Enhancing CotA-Laccase Activity Via Site-directed Mutagenesis And Its Degradation Mechanism To Malachite Green

Malachite green which has teratogenic and carcinogenic effects is considered as one of the most difficult triphenylmethane dyes to degrade.Malachite green is a cheap and special medicine for the infection of water mould and parasite.It is often used illegally in aquaculture.Malachite green has a serious pollution effect on water environment and aquatic organisms.It is urgent to find an effective way to degrade malachite green in water.CotA-laccase from Bacillus sp.has been proved to have good decolorization and degradation effects on many dyes in dyeing wastewater.Cot A-laccase is a green natural catalyst,which has a good prospect of industrial application.A strain of Bacillus pumilus W3 with high permeability and alkali tolerance was screened from honey source in the early stage of our laboratory,and its CotA-laccase was successfully cloned into Escherichia coli.However,the catalytic activity of wild-type CotA-laccase is low and it is intracellular enzyme that is not conducive to industrial production.In this work,the catalytic specificity of CotA-laccase to ABTS was improved by site directed mutagenesis.And the mutant S208G/F227A?SF?which increased the catalytic specificity of ABTS by 4.13 times was screened out.The mutant CotA-laccase SF could tolerate acid and alkaline environment and has good resistance to most organic solvents and metal ions.CotA-laccase SF was a thermophilic enzyme which can decolorize a variety of different dyes?methyl red,methyl blue,acid blue 129,malachite green?to varying degrees.In order to produce CotA-laccase SF in industry,the co-expression strain p RSF-SF-PLC of CotA-laccase SF and phospholipase C was constructed.The secreted expression strain pRSF-SF-PLC was induced with IPTG(0.4 mmol·L^-1),Cu²⁺(0.5 mmol·L^-1)and glycine(10 g·L^-1)at OD₆₀₀?0.6.The highest extracellular CotA-laccase SF activity was 1257.22 U·L^-1 after induction at 15°C for 24 h.The CotA-laccase SF could decolorize malachite green in both acidic and alkaline conditions.The best decolorization of malachite green was at pH 7.0.The decolorization rate of malachite green reached more than 90%after incubated at 25°C for 1h.The results of bacteriostatic test and micronucleus test showed that the toxicity of malachite green solution?decolorized by CotA-laccase SF?decreased.The mechanism of decolorization and degradation of malachite green by CotA-laccase SF was studied by HPLC-MS.The results showed that the carbon-carbon bond in the central region of malachite green was firstly broken under the catalysis of Cot A-laccase.And then it was degraded to 4-aminobenzophenone and 4-aminophenol after several demethylation.