In Vitro Molecular Directed Evolution Of Bacillus Subtilis Laccase BsCotA

Laccase（EC1.10.32）is a polyphenol oxidase with four copper ions in the form of a monomeric glycoprotein.Laccase has a wide range of substrates.It can oxidize aromatic compounds,metal ions,organic metal compounds,and even some steroid hormones,biological pigments and lignin.Laccase has great application potential in a series of industrial fields such as textile and dye degradation,paper and pulp industry,and food industry.Bacterial laccase has advantages in glycosylation,thermal stability,organic solvent resistance and preferring for neutral to alkaline conditions,thus endow it good prospect for neutral or alkaline industrial application environment.In order to obtain the bacterial laccase with improved activity and thermal stability,the error-prone PCR technique was used to modify the laccase Cot A from Bacillus subtilis in vitro and achieve heterologous expression in Escherichia coli.Four laccase mutants with improved enzyme activity and protein expression were acquired.The enzymatic properties of laccases were determined,and the three-dimensional structure and mutation sites of laccase of wild type and mutants were analyzed.The decolorization effects of the wild type and mutant laccase on indigo,denim and wood chips of paper mill were compared and analyzed.The main research results of this paper are as follows:（1）Directional evolution of laccase Cot A gene from B.subtilis was carried out by error-prone PCR,and four laccase mutants with improved enzyme activity were screened.The protein content of mutants A2,A3 and A4 was increased by more than4.9 times compared with that of the wild type.The enzyme activity of the four mutants was increased by more than 50%compared with that of the wild type.The optimal p H and temperature of the wild-type laccase and the four mutants were 5.5 and 70^oC,respectively.The residual activity of wild-type laccase and 4 mutant laccases was above 50%when incubated at p H 8-12 for 30 min.Wild-type laccase and 4 mutant laccases were incubated in the range of 43-63^oC for 30 min,and the remaining enzyme activity was still more than 50%.（2）The determination of enzymatic kinetic parameters of laccases showed that the Kcat values of mutant A1,A2,A3 and A4 were 80.5 s^-1,87.71 s^-1,79.55 s^-1 and71.57 s^-1,which were 2.74,2.99,2.72 and 2.44 times of those of wild type,respectively.The conversion numbers of laccase A1,A2,A3 and A4 in the mutant were significantly higher than those in the wild type.The Kcat/Km of mutants A1,,A2,A3 and A4 were 46.97（mol/L）^-1·s^-1,28.64（mol/L）^-1·s^-1,26.77（mol/L）^-1·s^-1,30.13（mol/L）^-1·s^-1,respectively.2.46 times,1.5 times,1.4 times,1.58 times of wild-type[19.13（mol/L）^-1·s^-1],respectively.（3）The decolorization rate of indigo by wild-type and mutant A1,A2,A4laccases at 12 h was more than 60%;When the concentration of A2 enzyme in the reaction system was 2361.2 ng/m L,the decolorization rate was as high as 90.26,and the saturation state was not reached.The concentration of A2 enzyme in the reaction system could be further increased to improve the decolorization efficiency of indigo.A2 enzyme decolorized indigo at different temperatures of 30-80^oC,and the decolorization rate at 80^oC was 90.26%,indicating that A2 enzyme is a high temperature resistant enzyme.A2 enzyme has obvious decolorization and bleaching effect on sawdust of paper mill.（4）The four mutant laccases screened in this study formed a total of 6 mutation sites（N67P,I149T,S278T,S448R,G109E,P458G）.Three-dimensional structure analysis of laccases showed that mutation sites S278T,S448R and I149T introduced new hydrogen bonds in the overall structure of laccases,thus improving the stability of the protein.Because of the introduction of proline in the loop position of the flexible region,the rigidity of this region was improved at the mutation site N67P,and then the activity of the enzyme was improved.Mutation site G109E introduced the carboxyl group of glutamate into the structure of laccase,which affected the electron transport of laccase,and thus appeared to have a negative effect on the catalytic efficiency.The mutation site P458G reduced the thermal stability of the whole protein due to the mutation of P458 to glycine without side chain group.