Study On The Mechanism Of Synergistic Action Of Poly-enzyme On The Lignin Oxidation Degradation In The Penicillium Sp’s Fermentation Substrate

The straw is the complex structure composed of cellulose,hemi-cellulose and lignin.The key step to the development and utilization of straw is to high efficiently and environmental friendly degrade lignin.In various methods for degrading lignin,the biological methods,especially enzymatic methods,have attracted extensive attention due to their mild conditions,short time and little coverage site.Many enzymes involve in the degradation of straw.The synergy action mechanism of various enzymes in straw lignin degradation process is helpful to improve the lignin degradation efficiency.In this study,the fermentation media components and fermentation conditions using the Penicillium sp.JSU-003 strain with high lignin peroxide（LiP）active as the tested strain were optimized by mono-factor-at-a-time and orthogonal design in the present dissertation.The regression analysis was performed between cellulase active and the yield of the total reducing sugar(Y_trs)after saccharification of the pre-treatment substrates and gave the affinity for enzymatic to the substrate and the maximum reaction velocity.The synergistic action of cellulase in the process of H₂O₂ oxidation degradation of lignin catalyzed by LiP was studied.The dissertation’s study will lay the foundation of the biodegradable straw,screen and isolation of high-yield LiP producing strain.The results from the study are listed as follows:（1）The JSU-003 isolated was identified as Penicillium by morphology and ITS.Mono-factor-at-a-time experiment showed that the optimum carbon and nitrogen sources for LiP synthesis by Penicillium were maltose,glycerol,corn protein powder,potassium nitrate,and fish meal protein.The optimum medium compositions were composed of maltose 1%,glycerol 1.5%,fish meal peptone 1.5%,potassium nitrate0.5%,corn protein powder 1.5%,and optimum fermentation conditions included material concentration 50%,culture temperature 27℃,straw pretreated with 6%sodium hydroxide for 28 h and then at 113℃for 24 minutes.Under these conditions,the highest LiP enzyme activity was 18.04 U/g on the fourth day of fermentation.（2）A comprehensive hydrolysis index（CHI）including single action and interaction of three components of cellulase:carboxymethyl cellulose sodium enzyme,filter paper enzyme andβ-glucosidase,was constructed by the principal component analysis（PCA）.The correlation between Y_trs of saccharification after oxidation degradation substrate and CHI was regressively analyzed using SPSS software.The results indicated that Y_trsrs was negatively correlated with CHI,that is,the saccharification of cellulose could inhibit the oxidative degradation of lignin in the process of oxidative degradation of straw lignin.（3）LiP was purified by DEAE-Sepharose Fast Flow anion exchange chromatography and SephadexS-100 gel filtration chromatography.After the first purification,the LiP was purified 17.9 fold with a 64%enzyme activity recovery and a 3.6%protein recovery.The LiP was further purified at 21.5 fold and a 17%enzyme activity recovery and a 0.7 protein recovery.SDS-PAGE electrophoresis showed a single band.The results of protein mass spectrometry and X ray diffraction showed that the molecular weight of the LiP was 45.4 kDa,and the peptide sequence was lsahsvaavndvdptvqglp.（4）After comparing the kinetic parameters of purified LiP and LiP in fermentation broth,μ_max OF the purified LiP（1.15 mg/mL/h）was higher than theμ_max（0.86mg/mL/h）of LiP in the fermentation broth,and the K_m（3.02 mg/mL）was less than the K_m（4.74 mg/mL）of LiP in the fermentation broth.The results demonstrated that purification increased the affinity of the LiP to lignin of the substrate and the maximum reaction rate compared with LiP in the fermentation broth.This conclusion further proved that glucose from saccharification cellulose inhibited the H₂O₂ oxidation degradation of lignin catalyzed by LiP.