Extracellular Expression Of CotA Laccase In Escherichia Coli:Enzymatic Characteristics And Application

The study employs two secretory expression systems to achieve extracellular expression of CotA laccase from Bacillus subtilis in Escherichia coli.The secretion efficiency of the two secretory expression systems was compared.The result indicates that the secretion efficiency of expression strain mediated by ?-hemolysin expression system was the highest under microaerobic conditions,with the efficiency of 78.75%.Different backbone vector can affect the secretory level;this may be dictated to the distinction of expression element.The activity of CotA laccase fused with carrier protein YebF cannot be detected in the culture medium under microaerobic condition,but can be detected in the medium after auto-induction cultivation,which means the secretion efficiency of different secretory system depends on the induction condition.Surprisingly,CotA laccase can be "extracellularly" expressed by PSD strain without any secretory element or signal.The secretory level was significantly higher than other enginnering strains.We optimize the induction condition of PSD strain for secretory expression of recombinant CotA laccase,and found that induction temperature and the concentration of CuSO4 are two crucial factors during strain induction.Hignest extracellular laccase activity was reached(167.2 U/L)when the shake temperature and microarobic culture temperature was 25? and 30?.No laccase activity was detected in the culture medium in absence of Cu2+ions.Increasing addition of CuSO4 significantly enhanced the production of extracellular laccase.The extracellular laccase was the highest when the concentration of Cu2+ions was 2.1 mM.Over dosage of Cu2+ions can severly inhibit the expression level of PSD strain,which is probably related to the copper resistance of E.coli.The induction happened in log phase can improve the secreotory expression of recombinant laccase.Moreover,the standard of culture flask can also affect the secretion efficiency of strain.Recombinant CotA laccase was successfully purified by Ni2+ affinity chromotography in one step.SDS-PAGE analysis revealed that the molecular weight of purified CotA laccase was around 63 kDa.The Mass spectrometry result indicates that target enzyme was most highly homologous with Spore coat protein A.The optimal pH for purified laccase to oxidize ABTS,SGZ and 2,6-DMP was 4.2?6.6 and 8.2;while the optimal temperature for CotA laccase to oxidaze the three substrates was 80?,60? and 70?.Recombinant CotA laccase showed strong thermostability under elevated temperatures and maintained high activity in natural and alkaline environment.The purifed laccase remained 104.67%,243.56%and 175.94%residual activity after incubation under pH 7.0,pH 9.0 and pH 10.0 for half month.Recombinant CotA laccase showed high resistance towards metal ions,except that Mn2+ and Hg2+can severely inhibit the activity of CotA laccase.Most of the tested inhibitors can severly inhibit the activity of CotA laccase.In comparison,chelating agent EDTA produced little inhibition on rCotA laccase oxidation.The CotA laccase only lost half of its activity when the concentration of EDTA was as high as 100 mM.Low amount of organic solvents have little effect on the activity and stability of recombinant CotA laccase.The inhibitory effect was increased when the concentration of organic solvents was higher.With the assistance of acetosyringone,CotA laccase showed high decolorization ability towards azo,anthraquinone,indigo and triphenylmethane dyes.The decolorization efficiency of puried laccase was higher than crude laccase.The decolorization rate of CotA laccase was effectively improved by changing the decolorization condition(including pH,temperature,mediator concentration and enzyme concentration).The ability of CotA laccase in simulated textile wasterwater decolorization was investigated.The decolorization efficiency after 48 h treatment was 68.06%,indicating that CotA laccase is a potential catalyst for practical dye effluent treatment.The study also explored the reason why the cytoplasmic CotA laccase was "secreted" by PSD strain.The phenomenon was most probably dictated by the permeabilization of PSD caused by Cu2+.Also,the study investigated the auto-induction culture of diffenret enginnering strains.PSD strain can also secrete CotA laccase under aerobic condition.Co-expression of molecular charptone FkpAs and cyto-FkpA can significantly enhance the secretory level of CotA laccase.After auto-induction,laccase activity was detected in the culture medium of YSD strain,which proves that YebF-mediated secretory system can also lead to the extracellular expression of CotA laccase.