Cloning And Modification Of Laccase From Bacillus Licheniformis And And Study On Decoloring Effect Of Dyes

Laccase（Laccase,Lac,EC 1.10.3.2）is a copper-containing oxidase produced mainly by microorganisms,which can decompose a series of substances such as phenols,polyphenols,aniline,lignin,polycyclic aromatic hydrocarbons and even inorganic substances.It is widely applyed in the treatment of toxic wastewater,decolorization of dyes and cosmetics synthesis,which has attracted wide attention of the scientific community.China is a large country of dyestuff production.Every year,10%-15%of dyestuffs are discharged directly into the water body to cause water pollution.The treatment methods of dyestuff wastewater include three kind of methods.Compared with the traditional physicochemical methods,biodegradation has become a research hotspot because of its advantages of freindly environmental protection,low operating cost and good decolorization effect.However,in the practical application of dye degradation,laccase mainly has the shortcomings of long fermentation period,low yield and poor stability,so it needs to be modified by genetic engineering or protein engineering technology.In this study,the laccase gene of Bacillus licheniformis was cloned,heterologous expressed,and further modified with site-directed mutagenesis and error-prone PCR,also decoloring effect of dyestuffs was detected.The results obtained as follows:1.The activity of crude enzyme from Bacillus licheniformis was determined,only laccase activity was detected,without lignin peroxidase and manganese peroxidase activity.The laccase gene of Bacillus licheniformis was cloned and expressed in E.coli.The activity of recombinant enzyme was 16.48 U,40%higher than that of Bacillus licheniformis,and the specific activity was 121.75 U/mg after purification.The optimum temperature of recombinant laccase was 65℃,the optimum p H was 4.0,and concentration of copper ion was 12 m M,presented with better stability.2.The recombinant laccase was modified by site-directed mutagenesis and error-prone PCR.The activity of mutant Lac^ep69and D500G were increased by 35%and250%respectively.In addition,the enzyme activities after purification were 170.45U/mg and 426.13 U/mg,respectively.The optimum temperature,p H and concentration of copper ion of the mutant Lac^ep69were 80℃,4.0 and 10 m M respectively,which showed better stability.The optimum temperature and p H of mutant D500G were increased,while concentration of copper ion was decreased,and its stability improved.We found that there was no significant difference in the secondary and tertiary structures of original laccase and its mutants.The reason of increasment in acitivity probably came from the changes of secondary bonds such as hydrogen bonds,the changes of surrounding electron clouds and the changes of hydrophobicity of amino acids.3.The laccase expressed in the purified recombinant E.coli and its mutants Lac^ep69and D500G was treated with 0.1 mg/m L for 6 h.The degradation rates of methyl orange,acid lake blue and acid violet were different,and D500G has the highest decolorization rate of up to 78%for acid violet.Modification of laccase from Bacillus licheniformis by site-directed mutagenesis and error-prone PCR,we obtained two strains with high enzyme activity and more stable enzymatic properties,which can provide theoretical guidance for the modification of laccase and the industrial treatment of dye wastewater.