Study On The Response Of Soil Intra-And Extracellular β-glucosidase To As(Ⅴ)

Nowadays,soil arsenic（As）pollution is one of the most serious environmental problems,and detect its’ecological toxicity rapidly and accurately is the key.to the studies.Soil enzyme is an important part of soil and one of the commonly used biological indicators for soil heavy metal pollution assessment.However,because soil enzymes and microbial enzymes are difficult to distinguish accurately,the response of soil enzymes to heavy metals are quite different.So it is urgent to carry out soil.The refinement of intracellular enzymes（mainly from microbial free enzymes）and extracellular enzymes（adsorbed and immobilized on organic matter and clay）at different sites will lead to a more accurate understanding of the effects of arsenic on soil enzymes.To explore the differences in the enzymatic reactions of different enzymes pools in soil,and finally provide an important basis for more reasonable and accurate evaluation of pollution levels and the construction of a scientific monitoring index system.In this paper,the characteristics of intra-and extracellularβ-glucosidase in soil were analyzed by chloroform fumigation method.And investigated the difference of intra-and extracellular enzymes in catalyze mechanism by enzymatic kinetics,and then,we tried to clarify the changes of the response of intra-and extracellular enzymes to arsenic,and studied the effects of different pollution time,soil properties,and field contaminated soil on the relationship between the two,preliminarily.The main results are as follows:1.Chloroform fumigation method is a good method to distinguish between intracellular and extracellular enzyme activities.The intracellularβ-glucosidase activity of the tested soil accounted for 81.06%of the total soil enzymes,which was significantly correlated with the microbial biomass carbon content（r=0.812^*）,while the extracellularβ-glucosidase activity accounted for only 18.64%,not related to microbial biomass carbon.Enzymatic reaction kinetics studies indicated that intracellular enzymes and substrate affinity,enzyme-substrate complex dissociation into enzymes and product rates are greater than extracellular enzymes,indicating that intracellular enzymes are more likely to complete enzymatic reactions.2.After 30 minutes of acute pollution,the total,intracellular and extracellularβ-glucosidase activities of the soil were inhibited by As（Ⅴ）,and the relationship between them reached a significant or extremely significant negative correlation(r=0.990^**、0.918^**、0.867^*,respectively),indicating that the soil enzyme activity can be used to characterize the degree of soil contamination by pentavalent arsenic;the calculated soil enzyme activity ecological dose ED₅₀（As concentration at 50%inhibition）were 28.79,26.81 and 98.97 mg kg^-1,respectively.Kinetic studies found that the effect of As on total enzymes and intracellular enzymes were anti-competitive inhibition,mainly on non-competitive inhibition of extracellular enzymes,kinetic parameters of total enzymes and intracellular enzymes(K_m,Ⅴ_max,Ⅴ_max/K_m)can also characterize the degree of soil As pollution to a certain extent.3.After As（Ⅴ）aging contaminated soil for three months,soil available As（Ⅴ）and its bioavailability coefficient（K）increased with the increase of exogenous As（Ⅴ）concentration.As inhibited soil total and intracellularβ-glucosidase activities,but had no effects on extracellular enzymes.ED₅₀ of soil total and available As for total enzyme are 32.59 and 4.38mg kg^-1,for intracellular enzymes are 29.91 and 4.72 mg kg^-1.Compared with acute pollution,the aging process reduced the inhibitory effect of As（Ⅴ）onβ-glucosidase（total,intracellular and extracellular enzymes）.Kinetic studies also obtained the inhibition mechanism of As（Ⅴ）for total and intracellular enzymes were similar to acute pollution.4.In the field As contaminated soil,the intracellularβ-glucosidase activity accounted for75.74%of the total enzyme activity on average,which was significantly correlated with soil microbial biomass carbon,and extracellular enzyme accounted for only 24.26%,significantly correlated with soil pH and amorphous iron content.The total enzyme activity/MBC was significantly correlated with the exchange state and the bioavailable As concentration.The intracellular enzyme/MBC was significantly correlated with all fractions of As.The complete inhibition model was well fitted to the relationship between As and enzyme activity,the ED₅₀of the bioavailable As for the soil intracellular enzyme response was 29.95 mg kg^-1,indicating that intracellular enzyme activity/MBC can be used as an indicator for soil arsenic contaminationIn summary,the chloroform fumigation method can initially distinguish between intra-and extracellularβ-glucosidase activities;the effect of As on total enzymes and intracellular enzymes are anti-competitive inhibition,while that of extracellular enzymes is mainly non-competitive inhibition.compared with extracellularβ-glucosidase,total and intracellular enzymes are more accurately in assessment the ecotoxicity of soil As contamination.The results will provide an important basis for the evaluation of soil arsenic ecotoxicity and the scientific construction of enzyme activity monitoring indicators,which are of great significance in theory and practice.