Heterologous Expression And Characterization Of Three Laccases Obtained From White Rot Fungi For Removal Of Environmental Pollutants

White Rot Fungi have enormous resources of laccase and laccase can act on a lot of substrates as well as environmental pollutants.In this study,the crude enzyme of Pleurotus ostreatus strain HAUCC 162?a white-rot strain with a strong ability for the degradation of xenobiotic compounds?were used to degrade different type of synthetic dyes.Three laccase isoenzymes obtained from Pleurotus ostreatus strain HAUCC 162were heterologously expressed and characterized with respect to their ability to degrade NPs,CPs,and SAs.All of these help us to futer know the characters and function of the recombination laccases.The main results are described as follows:The crude enzyme of Pleurotus ostreatus strain HAUCC 162 can degrade some different type of synthetic dyes?azo,triphenylmethane and anthraquinone dye?efficiently.In 24 hours,at 28?,MO?100 mg/L?,CV?100 mg/L?,MG?100 mg/L?,BB?100 mg/L?,RB4?800 mg/L?and RBBR?800 mg/L?can be degraded 81.3%,87.6%,85.1%,98.0%,64.6%and 89.1%,respectively.After adding the special inhibitor of laccase?kojic acid?,the degradation rate of synthetic dyes mentioned above were declined to 0.1%,12.9%,11.9%,18.9%,9.3%and 5.1%,respectively.From the results,laccase can be considered as the main element for the degradation of synthetic dyes.Three genes of laccase?lacc6,lacc9 and lacc10?from Pleurotus ostreatus strain HAUCC 162 were cloned and heterologously expressed in Points pichia.After fermental cultivation and purification,three recombination laccases?rLACC6,rLACC9 and rLACC10?were obtained.The apparent molecular weights determined by the mobility in SDS–PAGE were 64?rLACC6?,75?rLACC9?,and 64 kDa?rLACC10?.The specific activities of each isoenzyme were 45.50,9.36 and 8.56 U/mg,respectively.The optimum pH values of rLACC6,rLACC9 and rLACC10 are 2.5,3.5,and 4.0.The optimum temperatures of rLACC6,rLACC9 and rLACC10 are 60,40,and 40°C,respectively.The thermostability and pH stability were better under the low temperature and alkalescence.The recombinant laccases were found to be mostly inhibited by DTT and sodium azide.rLACC6 had better tolerance for metal ions and organic reagents than rLACC9 and rLACC10.All the purified recombinant laccases exhibited high better tolerance for Cu²⁺,K⁺and Na⁺.Methanol,glycol and glycerin are respectively most favorable to rLACC6,rLACC9 and rLACC10.Furter investigate the function of the recombinant laccases in degarding different type of environmental pollutants?NPs,CPs and SAs?under a laccase-mediator system?LMS?.Their best specific efficiencies for the removal of 2-NP,3-NP,4-NP,2-CP,3-CP,4-CP,2,4-DCP,2,6-DCP,sulfadiazine?SDZ?,sulfamethazine?SMZ?and sulfamethoxazole?SMX?over 60 min were determined to be 59.21%,47.91%,60.24%,41.35%,32.68%,36.72%,37.82%,35.05%,98.1%,97.5%and 97.8%,respectively.All the degradation process were conformed to the first-order kinetic equation.The oxidation products of the CPs,NPs and SAs were identified by liquid chromatography and mass spectrometry?LC-MS?and gas chromatography and mass spectrometry?GC-MS?.Based on the observations,pollutant removal pathways are proposed,namely,the production of3-nitromuconate and 3-chloromuconate as the key intermediates of 4-NP and 2,4-DCP.To sum up,our study obtained three recombination laccases?rLACC6,rLACC9 and rLACC10?through clone,heterologously expresse in Points pichia and purification,the recombination laccases can used in the degradation of NPs,CPs and SAs.Furter proposed the possible pollutant removal pathways of 4-NP,2,4-DCP and SDZ.Our results will facilitate the obtaining and utilization of laccase resources in the future.