Sod tJd Biochunt. Vol. 14. pp. 63 10 65. 1982 Printed m Great Brttain. All rights reserved
003%0717/82/010063-03SO3.00/0 Copyright 0 1982 Pergamon Press Ltd
SOIL UREASE ACTIVITY AND KINETICS Y. M. NOR* School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia (Accepted 20 Frhruuty
Summary-The activity and kinetic properties of urease in several Malaysian soils were examined. The values for K, and V,.,, of the soils computed according to the Hanes equation were in general agreement with other reports as far as magnitudes were concerned. A significant correlation between K, and V,,,
was also obtained. The urease activity of the soils was variable, and it was noted that expression of the activity as the time required to hydrolyze half of the applied urea has limited use in soils of low activity. In all soils studied, inhibition of urease activity was effectively achieved using Ag+, while Cuzc was only effective in two soils, and marginally effective in the other two soils. Urease inhibitors have potential appiications in reducing volatilization losses of ammonia derived from urea applied to soils. INTRODUCTION
The conversion of organic N to inorganic N through hydrolysis of urea to NH, and CO2 is an essential part of the N cycle and represents in important pathway in soils receiving applications of urea, urine and dung (Gasser, 1964). Compared to urease kinetics, urease activity, urea transformation and NH, volatilization in soils from various parts of the world have been more vigorously investigated. Although urea transformation and NH3 volatilization in Malaysian soils have been studied earlier (Watson et al., 1962; Rajaratnam and Purushothaman, 1973; Purushothaman and Joseph, 1975; Ng and Tay, 1977; Nor, 1981b) there is no report of any work on the kinetics of soil urease. Studies involving estimates of V,,.,, and K, for enzyme catalysed reactions obeying Michaelis-Menten kinetics apparently lack precision and accuracy and values obtained could vary according to the methods employed for computing (Roberts, 1977; Van Faassen, 1979). It is convenient to plot experimental data in one of the linear transformations for example the Lineweaver-Burk or double reciprocal plots. But because it is difficult to maintain both V and [S] free from errors in practice, selecting the most suitable linear transformation requires great care and necessitates application of the relevant statistical analysis for computation. Numerous methods for assaying urease activity are available. Some investigators have used techniques involving buffers, e.g. Tris-HCI, phosphate, etc., for incubation (Wall and Laidler, 1953; Tabatabai, 1973; Pettit et ai., 1976), while others have not (Paulson and Kurtz, 1969; Gould et al., 1973). However, the merits of either option have not been clearly established. I have studied soil urease activity and kinetics in some Malaysian soils.
1:2.5), cation exchange capacity by an ammonium acetate method (Chapman, 1965), organic C content as described by Allison (1965) and texture was determined by the hydrometer method (Day, 1965). The Hanes plot of [S]/o against [S] in Fig. 1 was obtained using the formula: [Sl/r = I&lo + [S]/v which gave a straight line with a slope of l/k’ and ordinal intercept of KJV (Roberts, 1977). The data obtained was fitted by the least squares method using unweighted linear regression analysis as per equation:
where b, the slope of the line gave the best fit to the data. The procedure to determine of K, and k’,,,,, involved addition of 1, 2, 3, 4 and 5 ml of urea substrate to log samples of soil giving urea concentrations on a dry soil basis of 1.7; 3.3; 5.0; 6.7 and ing determined (Nor, 1979). In the urease activity experiment, urea substrate (5 mg urea) was added to 10 g samples of soil and incubated at 37°C for various times, and the urea remaining then determined. To study the effect of Cu2+ and Ag’ on urea hydrolysis, 10 g samples of soil were treated with 120 ng g- ’ (soil basis) of either Cu2+ or Ag+. The substrate was then added and samples were incubated and urea determined. The controls consisted of only samples and the
Table 1. Analysis of soils Organic C CEC (m/e/100 g soil) (%)
Soil type” sii SC1 cl
1.23 0.98 2.25 1.68 1.47
25.3 23.6 20.5 15.0 17.5 10.4 4.0
pH 5.4 7.1 4.3 3.8 5.1 4.8 5.1
The soils used were surface samples (O-15 cm) selected to give a wide range of characteristics (Table 1). pH was determined with a glass electrode (soil: HZO,
Kanggar Keranji Selangor L. Itik B. Lima Kundor Hutan
* Present address: Department of Agronomy and Soils, Washington State University, Pullman, WA 99164, U.S.A.
