Gene Mining,Characterization And Structural Analysis Of Novel Bacterial Laccase

Laccases?EC1.10.3.2?,a type of copper-containing polyphenol oxidases,belong to the family of multi-copper oxidases?MCOs?.These enzymes couple the four-electron reduction of molecular oxygen to water with the oxidation of a wide variety of phenolic and non-phenolic compounds.Because of their wide spectrum of substrates,laccases could be good candidates in the dye bleaching,paper industry,food industry,biosensors and other fields.Therefore,it is of great significance to explore laccases with excellent properties and high activity.In contrast to fungal laccase,bacterial laccases often has high temperature resistance,alkali resistance and salt tolerance and other advantages.In this study,three novel bacterial laccases,named LacTT,LacCT and Lac BE,respectively,were screened from GenBank by genomic data mining technology.LacTT has outstanding enzymatic properties and was heterologous expressed,enzymatic properties and decolorization ability studied in Pichia pastoris.Then,the amino acids and their functions related to the electron transfer and substrate binding of the selected laccase were studied by homology modeling and site-directed mutagenesis.The main contents and results of this study are as follows:?1?Three novel bacterial laccases LacTT,LacCT and Lac BE from Thermus thermophilus SG0.5JP17-16,Caldalkalibacillus thermarum TA2.A1 and Bacillus endophyticus,respectively,were discovered from GenBank by genomic data mining technology.The results showed that LacTT had the highest reaction temperature?90 ??in the three bacterial laccases,and it had the highest enzyme activity at high temperature and neutral and alkaline condition.LacBE has a wide range of reaction temperature and pH.While LacCT showed the highest optimum pH,its reaction temperature and pH range were narrow.The enzymatic properties of LacTT show that they have high application potential.Recombinant expression in E.coli showed that LacTT had high expression level and was easy to be purified.?2?Then,LacTT were heterologously expressed in Pichia pastoris with the yield of 709 U/L in shake flask culture conditions.LacTT had a relatively high activity and stability in the neutral and alkaline condition with the optimum pH 4.5,6.0,7.5 and 8.0 for the substrates of ABTS,SGZ,guaiacol and 2,6-DMP,respectively.The optimum temperature of LacTT was 90?,with a half-life of about 4h at 90?.LacTT was more tolerant to halides than most other laccases,and NaCl and NaBr activated LacTT to varying degrees in less than 1 M concentration.The decolorization efficiency of LacTT was between 73%-94% for four synthetic dyes.In addition,NaCl can improve the decolorization efficiency of LacTT.These results suggest that LacTT is a good candidate for decolorization of dyes in textile wastewater.?3?Three charged amino acids?K428,R290 and D394?were predicted to be essential on the substrate binding and chosen as mutation targets.Site-directed mutagenesis including K428 M,K428L,K428 R,K428E,R290 A,R290H,D394 A and D394 R were performed with site-directed mutagenesis and were then heterologously expressed in E.coli and purified.The characteristic absorption peaks at 600 nm and 330 nm of the mutants were different from wild type,indicating that the mutation site had some perturbation to the structure of copper center.The k_cat/K_m value of K428 M was about 2 times but K_m value was about 1/2 of wild type,presumed that the increased K428 M enzyme catalytic efficiency may be due to the increased affinity of the mutant with the substrate.The k_cat values of R290 A and R290 H were about 2/3 of the wild-type,and D394 A and D394 R were almost inactivated.Comparison of their binding constants and spatial structures with LacTT showed that the side chains of R290 and D394 of LacTT may interact with the substrate,which may disappear after the mutation,thus affecting the substrate binding or even the enzyme catalytic efficiency of the substrate.?4?Based on sequence alignment,homology modeling and site-directed mutagenesis techniques,the essential residues of electron transfer of LacTT were studied.The homology modeling indicates that LacTT is a typical three-domain laccase with conserved spatial structure and active center structure.After comparing with other bacterial laccases and analyzing of the homology model of LacTT,four negatively charged amino acids?D106,E456,E356 and D423?were predicted to be essential on the electron transfer and chosen as mutation targets.Site-directed mutagenesis including D106 L,D106R,D106 E,E456L,E456 R,E456D,E356 A,D423A were performed with site-directed mutagenesis.Wild-type and its mutants were heterologously expressed in E.coli and purified.All mutants above showed significant decreased activities compared to the wild-type LacTT,indicating that D106,E456,E356 and D423 were essential residues for activity of LacTT.The catalytic efficiency(k_cat/K_m)of D106L?D106R?D106E were only 27%,20% and 30% of the wild type,respectively,presuming that because of the failure of the 106 th residue side chain to provide proton to form the correct water molecule network with T2 Cu,the reduction of oxygen in TNC was blocked and the catalytic activity is decreased.When E456 mutated to Leu,Arg or Asp,this site can not normally provide protons to the TNC,making the TNC reduction reaction of oxygen to be unnormal and almost lost of catalytic activity,indicating that E456 may be the only residue to transfer protons to the TNC at this position,but also stabilize the region.E356 was located in the substrate binding domain and very close to T1 Cu,with the carboxylic acid group of the side chain pointing to T1 Cu.The catalytic efficiency(k_cat/K_m)of E356 A was only 7% of the wild type,but the K_m value 3.8 times that of wild-type,indicating that E356 was likely to participate in the electron transfer between substrate and the T1 Cu,but also played an important role in substrate binding.D423 was located near the TNC,the side chain carboxylic acid groups pointed to the TNC,so D423 A may not be able to provide protons,thus affecting the TNC oxygen reduction,the k_cat/K_m value is about half of the wild type.Therefore,D106,E456,E356 and D423 may all be the key amino acid sites for LacTT during electron transfer.?5?The electron-transfer mechanism of LacTT is speculated as follows:when substrate was oxidized,the electrons of the substrate are captured by the histidine coordinated with T1 Cu,and E356 may accept protons from substrate.In the process of oxygen reduction,the role of E456 may provide protons to the TNC,while D106 and D423 may assist in the O-O bond cleavage in TNC.