Directed Evolution Of Laccase And Its Application In Dye Decolorization

Laccases?EC 1.10.3.2?that are polyphenol oxidases containing four copper ions belong to the copper blue oxidase family.Due to their broad substrates spectrum comprising varieties of phenolic and non-phenolic compounds,they are widely used in environmental remediation,paper industry,food industry,biological detection and other fields.However,because of the low activity and expression level of laccases in nature,they cannot efficiently oxidize the substrates,thus limiting the extensive applications of laccases.Therefore,improving the properties of laccases?expression level,enzyme activity,selectivity,stability,and substrate spectrum?through directed evolution will expand the applications of laccases in different fields,especially in dye wastewater treatment.In this study,the gens of laccase Lcc2 from Moniliothora roreri and the laccase Lcc from Tremetes viscolor were selected as objects of this project.The main research contents of this research are as follows:1.To improve the expression of the Tremetes ciscolor laccase Lcc,the transcription regulatory regions of Lcc were engineered by directed evolution.The mutation library was generated using error-prone PCR with different concentrations of Mn²⁺and the Saccharomyces cerevisiae was used as host cell for heterologous expression of laccase Lcc.Totally 960 mutants were picked into 96-well plates for cultivation and screened using2-nitrogen-bis?3-ethylbenzothiazoline-6-sulfonic acid?diamine salt?ABTS?assay.The mutants with enzyme activity higher than wild type were selected and then rescreened by shaking flask cultivation.Finally,three mutants?M1,M2,M3?with higher total enzyme activity were selected.The crude enzyme activities of these three mutants are 1.73 times,1.46times and 1.37 times of that of the wild-type strain,respectively.The crude enzyme activity of the best mutant strain M1 was up to 35.4 U/L.2.By optimizing the transcription regulation region of Lcc,the total enzyme activity and expression level wasn't improved too much.Therefore,through the literature investigation,we selected laccase Lcc2 with a high enzyme activity derived from Moniliophora roreri as the research object for subsequent engineering experiments.Because the Pichia pastoris can perform high-density fermentation and express more exogenous proteins than Saccharomyces cerevisiae,this chapter has chosen the Pichia pastoris for expression of the codon-optimized Lcc2 gene.To improve the ability of laccase to catalyze high redox potential substrate HOBT,a random mutation library of Lcc2 was established using error-prone PCR with a concentration of Mn²⁺at 0.3 mmol/L.First,the mutation library was screened by ABTS assay to remove the inactive and low active mutant strains,and then rescreened using HOBT as the substrate.Finally,a mutant E3 with a 1.2 times higher enzyme activity to the substrate HOBT than the wild type was obtained.Subsequently the E3 mutant and wild-type Lcc2 were purified and the concentration of pure enzymes were quantified with a BCA kit.The enzymatic properties and kinetics of pure enzyme mutant E3 and wild-type Lcc2 were examined using ABTS as substrates.The results showed that the optimum pH and optimum temperature are the same.The optimum pH is 3.0,and the optimum temperature is 30?.When ABTS is used as substrate,the K_m value,K_catat value,and the catalytic efficiency of wild-type Lcc2 are 37.61?mol·L^-1,138.12 s^-1,and 3.67 L·??mol·s?^-1,respectively,and the K_m value,K_catat value and the catalytic efficiency of mutant E3 are 50.5?mol·L^-1,133.65 s^-1,and 2.65 L·??mol·s?^-1,respectively.3.Wild laccase Lcc2 from Moniliophthora roreri expressed by Pichia pastoris cooperating with different mediators?HOBT,resveratrol,p-coumaric acid,NOP?was employed to decolorize five different types of dyes?azo,triphenylmethane,anthraquinone,indigo,phenothiazine?.The decolorization abilities of mutant E3 and wild-type laccase Lcc2with the HOBT mediator were compared.The results show that with the help of small molecule mediator HOBT,wild laccase Lcc2 can decolorize more than 95%of nine dyes respectively belonging to azo,triphenylmethane,anthraquinone and indigo.For the more complex heterocyclic phenothiazine dye azure I,the decolorization efficiency was low,the highest decolorization rate is only up to 40%under the mediation of p-coumaric acid.Through further optimization of the reaction system for the azure I decolorization,it was found that at pH 6.0,p-coumaric acid 2 mmol·L^-1,and laccase 125 U·L^-1,the decolorization rate for the azure I catalyzed by Lcc2 increased to 81%.In summary,the recombinant laccase Lcc2 shows a good application potential and prospect in the treatment of dye wastewater and the environmental protection.Compared with the wild-type laccase Lcc2,mutant E3 can improve the decolorization ability of dyes with the assistance of the HOBT mediator.The decolorization capacities of the mutant E3/HOBT mediator system to dye congo red,reactive black 5,reactive brilliant blue KN-R,phenol red,coomassie brilliant blue R,crystal violet,bromophenol blue,brilliant blue R,indigo blue,indigo carmine and azure I are 1.39 times,1.43 times,1.66 times,2.28 times,2.25 times,1.45 times,1.52 times,1.32 times,1.27 times,2.2 times and 1.21 times of wild type,respectively.