Relationship Between Structural Basis And Function Of The Bacterial Laccase

Laccase from Bacillus subtilis is a copper-containing polyphenol oxidase that catalyzes the oxidation of various small aromatic amines and phenolic substances.Laccase is able to participate in the polymerization and degradation of lignin.As a potential industrial enzymes,laccase has many advantages,such as its diversity substrate,wide pH range and high thermal stability.Laccase has a wide range of applications in food,paper,textile,biosynthesis,and biosensors.We found that a laccase,CAR2?R155?,from a original Bacillus subtilis was sensitive to the environment,but its activity was low.In order to achieve highly active laccase that meets the requirements of industrial applications,We made two rounds of screening by UV mutagenesis and obtained a second-generation strain ZXN4 and its laccase CAH4?H155,498LEAG501?.Furthermore,for the key site of laccase?502?,three groups of mutant laccases were achieved from the original laccases CAR2 and CAH4 by point mutation as follows: group one: CAR2?RF1?M502F?,RL3?M502L?;group two: CAH2?R155H,a control mutant?,HF3?R155H,M502F?,HL5?R155H,M502L?;group three: CAH4?HH5?498LEAG501,M502F?.Here the CAR2 and CAH2?R155H?laccases served as the shared laccase controls.The optimum pH of laccases?RF1,RL3?in the first group,second group?CAH2,HF3,HL5?,third group?CAH4,HH5?was increased from 4.0 of CAR2 to 4.6,and the optimum temperature was also increased from 50 ? to 60 ° C.It indicated that these laccases are more adaptable to the application environment.As a metal enzyme,different metal ions may have some effects on the activity of laccase.The activity and catalytic efficiency of the second group of laccases?CAH2,HF3,HL5?and the third group of laccases?CAH4,HH5?were higher than that of the first group of laccases?CAR2,RF1,RL3?,but the sensitivity of the first group of laccases?CAR2,RF1,RL3?and the third group of laccases?CAH4,HH5?to the environment was higher than that of the second group of laccases?CAH2,HF3,HL5?,especially to Mn²⁺.Among the three groups of laccases,CAH2 has the highest the catalytic activity and catalytic efficiency and has lowest sensitivity to environment.The catalytic activity and catalytic efficiency of CAH2 were about 4 times and 9 times higher than that of original laccase CAR2.The catalytic activity and catalytic efficiency of CAH4 were increased by about 2.7 and 4.1 times,respectively,compared with that of original laccase CAR2.For the sensitivity to the environment,CAH4 was also higher than the original laccase CAH2,and its catalytic efficiency was more significant in the presence of Mn²⁺,which almost reached the catalytic efficiency of CAH2.For the application of these laccases,the decolorization rates of indigo carmine?indigo?and Congo red?azo?were higher than 75%,but the decolorization rates of bright blue?Quinone?,methyl red?azo?were lower after 3h catalysis by these laccases.Especially,the decolorization rates of these dyes by the kinds of mutant laccases with M502 F,M502L is higher than those by other laccases.The decoloration of crystal violet by laccase is very dependent on the presence of mediator ABTS.In the presence of mediator ABTS,crystal violet?triphenylmethane?could be decolorized to 86% by all laccases except the original laccase CAR2,otherwise,the decolorization rate of crystal violet is very low,only about 10%.It was also found that in the presence of Mn²⁺,the decolorization rate of crystal violet was significantly increased to nearly 100% after 3h catalysis by all these laccases,while it reached to 100% after 1.5h catalysis by CAH4.The results above indicated that laccase could be significantly improved in the specific activity,the optimum temperature,the temperature stability,the decolorization effect and the sensitivity to the environment by site mutation,and the mutant laccases that were suitable to the industrial application could be obtained.The relationship between the structure and function of laccase could be further studied by changing the key amino acids in laccase molecule.The results in this thesis may lay the foundation for the application of laccase in all application fields.