Studies On The Degradation Of Hydrophobic Aromatic Compounds With Lignin Peroxidase And The Related Mechanism

Lignin is the second most abundant renewable aromatic polymer on earth.Its biodegradation is the rate-determining step in the carbon cycle.White rot fungi are the only known organisms that can completely break down the lignin to carbon dioxide and water.The ligninolytic system of Phanerochaete chrysosporium,a white rot fungus,has been widely studied as a model strain.White rot fungi was studied for the degradation of lignin at first.The degradation of lignin by white rot fungi mainly depended on the lignin degradative system,of which lignin peroxidase(LIP)was the most important enzyme.Veratryl alcohol(VA)was the physiological substrate of LiP. It is the secondary metabolite of Phanerochaete chrysosporium.The role of veratryl alcohol in the degradation of lignin has been the subject of numerous studies and considerabe debates.The degradation mechanism of white rot fungi was nonspecific and the nonspecific mechanism aroused the interest of the environment scientist.In 1985,Bumous et al.found that Phanerochaete chrysosporium could degrade polycyclic aromatic hydrocarbon and from then on,the degradation of aromatic pollutants using white rot fungi and their enzymes has been a hot topic in the fields of environmental science and technology.In this paper,by using LiP from Phanerochaete chrysosporium as the oxidant and phenolic or nonphenolic compound as the model compounds,we studied the LiP catalyzed oxidation of aromatic compounds and the related mechanism.Attempts have been made to improve the degradation efficiency of aromatic compounds with LiP,such as coupling glucose oxidase with LiP,medium regulation and addition of small molecular mediator.The mechanism of VA mediated LiP catalyzed oxidation of aromatic compound in different medium was also studied in detail to elucidate the role of VA in the degradation of lignin and aromatic compound.All these will not only help to clarify the role of VA in lignin degradation,but also contribute to find a new way to improve the degradation efficiency of aromatic pollutants with LiP.The innovative research results were acquired as following: â… .Improvement of the degradation efficiency of hydrophobic aromatic compounds by coupling glucose oxidase with LiP and medium regulation.1.High efficient degradation of aromatic dyes by coupling of glucose oxidase with LiP.The H₂O₂ supply strategy was one of crucial factors for high efficient degradation of pollutants with lignin peroxidase(LiP).In this section,an attempt was made to couple a H₂O₂ producing enzymatic reaction to the LiP catalyzed oxidation of dyes. H₂O₂ needed was generated by glucose oxidase(GOD)and its substrate glucose.The generation rate of H₂O₂ could be easily controlled by adjusting the pH of the degradation system and the amount of GOD added.Due to the controlled release of H₂O₂,a sustainable constant activity of LiP was observed.The inhibition of LiP by high level H₂O₂ supplied externally by a single addition at the beginning of the experiments could be avoided.Degradation of three dyes(xylene cyanol,fuchsine and rhodamine B)with LiP coupled with GOD indicated that the present H₂O₂ supply strategy was very effective for improvement of the efficiency of the decolourization of dyes.2.Improvement of the degradation efficiency of aromatic compounds with LiP by medium regulation.Anionic surfactant sodium bis(2-ethylhexyl)sulfosuccinate(AOT)had an inhibiting effect on lignin peroxidase(LiP).To improve the catalytic activity of LiP in an AOT reversed micelle in isoctane,a nonionic surfactant polyoxyethylene lauryl ether(Brij30)was incorporated into the interfacial membrance.H₂O₂ played dual roles in the LiP catalyzed oxidation of substrates.To obtain a sustainable high activity of LiP,a coupled enzymatic reaction,i.e.,the glucose oxidase(GOD)catalyzed oxidation of glucose was used an H₂O₂ source.Due to modification of the charge density of the interfacial membrane,the activity of LiP in an optimized AOT/Brij30 reversed micellar medium(xB(the molar percentage of Brij30)=0.53,ω₀([H₂O]/ ([AOT]+[Brij30])=23,pH=4.8)was ca.40 times that in a single AOT reversed micelle.Due to the controlled release of H₂O₂,the concentration of H₂O₂ in the mixed reversed micellar medium could be always kept at a moderate high level,which made the LiP catalyzed oxidation of substrates go at higher conversion than the counterpart in which H₂O₂ was supplied externally in one batch at the beginning of the reaction. Decolourization of two water-less-soluble aromatic dyes(pyrogallol red and bromopyrogallol red)using LiP coupled with GOD in the medium also demonstrated that a higher decolourization percentage was obtained if H₂O₂ was supplied enzymatically,our proposed measures(both physicochemicai and biochemical)were very effective for significant improvement of the catalytic performance of LiP in a single AOT reversed micelle in isooctane,which helped to degrade or transform hydrophobic aromatic compounds with LiP in reversed micelles more efficiently.