Effect of Agricultural Exploitation on the Activity of Alkaline Phosphatase and Its Kinetic Properties in Some Soils

In order to study the role of agricultural exploitation in the activity of alkaline phosphatase in the province of Diwaniyah, six sites that differ in some of their chemical, physical and biological properties were selected (Diwaniyah, Sunniya, Shamiya, Daghara, Afak and Al Budair). Three types of soils were chosen in each location, namely, orchard soil, field soil and jungle soil. The kinetic parameters of the enzymes maximum velocity (Vmax) and Michaelis constant (Km) were estimated in all study soils using increasing concentrations of the controlled substance. The results present that the highest efficacy of this enzyme is in the orchard soil of all sites except the location of the center Diwaniyah and Shamia. The average efficacy values ranged between (208.11 234.95) μg P-Nitro phenol. g -1 soil. 1 hour -1 . The highest value of maximum velocity (Vmax) recorded at the field soil of Al-Shamia site (108.57) μg P-Nitro phenol. g -1 soil. 1 hour -1 . The lowest value recorded at the soil of a jungle field at the Sunniya location (49.62) μg P-nitro phenol. g -1 soil. 1 hour -1 . While the orchard soil of Al-Budair had the highest value of the Michaelis constant (Km) of (85.90) mM, the orchard soil of the Sunniya location had the lowest value (24.34) mM. Keyword: Agricultural Exploitation, L-Asparaginase , Vmax, Michaelis constant.

QJAS Al-Qadisiyah Journal For Agriculture Sciences ISSN: 2618-1479 Volume 9, No.1 ,(2019), PP 120-135 http://qu.edu.iq/jouagr/index.php/QJAS/index Page 121 | http://qu.edu.iq/jouagr/index.php/QJAS/index Michaelis constant and the maximum velocity of the reaction vary depending on the enzyme surrounding conditions. The values of the ionization constant do not rely on the concentration of the enzyme but on its efficacy. Therefore, it is an important measure the enzyme dynamics. The Michaelis constant uses to measure the link to the substance of the interaction. The lower the Michaelis constant value, the greater the homogeneity between the substance of the reaction and the enzyme [17], clarified that high value of Km , which is also called the high affinity constant, means that there is poor indexation between the enzyme and the subject matter. This is due to the strong impedance of the enzyme by the soil components [18]. found that increasing Vmax values of the basal phosphatase in two different soils, the first is loamy clay and the second loamy sand, the maximum velocity values were (522.7 and 398.5) μg PNP-1 soil. 1 hour-1, respectively. The difference in these values is due to the high efficacy of the enzyme in the soil. Km values were low for both soils (1.3 and 1.2), respectively. The reason is either the way the enzyme is evaluated or the difference in the enzyme environment and the soil environment or both. Since there is a lack of studies on the role of agricultural exploitation in the activity of enzymes and kinetics, the study aims to the fellows: 1. Determination of the activity of Alkaline Phosphatase at different agricultural exploitation sites. 2. Determination of kinetic parameters of the (Vmax) maximum velocity and the Michaelis constant (Km) of the Alkaline Phosphatase.

II. Materials and Methods
Soil samples were collected from six locations that differ in some of their chemical, physical and biological characteristics in the Diwaniyah province (Diwaniyah, Sunniya, Shamiya, Daghara, Afak and Al Budair). Three types of soils within the same location that have been different exploited for agricultural purposes were selected, which are orchard, field, and bushes (table 1). Soil samples were randomly taken from the surface layer (0-30) cm from several different spots to each location and stored in polyethylene bags then transferred to the laboratory. They have undergone air-dried, grinded, and sifted with a 2 mm diameter sieve then mixed well for homogeneity. Some chemical, physical, and biochemical analysis have been carried out ( Table 2, 3, 4, 5, 6 and 7).

