Screening And Identification Of Laccase-producing Fungi And Its Application In Dyes Decolorzation

Laccase(Laccase EC 1.10.3.2)also known as enzyme and polyphenol oxidase,which is found in natural plants,fungi and insect tissues and organs.Laccase or laccase analogues can also be produced in some bacteria and higher animals.Laccase has strong ability to degrade natural and synthetic chemicals of various structures.At the same time,the byproducts of the reaction are only water,which is also called "green enzymes".At present,all the laccase producing organisms,fungi are considered to have the highest secretory efficiency and the best catalytic efficiency.Laccase is of great practical value in various fields,but laccase production is low and expensive at present.In addition,laccase from different sources has different enzymatic properties and catalytic capabilities,and the catalytic specificity for different substrates is also different.Therefore,on the one hand,improving laccase production and reducing production cost has become a hot research topic nowadays.Meanwhile,finding and developing new laccase resources is also of great significance.In this paper,an easy method was used to isolate laccase-producing fungi.A laccase-producing filamentous fungi was isolated from the dried and decayed plant samples.The strain was highly similar to the species in Genus Trametes by ITS sequence analysis and mycelia morphology,and was named Trametes sp.MA-X01.The addition of copper ions and aromatic compounds to the liquid medium could induce the laccase synthesis in Trametes sp.MA-X01.Copper-induced laccase activity increased in a dose-dependent manner.The highest laccase activity(2138.9 ± 340.2 U/L)was obtained by adding 2.5 mM Cu2+to the medium,which was about 7 times higher than that of the control group.The induction degree of aromatic compounds was different,and the highest laccase activities was obtained by adding the vanillin(981.6±77.2 U/L)or vanillic acid(1007.9±59.5 U/L)to the medium,the laccase activities increased by 3.4 times and 3.5 times separately compare to the control group.In this paper,the characteristics of a novel laccase from a newly isolated white-rot fungi Trametes sp.MA-X01 were studied,and the application in the dyes decolorization was also tested.The results showed that the molecular weight of laccase was produced by Trametes sp.MA-X01 under liquid culture condition was about 62 kDa.The enzyme belongs to high temperature reactive enzyme,the optimum reaction temperature is 60?,and the optimum pH is in the range of pH3.0-4.5.Among the tested metals,Mg2+,Mn2+,Zn2+,Cu2+can promote the enzyme activities.While a strong inhibitory effect was observed when ?-mercaptoethanol,L-cysteine,and dithiothreitol(DTT)was added to the reaction mixture.Trametes sp.MA-X01 produced laccase for different kinds of dyes on a large scale without the addition of reaction mediators.The results showed that the enzyme had good degradation ability for various dyes,but the degradation ability of different dyes was great.In azo dyes,the degradation rate of Evans blue is stronger,the degradation rate of 48h is more than 80%,and the degradation rate for trypan blue and orange yellow is more than 50%,but the degradation rate of two methyl yellow is only 5%.In heterocyclic dyes,48h degradation rate of tiger red sodium and azo carmine B is over 50%,and the degradation rate of 48h of Crocus T and fluorescein is over 40%.In the triphenylmethane dye,48h degradation of malachite green rate of more than 85%,the degradation of methyl green and light green SF rate is about 75%.The degradation rate of 48h for Prussian blue is about 40%,indicating that(CN-)can also serve as a catalytic substrate for this enzyme.In this experiment,3 types of 6 dyes were taken as the research objects,including azo dyes Evans blue and acid chrome blue K,heterocyclic dyes acid red 94 and basic red 2,triphenylmethane dyes light green SF and basic green 4,and the effect of different reaction conditions on the decolorization rate was preliminarily discussed.The results showed that under the same condition,adding mediator ABTS to the reaction system can improve the decolorization efficiency,and the decolorization effect is the best when the reaction temperature is 40 degrees.In the first 3 h of the reaction,the decolorization rate was higher,but the degradation ability of the enzyme to different dyes was different.The overall decolorization effect was triphenyl methane,azo and heterocyclic.The effect of increased enzyme dosage on decolorization of triphenyl methane dye(malachite green and bright green SF)was less,the amount of enzyme was increased from 1 U to 5 U,the decolorization rate increased by 10%.For two kinds of heterocyclic dyes(acid red 94 and alkaline red 2),the amount of enzyme was increased from 1 U to 5 U,and the decoloration rate of dye was increased by 51%and 40%,respectively.For several dyes with high decolorization rate,Evans blue,malachite green and bright green SF,the dye concentration has little effect on the decolorization rate,and the decolorization rate gradually decreases with the increase of the dye concentration in the lower decolorization rate.