Response Of Soil Intracellular And Extracellular β-Glucosidase To Pb And Its Mechanism

Lead（Pb）pollution has aroused globally public concerns due to its widespread distribution and environmental risks.Pb is easily accumulated in food which enters the human body and harms human health.Therefore,assessing of Pb contamination in soils in a rapid and timely manner is one of the crucial issues.Soil enzymes are considered excellent indicators of soil quality and health because of their sensitivity to heavy metal contaminations and their direct involvement with soil functions related to nutrient cycling.β-glucosidase（BG）,which hydrolyzes non-reducing sugars such as cellobiose and other water-soluble cellodextrins thereby releasing glucose,is widely used as a simple,sensitive and precise indicator to assess Pb pollution.It is well known that soil enzymes include extracellular and intracellular enzymes.The affinity between the enzyme and the substrate or inhibitor may differ among the extracellular and intracellular enzymes due to their different structural conformation and specific surface properties.The effect of Pb on soil enzymes may differ among various enzyme pools.Some studies have shown that there are differences in the responses of intracellular and extracellular enzymes to heavy metals.The inconsistency or heterogeneous responses of BG activity to Pb contamination may be attributed to the differences between intracellular and extracellular BG pools in the soil.However,soil extracellular and intracellular BG response to Pb is still poorly understood.Therefore,exploring how Pb affects soil enzymes across different enzyme pools is necessary.In this study,we hypothesized that intracellular and extracellular BG responses would differ under Pb contamination.The aims of this study were to（i）investigate the inhibition mechanisms of Pb on intracellular and extracellular BG;（ii）evaluate the dose-response models and elucidate the potential risks of Pb as influenced by soil properties;（iii）find a more suitable enzyme indicator for Pb evaluation;（iv）elucidate the toxicity evolution characterisitics of soil BG activity,microbial biomass carbon（MBC）during Pb aging process,which could provide rich microbial theory for evaluating the ecological toxicity and environmental risk of Pb contaminated soil.This study will provide a new perspective on assessing Pb contamination using soil enzymes as indicators.The main research contents and results of this paper are as follows:（1）The toxic effect of Pb on intracellular and extracellular BG activity in soils was investigated through acute contamination experiment.It was found that Pb had a strong inhibitory effect on intracellular BG activity in soil,which showed a tendency of decreasing BG activity with the increase of Pb concentration,and the inhibition rate varied with soil type.However,the inhibition of Pb on soil extracellular BG activity was weak.The ED₁₀（Ecological dose）values of total and intracellular BG fluctuated between 42.2～241.8 mg kg^-1and 40.1～193.3 mg kg^-1,respectively.The results showed that the intracellular BG could be used as sensitive indexes to evaluate the toxicity of Pb but extracellular BG were less sensitive to Pb stress.In addition,correlation analysis results showed that soil total nitrogen content and ED value of total and intracellular BG were significantly positively correlated,indicating that soil with a higher total nitrogen content had a stronger buffer capacity to Pb toxic stress.（2）The mechanism of Pb inhibition on soil BG was discussed through enzyme kinetics analysis.For extracellular BG,K_mremained unchanged and V_maxdecreased with Pb addition,which belonged to non-competitive inhibition.For the total and intracellular BG,the K_mand V_maxof chestnut soil,grey desert soil and irrigation soil decreased with the increase of Pb concentration,showing uncompetitive inhibition,but the K_mremained unchanged and V_maxdecreased in the three fluvo-aquic soils,which belonged to non-competitive inhibition.In addition,the ecological dose value calculated by V_maxcould be used as a sensitive index to evaluate the toxicity of Pb to soil BG.To be specific,the ED₁₀values of total and intracellular BG were42.24～241.76 mg kg^-1and 40.11～193.20 mg kg^-1,respectively.The results showed that V_maxof intracellular BG was more sensitive to Pb contamination.（3）The aging study of Pb in grey desert soil and fluvo-aquic soil showed that Pb had significant adverse effects on soil BG activity and microbial biomass carbon,but this negative effect decreased with the extension of culture time.The results indicated that both Pb concentration and culture time had significant effects on microbial activity.In the two soils tested,ED₁₀of intracellular BG in the early stage of culture was smaller than that of extracellular and total BG,so intracellular BG was more sensitive to Pb stress.In the aging process of two different types of soil,it was found that the performance of soil microorganisms in the face of Pb toxicity stress was different under different fertility conditions.Soil with higher organic matter content had stronger buffering ability to heavy metal pollutants.In addition,the microbial biomass carbon（MBC）and BG activities increased in the middle and late period of culture for the low fertility of fluvo-aquic soil.The ED₁₀values of total BG were171.48～452.97 mg kg^-1,and ED₁₀values of intracellular BG were 57.27～228.75 mg kg^-1in grey desert soil.The ED₁₀values of total and intracellular BG of the first day were 110.01 mg kg^-1and 91.07 mg kg^-1in fluvo-aquic soil,respectively.Which indicates that the ecotoxic effect of Pb on intracellular BG was more obvious.The performance of Pb toxicity stress on MBC was different in the two soils with different fertility levels.The grey desert soil with higher organic matter content had stronger buffered capacity on heavy metal pollutants,and MBC was less affected by Pb.For low fertility soil,MBC and BG activities increased and recovered in the middle and late period of culture.（4）It was found that aging pollution of Pb had different kinetic mechanisms on grey desert soil and fluvo-aquic soil.The inhibition of Pb on total and intracellular BG in grey desert soil were uncompetitive inhibition,while for the total and intracellular BG in fluvo-aquic soil were uncompetitive inhibition in the early stage and had no effect in the late stage.There was no inhibition of extracellular BG in grey desert soil,but the inhibition of extracellular BG in fluvo-aquic soil was linear mixed in the early stage and non-competitive in the late stage.Compared with acute pollution,the aging process reduced the ecotoxicity of Pb to total and intracellular BG,and intracellular BG were more sensitive to Pb stress in general.Correlation analysis showed that the contents of total and intracellular BG K_m,V_maxand MBC were significantly positively correlated,which confirmed that the increase of enzyme quantity during the aging process was due to the increased secretion of microorganisms.The results showed that the grey desert soil with higher fertility level had a better buffering ability to Pb toxicity stress during Pb aging.In summary,the effect of Pb pollution on intracellular BG activities was significantly stronger than that of extracellular BG.The main mechanisms of Pb pollution on total,intracellular and extracellular BG activities were uncompetitive inhibition,non-competitive inhibition and linear mixed inhibition.Compared to extracellular BG,intracellular BG are more sensitive to toxic stress of Pb,and are more suitable to be used as enzyme indexes for monitoring and evaluating Pb pollution.In addition,it was found that soil with a higher organic matter or total nitrogen content has a stronger buffer capacity to Pb toxic stress.The results will provide an important basis for the toxicity evaluation of Pb and the scientific construction of enzyme indexes,which is of great significance in theory and practice.