Study On Preparation Of Inorganic Salt Immobilized Laccase And Its Degradation And Decolorization Performance

Laccase is an oxidase with abundant substrates,which has the advantages of high efficiency,specificity,mildness and no secondary pollution in wastewater treatment.However,free laccase is limited in its application fields due to lack of stability and reusability.Immobilized enzyme technology is used to improve the stability of enzymes so that free enzymes can be recycled and reused.In this paper,mono/bimetal inorganic salt immobilized laccase was prepared by biomimetic mineralization method.In addition,the bimetallic inorganic salt immobilized laccase is coated with calcium alginate hydrogel to improve stability,and these immobilized laccases are used to decolorize bisphenol A and various dye wastewater.The main work is as follows:1.Aspergillus laccase was selected as the research target,and the enzyme activity of the free enzyme was determined with ABTS as the substrate.In this chapter,the definition of laccase enzyme activity and the determination method of enzyme activity are determined,and the basic enzymatic properties of free enzyme are investigated.The optimum pH of free enzyme is 2.5,and the optimum temperature is 50℃,it has poor stability when stored in alkaline and high temperature,the remaining enzyme activity is 12.8% when stored at room temperature for 8 days.2.Monometallic inorganic salt immobilized laccase（MM-Lac）was prepared by biomimetic mineralization method and ultrasonic assist.After screening of metal ions,Cu₃（PO₄）₂ immobilized laccase（Cu-Lac）has the highest enzyme activity.In the 30 mL 0.2 mg/mL laccase PBS buffer system,the optimal process parameters of Cu-Lac are as follows:Cu²⁺is 0.06 mmol,the pH of PBS is 7.5,the ultrasonic power is 43 kHz,the ultrasonic time is 16 min,and the incubation time is 24h.The basic enzymatic properties of Cu-Lac are compared with free laccase:the optimal pH and temperature range is widened,and the storage stability is significantly enhanced,the remaining enzyme activity after 8 days of storage is 57.4%,the affinity and reaction velocity between Cu-Lac and substrate were higher than free laccase.3.The preparation of Cu/Zn bimetallic inorganic salt immobilized laccase（Cu/Zn-Lac）was studied.The optimal process parameters of Cu/Zn-Lac:The volume ratio of copper zinc salt solution is 1:1,and the standing incubation time is 48 h.It is proved that Cu/Zn-Lac was successfully prepared and characterized by SEM,EDS and FT-IR.Compared with Cu-Lac,Cu/Zn-Lac is more stable,77.2%of the enzyme activity remains after 8 days of storage at room temperature,and the affinity and reaction velocity between Cu/Zn-Lac and the substrate are slightly less than Cu-Lac.Cu/Zn-Lac is used for the degradation of bisphenol A,and the optimal conditions are as follows:the initial concentration of bisphenol A is 50 mg/L,the pH is 7.5,and the temperature is 50℃.Comparing the degradation effect and reusability of immobilized laccase and free enzyme,the result is Cu/Zn-Lac>Cu-Lac>free enzyme.After 6 recycles,the degradation rates of Cu-Lac and Cu/Zn-Lac were 35.5%and 62.7%.4.Alg@Cu/Zn-Lac was prepared by coating Cu/Zn-Lac with calcium alginate gel.The optimal process parameters were as follows:The volume ratio of buffer to sodium alginate was 2:1,the addition amount of Cu/Zn-Lac was 3 mg/m L,and the concentration of CaCl₂ was 0.02 g/m L.SEM characterization showed that Cu/Zn-Lac was embedded in the grid structure inside the gel microspheres.Compared with Cu/Zn-Lac,Alg@Cu/Zn-Lac had wider optimum pH range,better adaptability to acid and alkali changes,and further improved thermal stability and storage stability.The residual enzyme activity was 87.9%after 8 days of storage,but the degree of affinity and reaction velocity with the substrate are small.Subsequently Alg@Cu/Zn-Lac was applied to the decolorization of Congo red,malachite green and Coomassie brilliant blue R.The optimal degradation conditions were as follows:the initial dye concentration was 60 mg/L,60 mg/L and 40 mg/L,pH was 7,9 and 8,and the temperature was 50℃.The comparison of decolorization effects of Alg@Cu/Zn-Lac,blank carrier and free enzyme showed that Alg@Cu/Zn-Lac>calcium alginate>free enzyme.After 4 recycles,the decolorization rates of Alg@Cu/Zn-Lac for the three dyes were 53.6%,61.9%and 13.1%.