Purification And Characterization Of Three Laccases From Pycnoporus Sanguineus MX5 And Their Decolorization Of Industrial Dyes

Laccase(EC 1.10.3.2,also known as benzenediol: oxygen oxidoreductase)is a class of copper-containing polyphenol oxidase,which has a broad application prospect in bioremediation of environmental pollutants,bioenergy production,compounds synthesis and biometric detection,etc.In this paper,the white rot fungus of Pycnoporus sanguineus MX5 was used as the strain for laccase production.For the purpose of exploring the unknown species of laccase and discovering their potential for industrial applications,the fermentation conditions for increasing laccase production were partially optimized.Purification and characterization of laccase from P.sanguineus MX5 and its decolorization for textile dyes were also investigated.The main results were as follows:The conditions of laccase production by P.sanguineus MX5 in submerged fermentation were partially optimized by using inducer,surfactant and fungus co-culture system,respectively.Firstly,different concentration gradients were set up,followed by multiple comparisons for statistical analysis,and it was finally found that the optimal inoculum volume(v/v)of o-toluidine,Tween 80,and Gongronella sp.w5 was 0.006%,0.04%,and 2%,respectively.The yield of laccase at the peak was 20980.55,12724.07 and 9389.81 U/L,respectively,increased by 36.25%,119.93% and 42.13% compared with the control.Three laccase isoenzymes(Lac1,Lac2 and Lac3)were purified by using ultrafiltration,ammonium sulfate fractionation,dialysis,DEAE FF ion exchange chromatography and Sephacryl S-200 HR gel filtration chromatography.The recovery of total laccase activity was 5.21%,including 2.01% Lac1 with a 3.49-fold purification,1.29% Lac2 with a 2.93-fold purification and 1.92% Lac3 with a 0.86-fold purification.SDS-PAGE and Native-PAGE showed that they were all monomeric proteins with apparent molecular weights of 61.8,61.8 and 60.6 k Da,respectively.The UV–vis spectrum of three laccase isoforms showed a broad absorption peak around 330 nm,and they were all identified as P.sanguineus laccase via nano LC-MS/MS and amino acid sequence analysis.The optimal p H values of three laccase isoenzymes were acidic from 2.5 to 5.0,and the reaction temperature range was broad,with relatively high catalytic activities under high and low temperature.Meanwhile,isoforms displayed good p H and thermal stability under neutral condition,of which Lac3 was the best.Laccases had a wide range of catalytic substrates,and ABTS was found to be the most suitable substrate among three substrates in this study.The Km values of the three purified laccases were 0.0093,0.0143,and 0.0230 mmol/L,respectively,and the Kcat/Km values were 64343.43,38586.04,and 4148.67 S-1· m M-1,respectively.The laccase activities were strongly inhibited by Fe2+,but slightly enhanced by Na+?Mn2+ ?Cu2+?Zn2+ at low concentration.Except for chelating agent EDTA-2Na,inhibitors SDS,?-ME,L-cysteine,DTT,Na F had a strong inhibitory effect on laccase.The decolorization of industrial dyes by P.sanguineus MX5 laccases was invistegated.The results showed that Remazol Brilliant Blue R(anthraquinone),Acid Red 1(azo)and Active Black 5(azo)were the substrates of the isoforms,and could be directly decolorized by P.sanguineus MX5 laccases.However,Crystal Violet(triphenylmethanes)and Neutral Red(heterocycles)were not the substrates,and could not be decolorized with the absence of redox mediator ABTS.The decolorization percentages of dyes were promoted by adding ABTS,among which Acid Red 1(azo)increased the most.After 180 min of reaction,the decolorization rate of dye AR1 by Lac1,Lac2,Lac3,and crude enzyme solution reached 65.33%,70.04%,70.09%,and 69.84%,respectively.Among the laccases,only Lac3 and crude enzyme solution could decolorize Neutral Red,but the effect is not good.In general,the decolorization of dyes by LMS was ranked in descending order as AR1,CV,RB5,RBBR,NR.