The Mechanism Of Laccase Modification And Its Catalytic Synthesis Of Aromatic Compounds

Laccase,one of the well-known multi-copper oxidases,which catalyze the oxidation of phenols,aromatic amines,aromatic carboxylic acid and other compounds using oxygen as the terminal electron acceptor.As an environment friendly biocatalyst,laccase has shown great potential applications in many fields,like papermaking industry,textile industry,biological resource utilization,environmental pollutant conversion and other areas.For the polymer fabricating area,enzyme catalyzed reactions have been widely investigated.However,the mechanism of laccase catalyzed aromatic compounds is still need to be further explored.The research and development of laccase on the polymer making study meet with requirement of the green chemistry,which will promote the application of the synthetic polymer materials.In this study,aromatic polymers were prepared based on the catalytic characteristics of laccase from Myceliophthora Thermophila.Different modifications of laccase were designed to obtain excellent performance.Molecular simulation technology was used to predict the geometry and structural changes of laccase.The regulation of polymerization in the aqueous system was achieved.Three reactors with different energy environment were applied in the polymerization procedure,and the function of high-energy environment was investigated using the homology modelling and molecular simulation.The polymers obtained via different reactors were characterized and applied onto different fabrics to achieve multi-functional fabrics.This study will provide fundamental knowledge for the polymer synthesis by laccase,and provide the theoretical and technical support for the establishment of high efficiency laccase-catalyzed synthesis system.The main content and results are as follows:1.Laccase was used as catalyst to perform the polymerization of catechol.The noncovalent and covalent modification of laccase using polyethylene glycol(PEG)was studied and the performance of modified laccase was evaluated.The catalytic performance of different laccases was compared in both aqueous and gel phases,and the interactions among laccase,substrate and PEG was simulated by molecular simulation.The results show that both noncovalent and covalent modification of laccase using PEG(PEGylated laccase)promote the polymerization and improve the polymerization yield and the degree of polymerization.The polymerization yield of PEGylated laccase is 2 times higher than native laccase,however,this promotion only happened in aqueous state.Molecular simulation shows that with the presence of PEG,it slows down the inactivation of laccase.2.The immobilization of laccase/PEGylated laccase was performed with the epoxy resins as the carrier,and PEG was used as bridging compound to connect laccase and epoxy resin.The performance of immobilized laccase in aqueous solution for polymerization was explored and the structures of polymer formed was proposed.After immobilization,the half-life time of laccase was improved,as well as the stability.The polymerization yield and the degree of polymerization were enhanced with the presence of immobilized laccase.The MALDI-TOF MS analysis showed that,when epoxy-native laccase,epoxy-PEGylated laccase and epoxyPEG-laccase used,the degree of polymerization are 10,14 and 15,respectively.Different connections were found when different laccase forms used.The particle size of polymers ranges from 100 to 200 nm with uniform dispersion.3.Three reactors,namely water bath,ultrasonic bath and high-pressure homogenizer were applied to perform the polymerization of catechol.The activity and stability of laccase in different reactors were discussed.The mechanism of laccase synthesis in high-energy environment was investigated.Polymers were characterized to speculate their structures.The results showed that high-energy environment promote the interaction between enzyme and substrate,which promote the polymerization yield,and the yield in ultrasonic bath and highpressure homogenizer were 83.6% and 86.2%,higher than water bath,73.75%.MALDI-TOF MS spectra showed different molecular weight,and the ranges are 500-700 Da,500-1250 Da,and 500-2350 Da,respectively.Laccase under ultrasound and high-pressure homogenization showed less stability compared with normal water bath,however,the polymerization proceeds earlier than this inactivation,thus no obvious negative effect on the synthesis.4.Three different laccases namely native laccase,PEGylated laccase and Epoxy-PEGylated laccase were used in high-pressure homogenizer.The activity and stability of three laccases were compared in high-pressure homogenizer and their corresponding polymers were evaluated.After incubation for 2 h,the residual activity of three enzymes are 55%,75% and 73%.With three enzymes,the polymerization yield of poly(catechol)are 60.8%,63.0% and 72.8%,and for poly(p-phenylenediamine),are 72.8%,84.8% and 71.0%,respectively.Both PEGylated laccase and Epoxy-PEGylated laccase showed the great catalytic property and stability.5.Catechol and p-phenylenediamine were synthesized using polyester(PET),cotton and wool as enzyme container in high-pressure homogenizer,to obtain polymers as well as the functionalization of fabrics.When native laccase,PEGylated laccase and Epoxy-PEGylated laccase were used as catalyst,the DP of poly(catechol)and poly(p-phenylenediamine)are 8,10,13 and 9,12,13,respectively.The particle size of poly(catechol)and poly(p-phenylenediamine)ranges from 30 to 70 nm and 400 to 460 nm.Both polymer present good thermal stability and good resistance to thermal degradation,as well as free radical scavenging ability.6.The functionalization of fabrics were achieved via in-situ polymerization of aromatic substrate onto fabric containers.Colored polymers were generated and the fabrics were dyed.The SEM observation shows uniform distribution of polymer on the surface of cotton,wool and PET.All the fabrics show certain color fastness to washing,sunlight and rubbing.The conductivity of fabrics was determined by the polymer property more than fabrics,after treatment of poly(p-phenylenediamine)all the fabrics show good conductivity.The antibacterial property of the fabrics depends on both fabric and polymer properties.The potential cytotoxicity test of fabrics showed both polymer diffusion and porous fabric structure may affect cell viability,which could be avoided by adjust the concentration of polymer.