Site-directed Mutagenesis And Enzymatic Properties Of Bacillus Sp.Laccases

Laccase is a polyphenol oxidase with phenols and arylamines as substrates.Laccase has potential application in various industries such as pulping and papermaking and environmental pollutant degradation.Although fungal laccase has been commercialized,it is unstable under high temperature and alkaline conditions which limits its application.In contrast,bacterial laccase often demonstrates good stability towards high temperature and high pH.Meanwhile,its short production period makes it more suitable for industrial application.In this work,we tried to improve the catalytic activity of laccases from Bacillus sp.by site-directed mutagenesis.We hope to obtain bacterial laccase mutants with good thermal stability and strong resistance to alkaline environment.In addition,dye degradation abilities of the mutants were also tested.The main results are as follows:The laccase gene of Bacillus licheniformis LS04 was site-directed mutated and a D500G mutant was obtained and heterologously expressed in Pichia pastoris.The activity of D500G was increased by 2.1 times.After optimization of the medium and culture conditions,the expressed laccase activity was 347 U/L after induction for 3 d,which was 9.3 times higher than that of the wild type.The D500G mutant laccase retained similar catalytic properties to the wild-type laccase.The optimum pH was slightly shifted towards alkaline range.The pH stability in the first 3 days was significantly higher than that of the wild-type,and the D500G was highly efficient in decolorization of synthetic dyes under alkaline conditions.The D501G mutant of B.amyloliquefaciens LC02 was obtained by site-directed mutagenesis and was over-expressed in Escherichia coli.The expression level of the mutant was 3 374 U/L.Compared with the wild-type laccase,the mutant showed higher pH and temperature stabilities,and exhibited higher catalytic efficiency.Indigo carmine is a synthetic dye widely used in textile processing.The D501G mutant laccase can degrade more than 92%of indigo carmine in 5 h at pH 9.0 without adding any mediators,which was 3.5 times higher than that of wild-type laccase.According to the UV-vis spectra and LC/MS analysis,the main degradation product of indigo carmine was assumed to be isatin sulfonic acid.The D500G and D501G mutated laccases obtained by site-directed mutagenesis maintained good stabilities under high-temperature and alkaline conditions,and they had potential in dye decolorization and other industries which provided a certain reference for further research and industrialization of bacterial laccase.