“sil, silt loam; scl, sandy clay loam; cl. clay loam; Is loamy sand.
scl SC1 IS
6.2m~ urea-N s&I-’ for soil urease. Petitt et ~11. (1976) in a similar study obtained a I(, value of 52.3 mM urea in Tris-HCl buffer while a K, of 62.5m~ urea was recorded in the presence of phosphate buffer. Tabatabai (1973) reported lower K, values ranging from 1.>7.0mM urea in the presence of THAM buffer and gave Michaelis constants for ureases from various sources. Thus, it can be seen that the data from mv study were in general agreement with earlier investigation as far as magnitudes of K, and V,,, are concerned. But, it should be emphasized here that strict comparisons do not appear meaningful nor justified because of disparities in experimental approach, e.g. buffers, soil types, duration of incubation. Figure 2 shows reIationship between urea hydrolyzed (%) and time of incubation in 3 soils. In Selangor and Keranji soils, 28 and 91% respectively of added urea was hydrol~ed after 72 h incuba?ion. In Kanggar soil, however, almost all the urea was hydrolyzed after only 24 h. Thus, considerable differences in S(mM Ursa,Dry soil basls) urea hydrolyzing capacity were exhibited by the soils Fig. 1. Hanes plots of [S]/D against [S] for 3 soils in I: 1.5, studied. The capacity of the soil to hydrolyze urea is soil: solution. often referred to as the urease activity. Conversely, the urease activity has been defined as the time required substrate. Each value reported is the mean of duplito hydrolyze 50% of applied urea or t+ (Beri et al., 1978). When the experimental data was expressed in cate analyses. this manner, the calculated r$ values were 13.7, 39.9 and 132.5 h for Kanggar, Keranji and Selangor soils, RESULTS AND DISCUSSIONS respectively. The slopes of the curves, incidentally, Figure 1 shows a Hanes plot [S]/c against [SJ in were determined by application of the appropriate three soils-Kanggar, Keranji and Selangor soils. The statistical methods. It is evident that the urease activity of Selangor soil was very low as shown by the calculated K, values for the soils were 2.06, 1.73 and large projected value of t$ Expression of urease ac1.04m~ urea respectively, while that of I’,,,,, were 0.40, 0.22 and 0.14 mM urea hydrolyzed g soil - ’ h- ’ . tivity in this form is only useful in so far as soils with rather substantial urease activity are concerned. But The correlation coefficient between K, and V,,, was unfortunately, for those that have low activity its use0.96 (P < 0.05). fullness is limited because of the need for Ionger incuBeri et af. (1978) obtained K, values ranging from bation. It should be remembered that long incuba10.4 to 22.2 mM urea-N and IJ’“,:,,:,, ranging from 2.0 to tions are undesirable because of possible interference from microbial activity (Skujins, 1967). Many compounds both organic and inorganic have . Kanggar been patented as inhibitors of urease activity (Kiss et o Keranji af., 1975). In my study, two cations, Cu*+ and Ag’ 0 Salangor were tested with four soils. Ag+ was an effective inhibitor of urease activity as shown by the absence of any urea hydrolyzed under the ex~rime~tal conTable 2. Effects of Cu’+ and Ag’ on urea hydrolysis
Soil L. Itik B. Lima Kundor 6
Time of incubation,h Fig. 2. Plots of Y0urea hydrolyzed against time of incubation from G-72 h in 3 soils.
?; of added urea hydr~lysed
Control cu2 + Ag+ Control CU2’ Ag’ Control Cu2 + A%+ Control Cul’ Ag+
IO0 0 0 100 0 0 100 22.5 0 100 8.3 0
Urease activity and kinetics
ditions (Table 2). Other investigators, too, have reported a similar observation (Douglas and Bremner, 1971; Tabatabai and Bremner, 1972). Cuz+ on the other hand, achieved looO/, inhibition only with two soils, and recorded a slightly reduced inhibitory effect on urease activity in the other two soils. Compounds that inhibit urease activity may have a practical application in agriculture. This is related to the latter’s influence on volatilization losses of NH3 from urea especially when applied superficially to soils. Work (Nor, 1981a) on the effect of Cu2+ and Ag+ on volatilization of ammonia under laboratory conditions has shown that both cations effectively controlled volatilization losses in some soils. Thus, urease inhibitors have potential in controlling volatilization losses of NH3 from urea applied to soils.
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