine produced (Goldbarg and Rutenburg, 1958). This method involves diazotization of the ß-naphthylamine released with NaNO2, decomposition of the excess NaNO2 with ammonium sulfamate, and conversion of ß-naphthylamine to a blue azo compound at pH 1.2 with N-(1-naphthyl) ethylenediamine dihydrochloride solution. The absorbance of the blue azo compound is measured at 700 nm. This method, however, is complicated and tedious. Therefore, we evaluated the optimal conditions for development of the red azo compound. The color of the red azo compound produced from the reaction described is stable for at least 24 h. The results obtained will be discussed under subheadings according to the factors studied.
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Results and discussion
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Buffer pH
To ascertain the optimal pH for arylamidase activity in soils, the activity was assayed at 2 mM of L-leucine ß-naphthylamide in the presence of THAM buffer at pH values ranging from 5 to 10. The rate of ß-naphthylamine produced was optimal at buffer
(Fig. 1)
. This value agrees with the range (6.1–8.0) reported by Appel (1974) for this enzyme purified from human and animal organs, plants, and microorganisms. The agreement of the optimal pH value (8.0) with the upper limit, but not with the lower limit, reported for this enzyme in other biological materials is expected because it is well known that the pH optima of enzymes in solutions are about 1.5 pH units lower than the same enzyme in soils (McLaren and Estermann, 1957). A shift in pH optimum to higher values in solution occurs because the Bronsted acidity at the clay surface is significantly greater than in the bulk solution (Boyd and Mortland, 1990).
Substrate Concentration and Amount of Soil
The initial rates of arylamidase activity in soils were measured at various substrate concentrations (Fig. 2)
. The reaction velocity increased with increases in the substrate concentration, and showed that the concentration (2 mM) adopted was satisfactory for the assay of arylamidase activity in soils. At this concentration, the reaction essentially followed zero-order kinetics. The rate of the reaction at this substrate concentration was, therefore, dependent on the enzyme concentration in the 1 g of soil used, evident from the levels of activity of the soils shown in Fig. 2.
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Fig. 2 Effect of substrate concentration on release of ß-naphthylamine in assay of arylamidase activity in soils
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Studies on the specificity of arylamidase showed that in addition to L-leucine ß-naphthylamide, this enzyme hydrolyzes L-alanine ß-naphthylamide. Results reported in Table 2 show that except for the activity of the Harps soil, both substrates gave similar activity values.
A linear relationship between the amount of soil and the amount of ß-naphthylamine produced showed that 1 g of soil was satisfactory for assaying the activity of this enzyme (data not shown). This is further evidence that the procedure described measures arylamidase activity and that neither the substrate concentration nor the products released influence the reaction rate of this enzyme in soils.
Time and Temperature of Incubation
The relationship between the amount of product formed and the time of incubation is usually linear in enzyme-catalyzed reactions in soils, as long as the enzyme is stable and retains its full activity (Frankenberger and Tabatabai, 1980). The observed linear relationship indicated that the method developed is not complicated by microbial growth or assimilation of enzymatic products by microorganisms (Fig. 3)
. Formation of ß-naphthylamine in the soil studied was a zero-order reaction for at least 4 h of incubation. The incubation time (1 h) allowed ample time for ß-naphthylamine to accumulate. It is not necessary to incubate the soil–substrate–buffer mixture for 4 h because results reported in Fig. 3 show that a much shorter incubation time can be adopted (e.g., 1 h).
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Fig. 3 Effect of time of incubation on release of ß-naphthylamine in assay of arylamidase activity in soils
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In general, enzyme-catalyzed reactions proceed at faster rates with increasing temperature until a temperature is reached above which the enzyme activity decreases due to denaturation. Optimal arylamidase activity occurred at 60°C under the assay conditions described (Fig. 4)
. Denaturation occurred at temperatures >60°C for both field-moist and air-dried soils, which is similar to those of asparaginase (Frankenberger and Tabatabai, 1991b), amidase (Frankenberger and Tabatabai, 1980), arylsulfatase (Tabatabai and Bremner, 1970), rhodanese (Tabatabai and Singh, 1976), and phosphodiesterase (Browman and Tabatabai, 1978) activities in soils. In general, the temperature needed to inactivate an enzyme in soil is about 10°C greater than the temperature needed to inactivate the same enzyme in the absence of soil (Skujins, 1967).
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Fig. 4 Effect of incubation temperature on release of ß-naphthylamine in assay of arylamidase activity in soils; (A) field-moist soils; (B) air-dried soils
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The activity of arylamidase was assayed at 37°C because this temperature has been used extensively for assay of other enzymes in many biological materials, including soils (Tabatabai, 1994), and because preliminary tests indicated that at temperatures <30°C, it could be difficult to quantitatively detect the low levels of activity exhibited by some soils, especially sandy soils. Tests with other soils showed that it was not necessary to use a higher temperature to obtain precise results under the conditions of the assay method described (Fig. 4).
The temperature used in drying field-moist soils and storing air-dried samples affect the enzyme activities (Tabatabai, 1994). The effect of temperature on the stability of arylamidase in field-moist and air-dried soils has not been studied. In this work, the soil samples were exposed to temperatures ranging from 20 to 120°C for 2 h, and the arylamidase activity was assayed at 37°C. Results showed that the activity of this enzyme was stable up to 40°C in field-moist soils and up to 60°C in air-dried soils (Fig. 5)
, suggesting that air drying of field-moist soil samples contributes to the stabilization of the enzyme protein. The rate of inactivation of this enzyme by temperature was much faster in field-moist than in air-dried soils. Arylamidase activity was completely destroyed in field-moist soils heated at 110 to 120°C, but the air-dried soils contained residual activity after heating at 120°C for 2 h.
