Structural Design And Functional Evolution Of Bacterial Laccase

Bacillus subtilis Laccase is a blue multi-copper oxidase that participates in the degradation of lignin and catalyzes the oxidation of various aromatic amine-like small molecules,and it is also an environmentally friendly enzyme.In this thesis,a bacillus subtilis strain ZNXH1 with high laccase activity was obtained by UV-induced mutagenesis from the original strain CAR2,its laccase,CAHH1?R155H,M157 H,A158T?had a significantly higher catalytic activity than laccase CAR2 from stain CAR2.In order to establish the correlation between the function and structure of laccase,we designed and constructed six mutant laccases by site-directed mutagenesis at three key loci of 155,157,158 sites in the second conserved region of laccase molecule: CAW?R155W?,CAWH?R155W,M157H?,CAWH1?R155H,M157 H,A158E?,CAHE?R155H,A158E?,CAHH?R155H,M157H?and CAH2?R155H?.Here the CAR2 and CAH2?R155H?laccases served as the shared laccase controls.The eight kinds of laccase above were effectively expressed in E.coli and their molecular weight was 66 kDa.The properties and functions of the eight laccases were compared and analyzed,and the following results were obtained:?1?CAHH1 and all six mutant laccases?CAW,CAWH,CAWH1,CAHE,CAHH,CAH2?,especially CAHH1 and CAHH,followed by CAH2 and CAWH,had a higher catalytic activity than wild-type laccase CAR2,indicating that His155,His157,Thr158 locus of bacillus subtilis laccase has a significant positive effect on laccase activity;?2?The optimal temperature was 50 ? for CAR2,but 60 ? for other seven laccase.Moreover,the third group of laccases?CAHH1,CAHH and CAH2?,followed by CAWH1 and CAHE showed a higher thermostability than CAR2,they could maintain the activity of 70% or more After incubation at 40-80 ? for 1h.These results indicated that His155 and His157 sites in laccase molecule had greater contributions to the thermal stability of laccase;?3?The optimal pH was 4.0 for laccase CAR2,but was 4.6 for CAHH1 and six mutant laccases,indicating a slight alkali shift for the catalytic conditions of CAHH1 and six mutant laccases,which was obviously beneficial for the application of laccase.All of mutant laccases containing W155?CAW,CAWH,CAWH1?showed a higher acid-base stability,indicating that W155 site has a significant contribution to improving the stability of laccase,especially in weak alkaline conditions;?4?Mn²⁺ as well as Mg²⁺ and Co²⁺ could increase the activities of these laccases,and the activation of Mn²⁺ is the most significant;?5?The kinetic analysis showed that His155,His157,Glu158 / Thr158 loci were beneficial or necessary to improve the catalytic efficiency of laccase;?6?In the presence of the mediator ABTS,both the original and the mutant laccases could effectively decolorize the Indigo carmine?anthraquinone?,Congo red?azo?and Crystalline violet?triphenylmethane?dyes to over 70% rate within 3 hours.Especially,the decolorization rate of Indigo carmine and Congo red was over 90%,of Crystal violet was over 80% by CAHH and CAHH1,indicating that His155,His157,Thr158 locus are also beneficial for improving the application of laccase.However,had lower decolorization rate of RBBR?anthraquinones?and Methyl red?azo?was less than 20% by these eight laccases,indicating that the second conserved region of laccase has a certain selectivity for its substrates.On the other hand,the decolorization of indigo carmine seem not to depend on the presence of the mediator ABTS,but that of Crystal violet was indeed dependent on ABTS by these eight laccases.In the presence of Mn²⁺,the decolorization efficiency of these laccases?except CAR2?,especially laccases CAHH and CAHH1,on Indigo carmine,Congo red and C rystal violet was improved obviously,it almost reached to 100% within 3 hours by these laccases,or it reached to 100% within 1.5 h by laccases CAHH and CAHH1.These results indicate that these laccases,especially the second and third groups of laccase?CAWH,CAWH1,CAHE,CAHH1,CAHH,CAH2?were expected to be the potential industrial enzymes.In general,the mutant laccase CAHH and CAHH1 were more prominent in terms of specific activity,temperature stability,decolorization rate,sensitivity to the environment and so on,both of them could better meet the industrial needs.Through the design and transformation of the amino acid residues at the key sites of the second conserved region of laccase,the differences in the activity,the temperature stability,the decolorization rate and the sensitivity to the environment were examined.The relationship between the structure and function of laccase in this region had laid a foundation for improving laccase and designing efficient laccase for different purposes and application conditions.