Optimization Of Fermentation Process For Laccase Production From Trametes Versicolor

Laccase(benzenediol:oxygen oxidoreductase,EC 1.10.3.2),a polyphenol oxidase containing copper atoms in the catalytic centre,is one of important blue oxidases in the multicopper oxidases family.It has attracked attentions for its wide industrial application in food,paper,textile,environmental protection and biosensor because of its broad substrate spectrum.In order to meet the increasing demand for laccase,fermentation process optimization of laccase production from Trametes versicolor by corn steep liquor(CSL)and farnsol was studied.In addition,laccase production from T.versicolor was scaled up in aerated stirred tanks by developing the optimization strategy.This work provided a base for the laccase fermentation industrialization from T.versicolor.CSL significantly promoted the laccase production.Under the optimized CSL concentration of 20 g L-1,extracellular laccase activity reached 633.3 U L-1 on the day 5 of the fermentation,which was 96.3%higher than that of the control cultures.The contents of five phenolic compounds existed in CSL,vanillic acid,caffeic acid,p-coumaric acid,ferulic acid and sinapinic acid,were analyzed.Caffeic acid and p-coumaric acid had no effect on laccase production.Vanillic acid and ferulic acid were efficient inducers,but sinapinic acid caused a reduction in laccase production.Vanillic acid and ferulic acid synergistically induced laccase secretion.Fungal signaling molecule-farnesol markedly enhanced laccase production from T.versicolor.At the optimal farnesol concentration of 60 ?M added at the beginning of the culture,the extracellular laccase activity reached 629.3 U L-1 after 6 days of cultivation,which was 92.3%higher than that of the control.The farnesol led to the formation of hyperbranched mycelia.The concentration of intracellular H2O2 was increased on the day 1 after the addition of farnesol,which was 2.7-fold compared with the control.Enhanced H2O2 can induce the cellular defense mechanism,and laccase plays an important role in resisting the oxidation stress.The quantitative gene analysis suggested that the expression level of laccase gene(lac)induced on the day 3 by farnesol was increased by 2.3-fold than that of the control.The transcriptional level of rhoA gene,that related to the mycelial morphology was also increased by the addition of farnesol and was 4.4-fold of that in the control.Enhanced expression levels of rhoA gene led to the hyperbranched hyphal morphology,which was beneficial for the laccase secretion.The farnesol optimization strategy was applied in 5 L and 2000 L aerated stirred bioreactors to scale-up laccase production.In 5 L bioreactor,laccase production reached 704.5 U L-1 when induced by farnesol,which was 11.9%higher than the flasks with farnesol,and 105.6%higher than that of control cultures.In 2000 L bioreactor,laccase yield was 543.6 U L-1 that was 53%greater than that of the control.Crude laccase liquids were obtained by filtration and concentration of fermented broth after scale fermentation.The purified laccase was prepared by the salting out-chromatography method and the magnetic separation method with magnetic mesoporous nanoparticles,respectively.The results indicated that the magnetic separation exhibited its priority in industrial application.The laccase was used for degradation of various phenolic compounds.The optimal degradation pH was in the range of 4.0-5.0 for various phenolic compounds.The optimal temperature was as follows:35 ? for 4-methoxyphenol and 2,4-dichlorophenol,25 ? for phenol.The laccase was used for decolorization of various dyes.The optimal decolorization pH and temperature was 5.0 and 40 ?.The decolorization rates of dyes were affected by the structures and characteristics.The optimal reaction pH and temperature for purified laccase was 4.0 and 25 ?,and it still maintained high activity at 40 ?.10 mM Cu2+ could upgrade the laccase activity by 10%compared with that of the control.The laccase was used for degradation of various phenolic compounds.The optimal degradation pH was in the range of 4.0-5.0 for various phenolic compounds.The optimal temperature was as follows:35 ? for 4-methoxyphenol and 2,4-dichlorophenol,25 ? for phenol.The laccase was used for decolorization of various dyes.The optimal decolorization pH and temperature was 5.0 and 40 ?.The decolorization rates of dyes were affected by the structures and characteristics.