Study On The Degradation Of Sulfamerazine By Horseradish Peroxidase

Sulfonamides are synthetic and widely-used broad-spectrum antibiotics.The amount of consumption and production of sulfa antibiotics in China are considerable.A lot of sulfonamides are used in clinical medicine and aquaculture.Since sulfa drugs can not absorb by human and animals completely,a part of antibiotics are excreted with excreta,and eventually enter into surface water and groundwater through the discharge of medical wastewater,urban domestic sewage and aquaculture wastewater.Some reports indicate that sulfa antibiotics can be enriched in living organisms,and the high concentration of sulfa antibiotics may cause toxic effects on microorganisms and other living creatures.Because of the adverse environmental effects of sulfa antibiotics,it is necessary to study how to remove these antibiotics from water.At present,the removal methods including advanced oxidation,electrochemical method,adsorption method are used most frequently.The use of enzymes to treat antibiotics in water is a new way compared with the method mentioned above.Compared with other methods,enzyme treatment is an environmental friendly and efficient method.Enzyme treatment is also a method with great development potential.In this study,a typical sulfa antibiotic-sulfamethazine was selected as the research object.Firstly,the degradation process of sulfamethazine by horseradish peroxidase was studied.Secondly,the effect of fulvic acid and protein on the removal of sulfonamide by enzyme were studied.The interaction mechanism between fulvic acid,protein and enzyme was explained.Finally,the ferrous salt(FeC12·4H2O)was added into the reaction system to form a Fenton reaction-enzyme coupling system,the addition of ferrous salt further improved the removal efficiency of sulfamethazine.The specific research contents were as follows:Firstly,according to the activity of horseradish peroxidase under different pH and hydrogen peroxide concentration,the most suitable reaction conditions were determined.Under the optimal conditions,the degradation of sulfamethazine at different initial concentrations(20mg/L,17.5 mg/L,15 mg/L,and 12.5 mg/L)by horseradish peroxidase was studied.Experimental results showed that the removal rate of sulfamethazine was as high as 65%after a 110 min reaction,and the degradation reaction is one of the first-order reaction.The possible degradation products and degradation pathway of sulfamethazine were analyzed by high performance liquid chromatography-mass spectrormetry.The removal of sulfamethazine by horseradish peroxidase was mainly achieved by the cleavage of the sulfa bond and the pyrimidine ring.Secondly,this study analyzed the effects of humic acid and proteinaceous substances(using fulvic acid and bovine serum albumin as model substanse)on the removal of sulfonamide by horseradish peroxidase.The effect mechanisms was analyzed as well.The results showed that fulvic acid and bovine serum albumin inhibited the reaction,and the inhibition effect became more significant with the concentration of fulvic acid and bovine serum albumin increased.By analyzing the enzymatic activity after adding fulvic acid;the ability of enzyme to bind hydrogen peroxide and fulvic acid'effect on the Michaelis constant of the reaction,we found that the inhibition effect of fulvic acid on the reaction is a kind of anti-competitive inhibition,fulvic acid can combine with the complex of enzyme and substrate and obstruct the generation of final product.The proteinaceous substances could combine with SMR,this might be the reason why proteinaceous substances could inhibit the degradation of SMR by horseradish peroxidase.Finally,the removal efficiency of sulfamethazine was enhanced by the construction of Fenton coupling enzymatic hydrolysis system,the pathway and mechanism of sulfamethazine degradation in this system were elucidated.The addition of ferrous ions reduces the enzyme activity by two ways:(1)ferrous ions could catalysis the reaction between hydrogen peroxide and SMR.this reduced the concentration of hydrogen peroxide which accessible to enzyme;(2)the Fenton reaction produced free radicals,these free radical had an effect on the structure of the enzyme.However,the introduction of the Fenton reaction improved the removal effect of the reaction system.Different degradation pathways were revealed by analysis of degradation products.This study not only provided a new degradation method for the removal of sulfa antibiotics,but also extended the application of Fenton system.This research showed a new path for water purification,especially for the tap water's purification;and also provided a reference for waste water advanced treatment.