Fermentation Process Regulation Of Laccase And Polysaccharides From Trametes Versicolor

Trametes versicolor(known as Yunzhi in China)is one of the most popular mushroom fungi with great application values.In submerged cultures,T.versicolor mainly produces laccase and polysaccharides.Laccase belonging to the blue multicopper oxidase is called green and environmental friendly catalyst.It has wide application in environmental protection,textile industry,biological detection and organic synthesis.The polysaccharides consist of extracellular polysaccharide(EPS)and intracellular protein-polysaccharide(IPS).EPS and IPS belonging to the?-D-glucans have various biological functions including antitumor,antioxidant,antiviral,immunomodulating,antidiabetic and hepatoprotective effects.They possess great application potential in food and medicine industries.Due to the increasing demand for laccase and polysaccharides,the enhanced production of laccase and polysaccharides has gained great interest.Therefore,vanillic acid as an aromatic phenolic compound was used to improve laccase production.The laccase in fermentation broth was directly captured by magnetic mesoporous silica nanoparticles for fabricating magnetic laccase catalyst.The catalytic oxidation of HMF was investigated by the magnetic laccase catalyst with TEMPO as the mediator.To further increase the laccase yield,farnesol as a fungal quorum sensing signal molecule was applied to regulate the T.versicolor morphology and stimulate the laccase biosynthesis.In addition,EPS production stimulated by farnesol was also studied.The physicochemical properties of EPS from farnesol-induced cultures were characterized,and its antioxidant and antitumor activities were evaluated.T.versicolor cell growth and IPS production were induced by the fungal quorum sensing signal molecule-tyrosol.The physicochemical properties of IPS from tyrosol-induced cultures were characterized,and its antitumor activity was also evaluated.An efficient strategy for laccase production in T.versicolor submerged cultures was developed using vanillic acid as the inducer.The optimized vanillic acid treatment strategy consisted of exposing 2-day-old mycelia cultures to 80.0 mg/L vanillic acid.After 6 days,laccase activity of 581.8 U/L and protein concentration of 96.7 mg/L were obtained,which represented 1.80-fold and 1.46-fold increase compared to control cultures,respectively.In 5-L aerated stirred bioreactor,the maximal laccase activity and protein concentration reached 740.2 U/L and 130.0 mg/L that were increased by 27.2%and 34.4%compared with those in culture flask,respectively.The magnetic laccase catalyst was prepared by directly capturing the laccase from fermentation broth using magnetic mesoporous silica nanoparticles.With TEMPO as the mediator,it has the remarkable capability of oxidizing HMF to FDCA.Under the optimal reaction conditions(pH 5.5,35?,10.0 mg magnetic laccase catalyst,24.0 mM TEMPO),90.2%FDCA was obtained after 96 h of reaction.Furthermore,the magnetic laccase catalyst exhibited good recyclability and stability,maintaining 84.8%of its original activity following 6 reuse cycles.In order to further improve the laccase productivity of T.versicolor,the effect of farnesol on mycelial morphology and laccase production was evaluated.Farnesol significantly promoted the laccase production by regulating mycelial morphology and physiological status of T.versicolor.Extracellular laccase activity and protein concentration reached a maximum of 2189.2 U/L and 145.0 mg/L in farnesol-induced cultures under optimal concentration(4.0 mM),which were 6.8-fold and 2.2-fold than those obtained in the control cultures,respectively.In 5-L bioreactor,the maximal laccase activity and protein concentration reached 3064.8 U/L and 196.5 mg/L that were increased by 40.0%and 35.5%compared with those in culture flask,respectively.SDS-PAGE and native-PAGE showed that farnesol mainly stimulated the biosynthesis of three laccase isoforms.Farnesol significantly enhanced laccase content in the secreted extracellular proteins,which was advantageous to the separation and purification of laccase in downstream processing engineering.Farnesol treatment resulted in a significant increase of oxidative stress level that significantly enhanced the expression of laccase genes for improving intracellular laccase biosynthesis.In addition,farnesol made T.versicolor develop into a hyperbranched morphology with short hyphae and bulbous tips by regulating several morphogenesis-related genes,which accelerated the secretion of intracellular laccase into culture medium.A novel strategy of exposing 2-day-old mycelia cultures to 0.8 mM farnesol was also developed to stimulate EPS production in T.versicolor submerged cultures.After 9 days,EPS yield reached a maximum of 2.56 g/L that was 2.7-fold greater than that of control cultures.In 5-L bioreactor,a peak value of 3.20 g/L was achived that was increased by 25.0%compared with that in culture flask.Farensol significantly increased EPS production by promoting polysaccharide biosynthesis and regulating mycelial morphology.EPS from farnesol-induced cultures(EPS-F)contained more uronic acid,glucan and high molecular weight polysaccharide fraction(134 kDa,85.0%).These physicochemical properties led to strong antioxidant and antitumor activities of EPS-F.T.versicolor cell growth and IPS production was stimulated by tyrosol that had the ability to promote DNA replication in dimorphic fungi.IPS yield was increased to 390.0 mg/L in tyrosol-induced cultures under optimal concentration(5.0 mM),which was 1.95-fold higher than that of control cultures.Moreover,the fermentation period was shortened by one-third.In 5-L bioreactor,IPS production achieved a maximum value of 450.0 mg/L that was increased by 15.4%compared with that in culture flask.Tyrosol significantly improved IPS production by promoting cell growth and stimulating IPS biosynthesis in T.versicolor submerged cultures.The IPS from tyrosol-induced cultures(IPS-T)contained higher contents of protein and glucan,which led to its strong antitumor activity.The anti-proliferative mechanism was identified as cell cycle arrest with cell accumulation in S phase and increase in apoptosis,which was the same as that of EPS-F.