Secretory Expression Of Cota-Laccase Mutant In Pichia Pastoris And Its Mechanism On Evans Blue Degradation

Laccase can catalyze the redox reactions of phenols and aromatic compounds,which use O₂ as an electron acceptor to catalyze substrate without the participation of cofactors.Water is the only by-product in these reactions,so laccase is a typical environmental-friendly“green catalyst”.Although the fungal laccase has been commercialized,its activity is very low or even inactivated in an alkaline environment,and its thermostability is also poor,which greatly limit its application in alkaline wastewater such as printing and dyeing wastewater.In contrast,CotA-laccase from Bacillus has high enzyme activity at alkaline pH and high temperature.Although the natural catalytic activity of CotA-laccase is slightly lower than fungal laccase,it has excellent pH and thermal stability and can tolerate higher salt concentrations and organic solvent.Therefore CotA-laccase is more suitable for application in the printing and dyeing wastewater treatment.However,CotA-laccase is an insoluble protein in spore coat,which results in the difficulties of purification and limits the application of CotA-laccase.In the work Pichia pastoris system was selected to achieve secretory expression of a D501G/L386W/G417L/G57F mutant of CotA-laccase from B.pumilus?GWLF?with highly improved catalytic effciency level.GWLF encoding gene was optimized according codon usage in P.pastoris,and the optimized gene was integrated into the genome of P.pastoris.Finally,GWLF was expressed extracellularly.The molecular mass of recombinant GWLF was estimated to be 75 kDa,and the active GWLF is a dimeric glycoprotein.Optimization of GWLF induction conditions were carried out by single-factor optimization in shake flask.The optimal strategy is formulated as induction at 26°C for 144 h in BMMY?pH 7.0?containing 0.25mmol·L^-1 Cu²⁺,1.0%?v/v?methanol and 0.5%?w/v?sorbitol,and the enzyme activity of recombinant GWLF was 4,092 U·L^-1.A maximal activity of 17,080 U/L in the culture supernatant was obtained by high cell density fermentation in a 5 L bioreactor.Biological toxicity test showed that GWLF can catalyze the detoxification of Evans blue efficiently.The mechanism for Evans blue decolorization and detoxification by GWLF was analyzed through liquid chromatography-mass spectrometry;the azo bond?-N=N-?was transformed into N2instead of toxic aniline compounds.This study is the first to clarify the mechanism of laccase-catalyzed degradation of Evans blue.