Study On Solid State Fermentation,Immobilization And Application Of Trametes Laccase

Laccase,as a green biocatalyst,has been used in many fields,such as paper production,textile processing,industrial wastewater treatment,etc.In this paper,the solid-state fermentation conditions for Trametes sp.were optimized by using bran and grapefruit peel as the main substrates.The three-phase partitioning was used to separate and extract the fermented laccase,and laccase was immobilized on macroporous resin by adsorption-crosslinking method and studied its clarification effect on thejuice.The laccase was used to degrade bisphenol A by purification to study the degradation performance of bisphenol A by laccase.The purpose of this study was to provide a certain theoretical basis for the efficient production and application of laccase.The main research contents were as follows:(1)For reducing the production cost of laccase,the solid-state fermentation conditions for Trametes sp.were optimized by using bran and grapefruit peel as the main substrates.As a result,by using the optimized medium containing components formulated in the propotion of bran and grapefruit peel ratio of 6:4(w/w),solid-liquid ratio of 1:2.5(w/v),copper ion 2%(w/w),sucrose 3%(w/w),potassium nitrate 2%(w/w),and rice husk 20%(w/w),the enzyme activity of the laccase product reached 38.4 U/gds in 11 days of fermentation.(2)To efficiently extract the laccase from the solid fermentation substrate of Trametes,improve the purity and recovery rate of the enzyme,the three-phase partitioning was used to separate and extract the laccase from the crude enzyme liquid,and the factors affecting the three-phase partitioning effect were investigated.Experiments show that the optimal three-phase partitioning conditions are:ammonium sulfate concentration 50%(w/v),volume ratio of crude extract and tert-butanol 1:1.5,system pH6,temperature 25? and 60 W ultrasonic power for 5 min.Under this optimized condition,the laccase purification factor and enzyme activity recovery rate reached the maximum,8.7 and 108.9%respectively.(3)To expand the application of laccase in clarified fruit juice,macroporous resin AB-8 was used as the adsorption carrier and TGase as the cross-linking agent to immobilize the laccase.The optimal conditions were:pH4 adsorption,4 h adsorption time,25?,10 mL/g carrier,200 U/ml TGase concentration,30?,crosslinking time 2 h,crosslinking pH4.Under the optimized conditions,the enzyme activity was 267 U/g,and the recovery rate was 86.2%.The enzyme properties of the immobilized laccase were studied.The immobilized laccase had better thermal stability and pH stability and remained 40%of the original activity after six times of repeated use.In the experiment,apple juice was used as a research model to investigate the clarification effect of immobilized laccase on fruit juice.The optimized juice clarification conditions were:the amount of laccase added was 8 U,and the total phenol degradation rate after enzymolysis at 55? for 150 min 53.2%.Compared with the storage conditions at 25?,the enzyme-treated juices had better stability when stored at 4?,with a total phenol content reduction of 8.1%,a color value change rate of 6.7%,and a turbidity value increase of 10.(4)To investigate the ability of laccase in the degradation of bisphenol A,the use of purified laccase to degrade bisphenol A was studied.Experiments show that when the pH is 5,the initial concentration of bisphenol A is 10 ?g/mL,and the concentration of laccase is 5.1 U/mL,after the system is hydrolyzed at 55? for 140 min,bisphenol A is basically degraded completely.