Preliminary Studies On The Isolation Methods Of Different Status Enzymes In Soil

Any overall activity of soil enzyme has related to the positions of enzyme existed. Soilenzyme has many exist location and status, where the intracellular enzyme living in the celland reflect microbial activity directly, therefore, intracellular enzyme was more sensitive toexternal stimulate; On contrary, the extracellular enzyme was associate with soil colloidstightly, the stable nature shows more regular response to agricultural practices, environmentalconditions and toxic. But the most serious problem in interpreting data on the determinationof enzyme activity in soil is to decide which combination of the various activities has beenexperimentally determined. Therefore, it is very meaningful to distinguish the intracellularand extracellular enzyme activity of soil.Whereas the soil enzyme have many sources, also the enzyme status is unstable, thestructure is complicated, and the knowledge related to soil enzyme is poor and the limits of instruments and reagents, which make us do not know the relationship between intracellular andextracellular enzyme well and the contribution to the microbial biomass was not very clear.So, the experiment of separate microbial activity from extracellular has not achieved yet.By simulative method, we attempt many method in differ soil intracellular andextracellular enzyme, investigated the nature and relationships of different parts of enzyme,studied the changing process and reaction mechanism of enzyme been released into soil byenzymatic kinetics. The results were as follows:1. The sterilized soil inhibited the kinetic parameter V0of pure arylsulfatase, theinhibition is getting stronger with the increasing content of clay. With the increasingconcentration of sterilized soil, Kmshow's increasing trend, Vmax, Vmax/Km, k was decreasing,the reaction mechanism between arylsulfatase and sterilized soil was mixed inhibition,indicating that both competitive and noncompetitive inhibition may be operative, That is, soilbinding to both active and non-active sites of the enzyme. The pure arylsulfatase kineticparameters reveals that the ability of enzyme absorbed by soil is red soil> lou soil>cinnamon> sandy soil, the mean factor impact enzymatic reaction was clay content.2. Toluene inhibited the pure arylsulfatase activity significantly, with the largestdecrement in activity being45.5%. The sterilized soil has great ability to absorb pure enzyme in solution.1.0μL g-1toluene could increase the soil arylsulfatase activity greatly, with theincreasing variation of9%～198%as that without toluene treatment. Increasing soilarylsulfatase activity became gently with increasing toluene concentration, Langmuir equationcould fit the variation of soil arylsulfatase activity well, and obtained the maximum activitywhich was significantly correlated with soil properties. It reveals that toluene affect soilenzyme activity mostly by killing the soil microorganism. The relationship betweenextracellular and intracellular enzyme in different soil was discussed, the extracellular andintracellular soil arylsulfatase activity were54.4%and45.6%at average, respectively intested soil sample. The soils with higher fertility have the greater ability to absorb enzyme,that makes the content of extracellular was higher in high fertility soil.3. Chloroform fumigation inhibited the pure arylsulfatase activity significantly,12hfumigation inhibit the most serious. Chloroform fumigation increased soil arylsulfataseactivity significantly, increase of25%～454%. Tradition of fumigate24h is too long, theoptimum fumigation time which calculate by regression equation is16～17h. Theintracellular and extracellular soil arylsulfatase activity were calculated by using Umax,contentof extracellular arylsulfatase activity is bigger than intracellular arylsulfatase activity in testedsoil sample.4. The induce matter increased soil microbial biomass C and soil arylsulfatase activity,change of arylsulfatase activity was significantly correlated with the soil fertility and numberof microorganism. The increase of induced activity was caused by the active ofmicroorganism, therefore, soil microorganism was more sensitive than soil enzyme tosubstrate inducement, thus the increase of soil microbial biomass C was bigger than soilarylsulfatase activity. The linear equation can describe the relationship between soil microbialbiomass C and soil arylsulfatase activity well, and obtained the relationship betweenintracellular and extracellular arylsulfatase which calculate by the intercept. Soilmicroorganism was also a part of soil fertility, thus the overall activity of enzyme was moreaccurate than intracellular or extracellular activity in evaluate soil fertility.5. The pure arylsulfatase has proper ability to resist microwave irradiation. Withouttemperature interference, when the microwave power was lower than the limit(240W), purearylsulfatase hardly affected, when the power was higher than the tolerance, with theincreasing power of microwave, the pure arylsulfatase shows decreasing trend. The inhibitionbecame stronger to soil arylsulfatase with the increasing time of irradiation, the main reasonof activity decrease after microwave irradiation was dramatic change of temperature in soil.ET50reveals that the soil with high fertility was more sensitive to microwave irradiation. Soilarylsulfatase have the most sensitive power to microwave irradiation which obtained the greatest change of enzyme activity, and the sensitive power was close to the limit power.Analyze shows that the sensitivity of soil is getting stronger with the increasing content of siltin soil to microwave irradiation, it reveals silt is the main part to absorb the microwave energyin soil.