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Fig. 5 Effect of preheating temperature on release of ß-naphthylamine in assay of arylamidase activity in soils; (A) field-moist soils; (B) air-dried soils
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In calculating the activation energy of soil enzymes, it is assumed that the incubation temperature (20–60°C for arylamidase, Fig. 4) has no effect on the stability of the enzyme. The results reported in Fig. 5B support the validity of this assumption.
Activation Energy and Kinetic Parameters
Temperature dependence of enzyme-catalyzed reactions is well documented. The dependence of the rate constant on temperature (below the inactivation temperature) of an enzyme-catalyzed reaction can be represented by the Arrhenius equation:
 | (1) |
where k is the rate constant, A is the pre-exponential factor, Ea is the activation energy, R is the gas constant, and T is the absolute temperature in K. The logarithmic transformation of the Arrhenius equation is expressed as follows:
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The activation energy can be calculated from a plot of log k (or apparent values or any parameter that is proportional to the rate constant) against 1/T (Segel, 1975, p. 932). The Arrhenius equation plot for arylamidase activity in the soils studied was linear between 20 and 50°C (Fig. 6)
. The activation energies of the enzyme reaction in the soils were obtained from the slope, and the values for field-moist and air-dried soils ranged from 30.6 to 49.8 kJ mol-1 and from 26.2 to 32.4 kJ mol-1, respectively (Table 3)
. These values are within the ranges reported for other soil enzymes (Tabatabai, 1994). The means of Q10 values for arylamidase in four soils for temperatures between 20 and 40°C ranged from 1.32 to 1.71 (
). These values are within the ranges reported for other soil enzymes, which are normally <2 (Tabatabai, 1994).
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Fig. 6 Arrhenius equation plot of arylamidase activity in soils; (A) field-moist soils, (B) air-dried soils (
)
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Three transformations of the Michaelis–Menten equation applied to arylamidase activity values obtained as a function of L-leucine ß-naphthylamide concentration are shown in Fig. 7
. The straight lines shown are those calculated by regression analysis. By using the Lineweaver–Burk plot, the Km values for arylamidase in four soils ranged from 0.19 to 0.35 mM (Table 4)
, suggesting high affinity of the enzyme for its substrate. These Km values are lower than those reported (6.7–17.9;
for eight soils) for linear amidase in soils by using formamide as a substrate (Frankenberger and Tabatabai, 1980). These values, however, are one order of magnitude greater than those reported (5.5–8.7 x 10-2 mM) for arylamidase extracted from several human organs (Hiwada et al., 1977). The greater Km values of arylamidase activity in soils compared with those reported for protein preparations is the result of enzyme adsorption on the clay–organic matter complexes (Tabatabai, 1994). That is because the interaction between the adsorbed enzyme and its substrate in solution is less than that of the free enzyme. Therefore, greater substrate concentration is required for an adsorbed enzyme to achieve the same reaction velocity as that of a free enzyme in solution. The Vmax values of the corresponding soils ranged from 15.4 to 69.8 x 10-2 mmol ß-naphthylamine kg-1 of soil h-1 (Table 4). Generally, the three methods of calculation showed similar results. Each transformation gives different weight to errors in the variables (Dowd and Riggs, 1965), and this is reflected in the variation of the estimated Km and Vmax values obtained for any soil by using the different plots. A similar observation has been reported for kinetic analysis of the reactions catalyzed by phosphodiesterase in soils (Browman and Tabatabai, 1978) and by linear amidase in soils (Frankenberger and Tabatabai, 1980).
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Fig. 7 The three possible linear plots of the Michaelis–Menten equation for arylamidase activity in soils. Substrate concentration (S) is expressed in mM and the reaction velocity in mmol of ß-naphthylamine released kg-1 of soil h-1
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Table 4 Km and Vmax values of arylamidase activity in soils calculated by using three linear transformations of the Michaelis–Menten equation
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Effect of Various Treatments
Tests were performed to identify possible inhibitors and activators of the reaction catalyzed by arylamidase because such tests would provide useful information about the structural requirements of the enzyme–substrate interaction. Treatment of soils with toluene, formaldehyde, dimethylsulfoxide, HgCl2, or iodoacetic acid inhibited arylamidase activity, and autoclaving completely destroyed the enzyme protein in soils (Table 5)
. The inhibition observed by treatment of soils with each of the last three inhibitors confirm the finding reported by Marks et al. (1968) that the active sites of this enzyme contain sulfhydryl groups.
Precision of Method
The method developed allowed for quantitative determination of the ß-naphthylamine produced. Arylamidase activity in soils ranged from 13.5 to 95.2 x 10-2 mmol ß-naphthylamine kg-1 of soil h-1. The coefficients of variation of the method described were generally 
4% (Table 6)
and comparable to other methods used for assay of enzymes in soils (Burns, 1978; Tabatabai, 1994).
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NOTES
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Journal paper J-18234 of the Iowa Agric. and Home Econ. Exp. Stn., Ames. Projects 3264, 3338, and 3391. This work was partly supported by the Biotechnology By-products Consortium of Iowa.
Received for publication January 21, 1999.
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REFERENCES
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ABSTRACT
INTRODUCTION
