Overexpression And Characterization Of Kurthia Huakuii Laccase From Pichia Pastoris

Laccase is a phenolic oxidase,belonging to the copper oxidase family.Four copper ions are combined with the catalytic activity center of the enzyme as a cofactor for Laccase.The substrate of Laccase contains a variety of phenols and non-phenolic substances,and the process of catalytic oxidation required less energy,no additional harmful by-products.As an environmentally friendly green catalyst,Laccase is widely used in environmental protection,fabric printing and dyeing,pulp bleaching,bio-energy,food industry,biosensors,chemical synthesis and many other fields.In this research,we studied the high expression of Kurthia huakuii laccase in Pichia pastoris and its enzymatic properties,and confirmed its possible application in dye decolorization.1.The laccase gene from Kurthia huakuii LAM0618 was codon-,GC content-and mRNA secondary structure-optimized before being cloned into the pPIC9K-NTS,derived from the pPIC9 K vector.The recombinant vector was linearized and subsequently transformed into Pichia pastoris GS115,and the yeast transformants were selected on the plates with gradient concentration of geneticin(G418).Further screening of transformants with high Laccase expression level was conducted through induction of protein expression and detection of enzyme activity to obtain a high expression strain.2.The optimal condition for Laccase production in flask was 28°C,1.0% Tween-80 in the induction medium,and 1.0% methanol added at every 12 h.At these conditions,the highest Laccase activity reached 6877.24 U/mL at 72 h,which was much higher than the highest level reported.3.The optimal reaction temperature was 65°C using 10 mmol/L 2,6-DMP(2,6-dimethoxyphenol)as the substrate.The enzyme was thermal stable that 70% activity retained after reaction at 90°C for 6 h.The optimal reaction pH was 7.0,but the pH tolerance was narrow that the enzyme activity was rapidly lost at pH < 7.0 or pH > 8.0.The optimal concentration of Cu2+ in the enzyme reaction was 0.04 mmol/L;1 mmol/L EDTA completely inhibited theenzyme activity,3 mmol/L PMSF to reduce the relative activity to 60%;1 mmol/L Mn2+can promote enzyme activity,and most other metal ions all have a negative effect on the enzyme activity,regardless of the concentration.4.In dye decolorization process,reactions were carried out at 65°C for 6 h,using malachite green(10 mg/L)and brilliant green(10 mg/L)as the substrates.The degradation rate was86.50% and 70.82%,respectively.Congo red(500 mg/mL),basic fuchsin(50 mg/mL)and methylene blue(10 mg/mL)were decolorized but not obvious,and the decolorization rates were13.95%,23.03% and 6.95%,respectively.There was almost no decolorization effect on crystal violet(10 mg/L)and basic Tibetan red T(200 mg/mL).In summary,we found that the Laccase of Kurthia huakuii LAM0618 was highly expressed in Pichia pastoris and the enzyme activity of the crude enzyme solution was 6877.24 U/mL(at65°C and pH = 7.0),and Cu2+ is essential.The enzyme have a good effect in the decolorization of waste containe malachite green and brilliant green.