The Recombinant Expression Of CueO Laccase Under Computer-aided Modification And Its Degradation Of PAHs

Known as liquid gold,petroleum has a high industrial value with a profound influence on international political forms.In the process of oil exploitation,transportation and refining,oil leaks into the environment,causing water,soil and air pollution and endangering human health.Among them,polycyclic aromatic hydrocarbons(PAHs),as the main pollutants of oil,are carcinogenic,teratogen and mutagenicity hazards.Due to strong hydrophobicity,it is difficult to degrade,and the more benzene rings are,the more difficult to degrade will be.Many studies have found that biological enzymes have the potential to repair environmental pollution.In addition,existing studies have applied laccase to the degradation of PAHs with good results.Bacterial laccase is highly adaptable to the environment with a wide spectrum of applicable substrates with low and expensive activity of wild laccase.In this study,wild-type laccase CueO was carried out allogenic expression by using Escherichia coli as the carrier.To further improve the activity of laccase and solve the problem that the weak stability of wild laccase which could not adapt to the needs of industrial use under extreme temperature conditions,it used computer-assisted means to carry out rational design transformation of CueO laccase,so as to study the optimum catalytic temperature and thermal stability of the modified laccase.To explore the catalytic oxidation ability of the modified laccase substrate,CHI660 e electrochemical workstation was used to analyze the modified laccase protein.In addition to the structure of laccase itself,other factors in the fermentation process also affect the activity of laccase.Therefore,the modified laccase would be optimized for fermentation conditions and finally used to degrade phenanthrene(PHE)and Benzo [a] pyrene(Ba P).By using Autodock Vina,Rosetta Fold,Py MOL and Procheck computer software,the activity of CueO laccase increased from 129.14 U/L to 155.9U/L,while the molecular weight was reduced by 30%,which reduced the burden of protein expression.Using ABTS as the substrate,by setting the catalytic temperature gradient of15℃-90℃ and different catalytic time of 1-48 hours,it was found that the optimal temperature of the modified laccase was 80℃,and the thermal stability was the best at55℃,and the activity remained 80% after 48 hours of catalysis at 80℃.The oxidation peak of the modified laccase was 0.78 v,which was consistent with that of fungal laccase.After the modification,the fermentation conditions of laccase were optimized in terms of mother liquor age,inoculation amount,medium liquid content,bacterial liquid OD600,induction temperature,p H value of medium and copper ion concentration.Finally,the enzyme activity was increased to 543.32±15.03U/L.In the experiment of PAHs degradation,5mg/L phenanthrene and benzo[a]pyrene were degraded by 66% and63%,respectively,under the time gradient for 48 hours.Under the laccase concentration gradient,benzo[a]pyrene and phenanthrene were degraded by 67% and 68%,respectively,after the degradation of 20% concentration of laccase in the solution for 6hours with a good degradation effect.