III. Results and Discussion
Physical properties volumetric distribution of soil separators Estimated according to the international pipette method according to the method in Black, 1965a. Bulk density Estimated according to the Core Sample method that mentioned in Black 1965a [19]. Chemical properties Soil pH Measure in 1: 1 (soil: water) extract using a pH-meter using the Black 1965a Method. Electrical conductivity (EC) It was estimated at 1: 1 (soil: water) extract using an ECmeter according to the method in Black, 1965a. Cation exchange capacity CEC Estimated by Papanicolaou method ,1976. Through soil saturation with calcium chloride (0.1) standard solution at pH = 7 and displacement with sodium nitrate (0.1) standard. CalciumCarbonate CaCO 3 Calcium carbonate was measured by calculating the loss of carbon dioxide by treating the soil with hydrochloric acid (3 standards), according to the method in Black, 1965a. Gypsum CaSo4 Estimated through sedimentation by acetone and by the method given in Black, 1965b. Positive and negative dissolved ions Estimated in 1: 1 (soil: water) extract according to the methods stated in Black, 1965b [20].
Sodium Na + and potassium K + Estimated by using a flame photometer device. Calcium Ca+2 and magnesium Mg+2 Estimated by the titration with Na 2 EDSA. Chloride (Cl) estimated by the titration with silver nitrate 0.005 standards. Sulfates (SO4-2) Estimated according to turbidity method by using barium chloride and through the Spectrophotometer device. Total nitrogen Estimated by digesting soil samples with concentrated sulfuric acid then using micro-Kjeldahl steam distillation device according to a method of Bremner, 1965. That mentioned in Black, 1965b. Available Phosphorus available soil phosphorus was extracted using 0.5 molars of NaHCO3 according to Olsen method.
The color was developed with ammonium polysaccharides and ascorbic acid and was evaluated using the Spectrophotometer according to Page et al, 1982. Available potassium Soil potassium was extracted by using (1) molar of ammonium acetate and then extracted potassium estimated by Flame-photometer device according to the method in Page et al ,1982. Organic matter Organic matter was estimated according to the method of Walkely-Black, Black ,1965b. by oxidation with potassium dichromate solution with a concentrated sulfuric acid, and reverse titration with ferrous sulfate using D-phenylamine. Determination of the number of bacteria and total fungi A total number of fungi in the soil were estimated by dilution method and counting. 10 grams of soil and were transferred to a dilution bottle containing 90 ml distilled and sterilized water. After that, 1 ml was removed and transferred to another bottle containing 90 ml distilled and sterile water. The dilution process continued to obtain a dilute chain from (10-1 to 10-7). Dilutions of 10-5, 10-6 and 10-7was used to estimate the numbers of bacteria, which were grown on Nutrient Agar medium in accordance with the [20] method.
To estimate the number of fungi, dilutions of 10 -3 , 10 -4 , and 10 -5 were taken was grown in Martin medium, according to Rashidi ,1987. Determination of the activity of alkaline phosphatase in soil The efficacy of the alkaline phosphatase was estimated according to Eivazi and Tabatabai ,1977 method. It was done by placing 1 g of soil in a 50 mL flask with 0.2 mL of toluene and 4 ml of Modified universal buffer (MUB) (boric acid, citric acid, salicylic acid, And THAM with pH= 11 and add (1) mL of P-nitro phenyl phosphate as an enzyme-dependent substance. After that, it incubates at a temperature of (37O) for an hour. After incubation, (1) ml of potassium chloride solution 0.5 molars and 4 ml of 0.5 mM sodium hydroxide were added and the soil suspension filtered. The efficiency of the enzyme is estimated by the amount of P-nitro phenol released, which is measured by the spectrophotometer at a wavelength of (420) nanometres. The efficacy of this enzyme is calculated according to the following equation: = p-nitro phenol (µg. g-1 dwt . h-1) Where: C = concentration of P-nitrophenol (μg.ml-1) V = soil suspended volume (ml) dwt = dry weight for (1) gram wet soil weight SW = weight of wet soil (g) t = incubation time (1 hour).
Study of the kinetic parameters of alkaline phosphatase Vmax and KM of the basal phosphatase enzyme were estimated by studying the effect of the difference in the concentration of the controlled substance in the activity of the enzyme. Seven concentrations of the controlled substance (12.5, 25, 50, 100, 125 and 150) mM were used, according to [21] method. The enzyme activity was estimated according to Eivazi and Tabatabai     μg P-Nitro phenol. g -1 soil. 1 hour -1 . Orchard soils had the highest values an all of the studied locations except for the location of Diwaniyah and Shamiya. Field soils that were cultivated with eggplant and cowpea plants had the height values in these two locations.  [22] who found that leguminous plants secrete more basal phosphatase enzyme than cereal crops. This because the high need for phosphorus in legumes, due to vagueness stabilizations of nitrogen compared to cereal crops , [23]. The results also indicate that the nature of agricultural exploitation has had a direct effect on the increase or decrease of the efficiency of the enzyme, [24] and [25]. Table (8) shows the average enzymatic activity. The highest values were recorded in the soils of the Shamiya location (234.95) μg P-Nitro phenol. g -1 soil. 1 hour -1 , and the lowest in the soils of the location of Afak (208.11) μg P-Nitro phenol. g -1 soil. 1 hour -1 . Comparing the values of this enzymatic activity in these soils, the highest efficacy values were found in soils with low electrical conductivity (EC) and the lowest in soils with high electrical conductivity (Tables 2-7). The effect of number total bacteria in the activity of alkaline phosphatase was varied among and within the location. Increasing in the number of bacteria in the soil will increase the activity of basal phosphatase enzyme and vice versa, as in the soil of Shamiya, which had the highest enzyme efficacy value (Table 4). This indicates that the production of alkaline phosphatase is from two sources are either microbiology or plant secretions implanted. This is consistent with [26] the results that enzymatic activity is influenced by several factors, including soil, biomass, climate factors, soil ecosystem functions and crop cultivars.
It also in line with what Oujda Bayerjee and Sanyal [3] found, which is that this enzyme is secreted by plants, microorganisms, and soil animals. Sinsabaugh and Moorhead , [27] stated that microbial microorganisms, which act as a double function by breaking down organic matter into simpler forms and then acquiring sources of produced enzymes, secrete enzymes. The effect of the fungus in the activity of alkaline phosphatase varies within and among locations. Enzyme activity increased by decreasing the number of fungi in some locations, but the reverse in some other locations. This variation in the efficacy values with the preparation of fungi may because the source that secretes the enzyme is innate rather than bacterial. The increase in the number of fungi increases the activity of the enzyme, and the fungi would increase the secretion and thus increase its activity. This supports what Fitriatin et al. [28] find, which present that the efficacy of phosphatase produced by certain species of fungi is higher than that produced by bacteria.
The difference in the values of enzyme activity is significant at the level of 5% within and among soils of different locations. The values of the alkaline phosphatase enzyme are generally higher in all studied sites and this is consistent with the findings of [5], who found that basal phosphatase is abundantly produced in neutral or alkaline soils. This is consistent with the results of the pH values in tables (2)(3)(4)(5)(6)(7). Plant type, root secretions, and a number of bacteria influence the efficacy of the enzyme. Electrical conductivity (EC) and the pH had a significant effect on its efficacy and secretion at all studied locations. | http://qu.edu.iq/jouagr/index.php/QJAS/index of the Sunniya location (24.34) mM. There is a difference in Vmax values within and among locations. This variation may due to the difference in the nature of agricultural exploitation and/or soil properties, table (2)(3)(4)(5)(6)(7). This is consistent with the Al-Taweel ,2001study of Amidase in different soils of northern, central and southern Iraq, as well as her study in ,2007. about the study of Vmax values of basal phosphatase in the two different soil textures, which attributed the increase in Vmax values to the increasing in the activity of soil enzyme. These results were less valuable than those found by [29] in their study of some soils in the Basrah province (not cultivated), which Vmax values ranged from (400) to (560) μg P-nitro phenol. g -1 soil. 1 hour -1 . Km values also differed within and among locations. This is due to the high or low affinity between the enzyme and the controlled substance, which is affected by the molecular structure of the enzyme, which varies according to the biological source, [30]. It is natural that the source of the enzyme and the molecular structure according to the dominant group will change the microorganisms, the soil animals and plants, which are affected by the conditions and properties of the soil, as well as the difference of the root plant secretions. In general, Km is an indication of affinity between the substances matter and the enzyme as mentioned. The low value of Km means that the affinity is high between the enzyme and the controlled substance. The enzyme needs a lower amount of the controlled substance to reach its maximum velocity, and the high-value Km means the weak affinity between the controlled substance and the enzyme, [31].

IV. Kinetics measures of alkaline phosphatase
Table (9) shows that the highest Vmax value in the orchard soils in all locations except for the locations of the Diwaniyah and the Shamiya, where the values of the field were superior. These results agree with the activity values of this enzyme in those soils. The factors that influenced the enzyme's effects are the same that affected the maximum velocity values of the enzyme. These results correlate with [32], found that soil fertility and crop diversity in soils significantly affect biological processes occurring in the soil, which affect soil enzyme efficacy.