â…¡.Mechanism studies on the VA mediated LiP catalyzed oxidation of aromatic compound in aqueous medium and reversed micelles1.Studies on the hydrogen peroxide regulated VA mediated oxidation of pyrogallol red(PR)catalyzed by LiPThe oxidation reaction of PR by H₂O₂ in the presence of LiP was studied at different concentrations of H₂O₂.Experiments showed that the oxidation products depended on the molar ratio of H₂O₂ to PR,suggesting that the LiP catalyzed oxidation products of PR should be controllable.This phenomenon was caused by the dual roles of H₂O₂;i.e.,at lower concentrations it was an activator of LiP,while at higher concentrations it was an inhibitor.VA could stimulate the oxidation of PR catalyzed by LiP,especially at higher molar ratios of H₂O₂ to PR,however,PR inhibited the LiP catalyzed oxidation of VA.The inhibition should be used to explain a phenomenon that no veratryl alcohol activity was detected in the culture of white rot fungi where dye was effectively decolorized.Kinetics analysis suggested that VA should accelerate the conversion of LiP(â…¡)and/or LiP(â…¢)to LiP,and therefore the catalytic cycle of LiP.Indirect oxidation of PR by the veratryl alcohol cationic radical was also contributed to the increase in the oxidation rate of PR.2.Mechanistic studies on the effect of veratryl alcohol on the lignin peroxidase catalyzed oxidation of pyrogaliol red in reversed micelles.The LiP catalyzed oxidation of PR in the absence and presence of VA was carried out in AOT/Brij30 reversed micelles to elucidate the role of VA.Results indicated that VA could accelerate the LiP catalyzed oxidation of PR,especially at low H₂O₂ concentrations.Unlike in bulk aqueous medium,the protection of LiP by VA in the present medium was relatively not prominent,even at high H₂O₂ concentrations. Analysis of data from a series of well designed experiments showed that the enhancement of the PR oxidation caused by VA was mainly due to the indirect oxidation of PR by VA^+·from the LiP catalyzed oxidation of VA.It was also found that at low concentrations,VA(the psychological substrate of LiP)was less effective than PR(a phenolic compound)in protecting LiP from the H₂O₂ derived inactivation. This novel phenomenon deserves further study.3.Effect of medium on the life time of VA^+·The life time of VA^+·was a key factor in the elucidation of the role of VA in the degradation of lignin and environmental pollutant.Previous studies on the life time of VA^+·were all carried out in aqueous medium.However,natural degradation of lignin by the fungus was carried out in a colloidal medium which is very similar to reversed micelles but quite different from bulk aqueous medium.Therefore,the difference of medium may cause the inaccuracy of the determining of the life time of VA^+·.In this section we studied the effect of medium on the life time of several radicals.Results indicated that the decay of all the radical studied in reversed micelles were slower that that in the aqueous medium.For example,the first-order rate constant of the phenol radical in aqueous and reversed micelles were k_{aqueous medium}=0.023s^-1and k_{reversed micelles}=5.8×10^-3s^-1,respectively.The difference of the dielectric constant between the two medium may be the reason that caused the above results.â…¢.Tryptophan enhanced oxidation of aromatic compounds with lignin peroxidaseTryptophan(TRP)could accelerate the LiP catalyzed oxidation of some aromatic compounds with redox potential lower than that of TRP.To explore the role of TRP and the related mechanism,PR was chosen as a model compound and the LiP catalyzed oxidations of PR in the absence and presence of TRP were investigated in detail.Our results indicated that the acceleration of the LiP catalyzed oxidation of PR was owed to the protection of TRP against the H₂O₂-derived inactivation of LiP and the indirect oxidation of PR by TRP^+·of high redox potential.TRP was a good substrate of LiP.In the presence of both PR and TRP,TRP was preferentially oxidized by LiP to form a TRP cation radical(TRP^+·).TRP^+·could oxidize PR and make the LiP catalyzed oxidation of PR proceed at a larger rate,but the LiP catalyzed oxidation of TRP be inhibited.The present study also showed that TRP had a better reactivity towards LiP(â…¢)than VA,the physiological substrate of LiP.By reacting with LiP(â…¢), TRP could convert LiP(â…¢)to its native state and therefore protect LiP from the H₂O₂-derived inactivation.The present study helps to find a new effective way to improve the degradation efficiency of aromatic pollutants with LiP.