Improving The Decolorization Activity Of CotA-laccase To Congo Red By Rational Modification And Its Decolorization Mechanism Analysis

Azo dyes（-N=N-）are the most widely used and the largest class of industrial synthetic dyes with the characteristics of stable structure,strong toxicity,strong stability and efractory degradation.It brings great risks and challenges to human health and environmental governance.Therefore,the decolorization and detoxification of azo dyes is the primary task of industrial dye wastewater treatment.CotA-laccase is a highly robust,heat-resistant and alkali-resistant enzyme that can catalyze the decolorization and degradation of dyes.As a green-friendly biocatalyst,it shows great application potential in dye decolorization.The decolorization and degradation of azo dyes catalyzed by CotA-laccase usually requires the assistance of the mediators,but the adverse factors such as toxicity and high cost of the mediators limit their practical application.In this study,the typical azo dye Congo Red was used as a model compound.The molecular docking of CotA-laccase and the substrate Congo Red was carried out,and the complex structure of CotA-laccase/Congo Red was analyzed rationally.The molecular modification of CotA-laccase from Bacillus pumilus W3 was carried out by site-saturation mutagenesis method to improve the decolorization activity of CotA-laccase to Congo Red without the addition of a meditor,and the degradation pathway and mechanism of CotA-laccase to Congo Red were analyzed.It provides a new enzyme tool for biological decolorization of dye wastewater.The main research results are as follows:（1）The complex structure of B.pumilus W3 CotA-laccase/Congo Red was obtained by homology modeling and molecular docking,and the interaction mode of CotA-laccase and Congo Red was analyzed.Three residues that may affect the action of CotA-laccase to Congo Red were rationally selected as candidate modification sites,namely Gly323 and Thr377 at both ends of the substrate binding pocket and Thr418 near the T1 Cu site;（2）A single-point saturation mutagenesis library of three target residues of CotA-laccase were successfully constructed,and single-point beneficial mutants G323S,T377I and T418G were obtained by screening.Combinatorial mutants were further constructed,and finally two mutants T377I/T418G and G323S/T377I/T418G with significantly improved decolorization activity were obtained.The two combinatorial mutants catalyzed the reaction for 24 h at p H 8.0without mediator addition,and the decolorization rates of Congo Red could reach 51.77%and41.34%,respectively,which were 3.01-and 2.40-fold higher than the wild-type CotA-laccase,respectively;（3）The analysis of the reaction kinetic parameters showed that the catalytic efficiency(k_cat/K_m)of the mutants T377I/T418G and G323S/T377I/T418G for Congo Red was 2.21-and2.01-fold higher than that of the wild-type,respectively.The enzymatic properties analysis showed that the two mutants still retained the excellent thermal stability and alkali tolerance of the the wild-type.The structural mechanism of the enhanced catalytic activity of the two mutants was analyzed by molecular docking;（4）By studying the decolorization characteristics of the optimal decolorization mutant T377I/T418G on Congo Red,the optimal decolorization conditions were established:the dye concentration was 25 mg?L^-1,p H 7.0,and the temperature was 60°C.Under these conditions,the catalytic efficiency of T377I/T418G to Congo Red reached 3.96 L·mmol^-1·s^-1,which was2.21-fold that the wild-type CotA-laccase,and the decolorization rate reached 86.47%after 8h;（5）The decolorization and degradation mechanism of Congo Red catalyzed by the optimal mutant T377I/T418G was studied by ultraviolet–visible spectroscopy（UV/VIS）,gas chromatography-mass spectrometry（GC-MS）and liquid chromatography-mass spectrometry（LC-MS）.The results showed that the asymmetric cleavage of the azo bond（-N=N-）of Congo Red firstly occured under the action of T377I/T418G.Then the carbon-nitrogen bond（-C=N-）was broken,and the substituents were oxidized to produce naphthalenesulfonic acid sodium salt,dihydroxybiphenyl and a hydroperoxide.These substances were further degraded into other small molecular compounds under the combined action of the environment and CotA-laccase.