Preparation Of Magnetic Cross-linked Laccase Aggregates And Their Application In Dye Degradation

Laccase is a copper-containing polyphenol oxidase with high catalytic efficiency and wide range of substrates.It has broad application prospects in many industries.In this work,laccase immobilization was carried out by cross-linking enzyme aggregates(CLEAs)based on the recombinant Bacillus amyloliquefaciens laccase obtained in the laboratory.The related factors affecting the enzyme activity were optimized during the immobilization process,and the enzymatic properties of free laccase and magnetic cross-linked enzyme aggregates(M-CLEAs)were analyzed.The laccase M-CLEAs were also tested for their ability in dye decolorization.The main results of this work are as follows:The optimal immobilization conditions are:saturated ammonium sulfate as precipitant,ammonium sulfate and laccase volume ratio of 3:1,precipitation temperature of 25?,precipitation for 3 h,using 0.16%glutaraldehyde for 1 h cross-linking,with optimal mass ratio of magnetic nanoparticles to laccase of 7:1.Under these conditions,the final enzyme activity recovery of the immobilized laccase was 37%.Compared with the free enzyme,laccase M-CLEAs had a wider pH range.It retained 50%relative activity at pH 8.6,while the free laccase only remained 25%of activity.In the presence of different organic solvents,lacease M-CLEAs was more stable.Its activity in 30%methanol was 1.5 times higher than that of the free enzyme.In addition,laccase M-CLEAs demonstrated higher activity 30%of ethanol and acetone.It also had better salt toleranee,remaining 70%of activity in 1 M NaCl,while free laccase only retained 20%of activity.Laccase M-CLEAs poorly decolorized dyes of different structures in the absence of mediators.However,the addition of acetosyringone significantly promoted the oxidation process of synthetic dyes.Indigo carmine could be completely decolorized.The decolorization percentage of reactive black 5 was more than 90%,while 64%decolorization of remazol brilliant blue R was observed in 6 h.Complete decolorization of reactive black 5 and indigo was also achieved in 6 h when their concentrations increased to 160 mg/L and 100 mg/L,respectively.Indigo carime was selected to test the reusability of laccase M-CLEAs.About 30%of indigo carmine could be decolorized after seven repeated use.In summary,laccase M-CLEAs show high potential in dye decolorizatin,which lays a foundation for future application in dye effluents treatement.