Purification,Mediator Systems,Nano-immobilization,and Decolorization Of Extracellular Laccase From Gymnopus Luxurians

Laccases(EC 1.10.3.2),polyphenol oxidases belonging to blue multi-copper oxidases family,usually catalyze the mono-electronic oxidation to degrade substrates and reduce the acceptor of O2 to H2O.They are efficient and eco-friendly catalyzers.The laccase substrate range and catalytic capability can be enhanced through the formation of laccase mediator systems(LMS)with small molecular weight redox mediator.The stability and reusability have been improved as well by nano-immobilization,hence,there is a huge application potential in degradation of environmental pollutants and lignin.The genus Gymnopus is an important group of Marasmiaceae,which plays an important role in carbon cycle of forest ecosystem.Gymnopus species demonstrate a great variety and very complicated phylogenetic relationship,with the result that related studies mainly focuses on system classification.However,the studies of their lignin-degrading enzymes are rare.In this study,fruiting bodies of Gymnopus,collected and separated from Chuxiong,Yunnan were used as material.Further studies were focused on strain isolation and identification,optimal culture conditions of mycelia.lignocellulose degrading enzymes activities,Cu2+ induced laccase production,purification of extracellular laccase.laccase mediator system.preparation of laccase immobilized by nano-SiO2,and decolorization of dyes,etc.The details are as follows:1.Based on morphology and internal transcribed spacer(ITS)identification,the present strain YAASM5159 was confirmed as Gymnopus luxurians.Mycelia optimum culture conditions by single factor experiments resulted in carbon source of starch;nitrogen sources of yeast extract,tryptone,and beef extract;C/N ratio range of 50-60/1;growth factor of VB1;temperature range of 26-28?;and pH value of 6.5.After a 7-day shaking cultivation of 150 r/min at 28? using PD media with 0.25 mmol/L Cu2+,laccase activity reached 18.73 U/mL which was 3.17 times as much as that in control group.2.An extracellular laccase(GLL)was purified from fermentation broth of the G.luxurians by three ion-exchange chromatography steps and one FPLC step.which resulted in the recovery rate of 42.05%,purification fold of 41.22,and a high specific activity of 306.73 U/mg.GLL was a monomeric protein of 64 kDa by SDS-PAGE and FPLC.The N-terminal sequence of GLL based on Edman degradation was AIGPV TDLHI,which demonstrated high similarity of 100%with that of Lentinula edodes,Hericium coralloides and Panus rudis,etc.GLL demonstrated an optimum temperature range of 55-65? and an optimum pH 2.2 towards ABTS.GLL was significantly enhanced in the presence of K+,Na+,and Mg2+ ions,while GLL was inhibited by Cu2+?Mn2+Ca2+?Cd2+?Co2+ and EDTA.It demonstrated a Km and Vmax of GLL was 539 pmol/L and 3.12 ?mol/min towards ABTS at pH 2.2 and 37?.3.Seven common small molecular mediators and GLL(0.45 U/mL)were used to screen of the best LMS.The result showed that Acetosyringone(AS)and 3,5-dimethoxy-4-hydroxybenzaldehyde(SA)were the optimal mediators for GLL.At 25? pH 4.0 and 0.1 mmol/L of mediator concentration.the two LMSs showed high efficient decolorization ability with the decolorization ratios towards coomassie brilliant blue(CBB),malachite green(MG),reactive black(RB),and tribenzene blue(TB).The optimal reaction condition of LMSs showed that the temperature range was 25-60?,pH was 4.0,and mediator concentration was 0.1 mmol/L.The decolorization ratio of these 4 dyes were in the range of 50-90%for 4 h incubation.4.Commercial SiO2 was modified using the magnetic stirring oil bath heating method with APTES as the modifier.The obtained nanoparticle,named as SiO2-H,was further used as the nano-carrier for nano-immobilized laccase(SiO2-H-GLL)using glutaraldehyde as crosslinking agent and pure laccase GLL(0.03 U/mL).FTIR results showed that GLL was combined with SiO2-H through these amino sites.The optimal immobilization condition showed that the optimal glutaraldehyde concentration was 1%(V/V),the optimal crosslinking time was 40 min,the optimal dosage of GLL was 0.04 U/m L,and the optimal immobilization time was 2 h.SiO2-H-GLL demonstrated an optimal temperature of 60? and an optimal pH of 2.2.Comparing with free GLL,the immobilized laccase possessed remarkable thermostability.SiO2-H-GLL possessed efficient decolorization capability towards MG with the maximal decolorization ratio of 91.1 8%at 4 h,and maintained decolorization ratio about 90%when reused four times,and about 40%when reused eight times.Briefly,it's the first time to purify a laccase of the genus Gymnopus(GLL).Immobilized laccase(SiO2-H-GLL)and LMS of GLL were established,which possessed efficient decolorization capability with dyes as MG.It suggests that they demonstrate great application potentials for biodegradation of environmental pollutants.