Enhancing CotA-laccase Decolorization Activity Via Site-saturation Mutagenesis And Its Degradation Mechanism To Reactive Black 5

Azo dyes(-N=N-)are synthetic dyes which were widely used in textile dyeing in the printing and dyeing industry.Reactive Black 5(RB5)is a typical refractory azo dye,which has carcinogenic,teratogenic and mutagenic effects.It will cause serious environmental pollution and health hazards if the printing and dyeing wastewater is pumped into the environment without treatment.Therefore,the decolorization and degradation of dyes is an important task in the treatment of printing and dyeing wastewater.CotA-laccase from Bacillus sp.has the advantages of environmental protection and cost competition.It is a green catalyst and has broad application prospects in the degradation of harmful dyes in printing and dyeing wastewater.However,the effective decolorization of CotA-laccase usually requires the participation of expensive toxic mediators,which limits its application in industry.In this study,a semi-rational method of saturation mutation was used to modify the Bacillus pumilus W3 CotA-laccase,and mutants with increased decolorizing activity on Reactive Black 5 without the participation of mediators were selected.The enzymatic properties of the mutants and the molecular structure mechanism of increased catalytic activity were analyzed,and the degradation mechanism of the mutants on Reactive Black 5and the toxicity analysis of the degradation products were further carried out.The main research results are as follows:(1)Tough homology modeling and molecular docking analysis,it was speculated that the amino acids(T415 and T418)near the type 1 copper site of B.pumilus CotA-laccase and the amino acids(Q442) at the entrance of the substrate binding pocket have significant catalytic efficiency of the enzyme influences.Therefore,saturation mutations were performed on these Tee sites.(2)Two double site mutants T415D/Q442A(DA) and T418K/Q442A(KA)obtained demonstrated 43.94% and 52.64% RB5 decolorization rates in the absence of a mediator at pH 10.0,respectively,which were about 3.70-and 4.43-fold higher compared with the wild-type CotA-laccase.The mutant has good stability in a wide pH range,and is resistant to alkalis and most metal ions.It is a thermophilic alkali-tolerant enzyme.(3)The catalytic efficiencies of the double mutants DA and KA on the substrate ABTS were 356.73 L�mmol^-1�s^-1 and 845.50 L�mmol^-1�s^-1,respectively.Compared with the wild-type CotA-laccase,the catalytic efficiency of DA and KA to ABTS were about 2.25-and5.33-fold.The catalytic efficiencies of the double mutants DA and KA on the substrate RB5are 18.67 L�mmol^-1�s^-1 and 22.54 L�mmol^-1�s^-1.Compared with the wild-type CotA laccase,the catalytic efficiency on the substrate RB5 were about 2.67 and 3.22-fold,respectively.(4)CotA-laccase mutant KA has the best decolorization effect on RB5 at pH 7.0,50?,and dye concentration of 50 mg�L^-1.The decolorization rate can reach 85.11%after 9 hours.Meanwhile,CotA-laccase mutant KA can also efficiently degrade synthetic dyes of different structures and types such as azo,an Taquinone and triphenylmethane.After CotA-laccase mutant KA decolorizes Evans Blue(EB) and Acid Red 1(AR1),the decolorization rate can reach 96.34% and 84.01%.(5)The decolorization solution of RB5 catalyzed by CotA-laccase mutant KA was analyzed by ultra high performance liquid chromatography-mass spectrometry(UPLC-MS).The results showed that the azo bond(-N=N-) was firstly broken under the catalysis of CotA-laccase mutant KA.And then it was further degraded into other possible compounds after desulfonation and deamination.Finally,the effect of RB5 decolorizing solution on wheat germination was studied.the results showed that compared with the original solution of the control RB5,the product after the catalytic degradation of the dye has reduced phytotoxicity.