Effect Of Rare Earth Elements On Laccase Production By Trametes And Immobilization Of Laccase

Laccase(E.C.1.10.3.2)is a polyphenol oxidase with copper ion as the active center.The enzyme can utilize oxygen as the final electron receptor to efficiently convert azo compounds,phenols,steroids and aromatic amines into quinones,carbonyl compounds and water.It has application value in degradation of phenols and azo dyes,bleaching of pulp and clarification of fruit juice.In the process of laccase fermentation,carbon nitrogen source and inorganic salt are usually used as the optimization objects.Due to the four copper ions in laccase structure,the research on divalent metal salt is more important in the optimization of inorganic salt.Rare earth elements are often used in agriculture,forestry,animal husbandry and fishery industry to promote the growth of animals and plants due to their hormesis effect.Taking advantage of the characteristic,it can also be applied in the field of microbial fermentation to improve the level of fermentation.In this paper,Trametes sp.LS-10C was used as the research object to investigate the effects of rare earth elements on the production of laccase by its fermentation.The proteomic changes of Trametes sp.LS-10C under the influence of rare earth elements were studied,and the functions and regulation of some differential proteins were analyzed.The sodium alginate and gelatin were used to immobilize laccase.The preparation process of immobilized laccase,the properties of immobilized laccase and the degradation of environmental phenolic pollutants(bisphenol A)were studied.The main research contents were as follows:1.The effects of different rare earth elements and its concentrations and its addition time on laccase production were studied.The results showed that rare earth Y promoted laccase production by Trametes sp.LS-10C fermentation more than rare earth Ce.When the concentration of Y3+ in the fermentation medium reached 12mM,the laccase activity reached the maximum of 70.25U/mL,which was 5.3 times that of laccase production without adding rare earth.Rare earth Y had a great promotion effect on the production of laccase during the logarithmic growth stage,and the rare earth Y had little effect on the biosynthesis type of laccase.2.The effect of Y on laccase production by Trametes sp.LS-10C was studied at proteomic level.The results showed that Trametes sp.LS-10C had 678 differentially expressed proteins caused by rare earth Y during fermentation,including 390 up-regulated proteins and 288 down-regulated proteins.Y3+ promoted the up-regulation of membrane component related proteins,which accounted for 70.5%of the total up-regulation of cell component enrichment proteins,including mitochondrial carrier proteins and transporters.The up-regulated oxalate decarboxylase in the process of oxalic acid metabolism had the highest differential ratio of 10.69.3.The laccase was pretreated with glutaraldehyde before immobilization with sodium alginate gelatin and the properties of the immobilized enzyme were studied.The optimal preparation conditions of immobilized laccase were determined as follows:the concentration glutaraldehyde was 2%,the concentration of sodium alginate was 2%,the concentration of gelatin was 2%,the amount of laccase solution was 20%(v/v),the concentration of calcium chloride was 1.5%,and the immobilized time was 1 h.The results showed that the optimum pH was 4.5 and the optimum temperature was 55?.The stability of immobilized enzyme was better than that of free enzyme;The degradation of bisphenol A by immobilized enzyme in a bubbling reactor showed that the degradation rate of bisphenol A reached 80.63%after 3 h under the conditions of bisphenol A concentration of 10?g/mL,temperature of 30?,and aeration rate of 500 mL/min.After 5 batches of continuous degradation,the degradation rate of immobilized enzyme still reaches 71.13%.This study improved the laccase production level of Trametes sp.LS-10C in shaking flask fermentation,and promoted the application of laccase in practice by immobilization of laccase.On the other hand,this study clarified the expression difference at the protein level under the influence of rare earth Y,as well as the functions of the different proteins in biological processes such as cell metabolism.