A Novel Laccase From White Rot Fungus Trametes Orientalis:Purification And Degradation Of Environmental Hormones

Endocrine disruptor chemicals(EDCs),as exogenous toxic agents,would interfere with human and animals’ health and reproduction when released to the environment.Recent few years,since laccase is found to be able to degrade various aromatic compounds including EDCs,enzymatic approaches to development of green chemical technologies have considerably aroused the interest of many researchers and industries.In the present study,the laccase Tolacc from white rot fungus Trametes orientalis was purified,immobilized,and applied to degradation of EDCs benzyl butyl phthalate(BBP),bisphenol A(BPA),4-nonylphenol(4-NP),and 4-octylphenol(4-OP).The main results are demonstrated as follows:Firstly,the variations of physiological indexes,as determined by viz.mycelial biomass,laccase activity,reducing sugar content,polyphenol content,MDA content,T-AOC,SOD activity,CAT activity,H2O2 level,AA content,RAHFR,and DPPH radicals scavenging ability,of T.orientalis were investigated during laccase-producing process under liquid cultivation.The results demonstrated that laccase could directly affect the nutrition utilization of white rot fungus,and its activity was closely related to the fungal growth and secondary metabolism.Laccase activity was positively correlated with polyphenol content,MDA content,CAT activity,and AA content.However,the laccase activity had a negative correlation between higher RAHFR and H2O2 level.When compared with the physicological indicators obtained from liquid medium Ⅰ(nutrient-rich),T.orientalis in liquid medium Ⅱ(nutrient-deficient)was shown to exert higher mycelial biomass,SOD activity,RAHFR,and DPPH radicals scavenging activities,as well as lower H2O2 level,suggesting that the liquid medium Ⅱ with absence of nutrition at a certain degree could stimulate the antioxidant capacity to protect the fungus from oxidative damage.A novel laccase(Tolacc)from T.orientalis was purified to apparent homogeneity using ammonium sulfate salting-out,anionic exchange column chromatography(DEAE-cellulose DE52),and agarose gel column chromatography(Sepharose CL-6B).It was enriched 20.282-fold,with a specific activity of 20.667 U/mg protein and recovery yield of 47.33%.The SDS-PAGE gave a single protein band indicating that Tolacc appears a monomeric protein with a molecular mass of 44.0 kDa.Optimal pH and temperature of Tolacc were 4.0 and 80℃,respectively,and it retained more than 80%of its original activity after 2 h incubation at 10℃ to 50℃.The enzyme exhibited strict substrate specificity towards ABTS but showed no or trace activities towards other tested substrates.Among the metals tested,Mn2+was proved to be the best activator for enhancing the laccase activity.A strongly inhibiting effect was found when NaN3,L-cysteine,and DTT were added to the enzyme.However,Tolacc activity was little bit inhibited in the presence of chelator EDTA.Furthermore,the purified enzyme was capable of degrading structurally different synthetic dyes in the absence of a redox mediator.Additionally,the purified laccase Tolacc was immobilized onto chitosan beads with the crosslinker glutaralydehyde,in order to improve the stability and recovery rate of Tolacc,and was applied in biodegradation of EDCs.The optimal conditions for Tolacc entrapped onto chitosan beads were 0.6%(v/v)glutaraldehyde concentration,3 h crosslinking time,3 mL enzyme,and 6 h immobilization time,respectively.The pH adaptability and resistance to thermal denaturation of immobilized Tolacc were considerly enhanced compared with free Tolacc,and both the operational stability and durability during multiple reuses were superior to those of free Tolacc;after five cycles of continuous use,the activity of immobilized enzyme remained above 45%.Also,the immobilized Tolacc was able to biodegrade EDCs,as evidenced by FT-IR and GC-MS.The enzyme obtained in this study possessed excellent activity and stability,thus supporting a reliable theoretical basis for laccase’s practical use,laying a foundation for non-pollution treatment of these compounds,and providing development and utilization of new fungal resources.