Study On The Characteristics Of Atrazine Degrading Enzyme And The Remediation Of Pollutant Soil By Immobilized Enzymes

In this study, based on analysis the basic characteristics of the crude enzyme solution，whichextracted from long residue herbicides atrazine degradation bacteria Arthrobacter sp. DNS10.Optimized and determined the immobilized conditions, in-depth discussed the feasibility ofimmobilized enzymes to eliminate atrazine in the soil. Hope for reasonable, effective, safe remediationtechnology to provide a theoretical basis. The main results were as follows:The degrading enzyme from efficient atrazine degrading bacteria DNS10was inducibleintracellular. The degrading enzymes solution (protein concentration was88μg·mL^-1) was inoculatedinto the sodium phosphate buffer (atrazine concentration was50mg·L^-1) by transferring1:10（v：v）,after reaction for2h in the most suitable conditions （45℃, pH8.0） the atrazine degrading rate was96.23%. Degradation kinetics equation was y=109.38x+280.18，free enzyme and substrate （atrazine） hasgood affinity, Km was2.56mmol·L^-1and Vmax was3.57×10-3mmol·min^-1. The thermal stability and pHstability of degrading enzyme. was poor. Several typical metal ions （Ca2+, Mg2+, Mn2+, K+, Na+, Fe2+）had little affect on the degradation ability of degrading enzyme.This study selected alginate sodium as immobilized matrix to immobilize the degrading enzymesextracted from strain DNS10. Took advantage of SAS software to chose the concentration of alginatesodium, the pH of immobilized matrix, the volume of degrading enzymes and the CaCl₂solutionquality scores as the typical factors which could affected the enzymatic activity significantly. In addition,the results of response surface optimization indicated the most immobilize coditions as following: thepH of the matrix was about8.5, the mass fraction of CaCl₂solution was2.7%, the mass fraction ofalginate sodium in immobilized matrix was1.93%. At last, the volume of degrading enzymes was thatthere was983μL degrading enzymes solution (the protein concentration was about88μg·mL^-1)in every10mL immobilized matrix.The most suitable reaction conditions of immobilized enzyme were50℃，pH=8.0, the degradationkinetics equation was y=128.57x+463.13Vmax was2.16×10-3mmol·min^-1，Km was3.74mmol·L^-1,which slightly bigger than the Km of free enzyme. In addition, the tolerance of the immobilizedenzymes was better than free degrading enzymes. The immobilized enzymes recycle used after3times,the atrazine degradation ability was not significantly reduced. Examing the storage stability of twoforms enzyme at room temperature and4°C conditions, respectively. However, the free enzymes had no degrading ability at the end of experimental period. During the35days experimental period, theimmobilized enzymes lost only73.90%and74.67%.Under laboratory conditions，the immobilized enzyme（dosing rate1:10（m:m））could speed up thedecomposition rate of atrazine in soil. After28days, remediation treatment was no atrazine could bedetected (the initial atrazine concentration of soil was10mg·kg^-1). While the final atrazine concentrationof polluted treatment was still5.02mg·kg^-1. During remediation process, comparing with the othertreatments（remediation treatment and CK treatment）the soil microbial biomass C and soil respirationintensity of the polluted treatment was increase obviously. At the same time, the soil bacteria diversityindex and soil bacteria evenness index was fall down during the experimental period. The remediationtreatment could relief the affection of atrazine on the three kinds of indexes mentioned above and theimmobilized enzymes also could make these indexes of polluted treatment approach to these indexes ofblank control treatment. The result shows that the preparation of immobilized enzyme in this article usingnot only could effectively degradation atrazine, and had a certain ecological safety.