Screening Of High-yielding Laccase-producing Strains From Ganoderma Species And Its Laccase Purification And Feature Characterization

Laccase is a kind of green catalyst with excellent properties.It can oxidize large amounts of substrates such as phenolic and aromatic amine compounds,coupled with a concomitant reduction of molecular oxygen to water and other final products.Owing to the up-mentioned characteristics,laccase is regarded as a promising alternative for environmental demand,and thus has great application potentials in various fields,including pulp bleaching,environmental protection,biological detection,and organic synthesis.Many biotechnologically worthy laccases are of fungi origin especially from white rot fungi and laccase has gained public interest after the beginning of studies on enzymatic degradation of lignin by white rot fungi.As a general name of a class of traditional medicinal fungi,Ganoderma species are also important white rot fungi.The genera Ganoderma species not only has various medicinal values such as antioxidant,anti-cancer,anti-bacterial,and anti-inflammatory activities,blood glucose and lipid suppression,and promotion of immunity,but also possess efficient laccase-producing potentials.However,comprehensive analysis on their laccase-producing abilites are still lack.In this study,the laccase-producing abilities of 51 strains of white rot fungus Ganoderma species were systematically studied,wherein a high-yielding laccase-producing strain G.australe Dai11646 was screened.Additionally,its laccase Galacc was purified and feature characterized.It was demonstrated that this study has realized the exploration of new resources,provided a reliable theoretical basis for selection,development,and utilization of high-yielding laccase-producing Ganoderma strains,and laid an important theoretical foundation for the application of laccase in industrial fields.Firstly,51 Ganoderma strains were accurately identified by molecular biological method based on ITS and n LSU sequences,belong to six species:G.applanatum,G.australe,G.brownii,G.fornicatum,G.lingzhi,and G.multiplicatum.Laccase-producing abilities of the 51 strain were systematically studied by preliminary screening and rescreening through guaiacol-containing plate and liquid fermentation,respectively,herein a high-yielding laccase-producing strain G.australe Dai 11646 was screened and obtained.For preliminary screening by guaiacol-containing plate,the laccase-producing rate of the tested strain was 98.04%,which showed a widespread laccase-producing characteristic.It was different for time,size,and color depth of the colorized zones generated by various strains.Generally,colorized zones were generated within 4-5 h,then their color gradually changed from water red to reddish-brown.After 1 d,the color was almost stable,and the halo lightened to light brown from the center outward.During the process of mycelial growth,the diameters of the colorized zone increased synchronously,and the laccase activities of most Ganoderma strains were higher in the first 10 d.By virtue of determination of the size of colorized halo,species name,and mycelial growth,Dai 11646,Cui 7048,Cui 7247,Cui 9164,and Cui 16779 were selected for rescreening.To liquid fermentation rescreening,the laccase activities of the five strains displayed upward-downward trends with incubation periods.Among most strains that reached the peaks for laccase activities on the 7^th d,G.australe Dai 11646 had the highest laccase activity,in correspondence to 0.809 U/m L.Secondly,the laccase Galacc from G.australe Dai 11646 was purified to homogeneity by salt precipitation,ionic exchange,and size exclusion chromatography with a final specific activity of 22.214U/mg,yielding a purification fold of 23.989 and recovery of 38.44%.During the laccase Galacc was partially purified by DEAE-cellulose ionic exchange column,three fractional peaks i.e.Galacc-F,Galacc-S,and Galacc-T eluted by 0.1,0.3,and 0.7 M Na Cl appeared.Among them,the fraction Galacc-F,amounting to a specific activity of 4.274 U/mg,was approved to be the main laccase portion secreted by G.australe Dai 11646.Lastly,this fraction was collected then further subjected to size exclusion chromatography.Lastly,features of the purified laccase Galacc-F from G.australe Dai 11646 were characterized.Its molecular weight was estimated to be 48.0 k Da by SDS-PAGE and native PAGE,which confirmed the monomeric and blue copper nature.Galacc-F exhibited high levels of activity and stability over a wide range of p H 5.0-8.0 and temperature 10?-60?,which are highly valuable in industrial processes.Broad substrate specificity was observed,wherein a better affinity was found for ABTS with a low value of K_m?164.137?M?and higher k_cat/K_m ratio(1.663 s^-1?M^-1).Among the additives tested,enzymatic activity was stimulated by Cu²⁺and?-ME but inhibited by EDTA,DEP,IAA,PMSF,and Hg²⁺,indicating that Galacc-F is belonging to a metalloprotease containing a typical His-Cys-Ser catalytic triad.It was shown to possess high tolerance to surfactants,oxidants,and salts.Most importantly,the enzyme was considered to be ideal strategy for efficient dye decolorization than the laccases requiring redox mediators.