Research On Anti-Nutritional-Repression Enzyme Production And Lignin-degradation Mechanism Of Phanerochete Chrysosporium

White rot fungi are the aggregation of filamentous fungi which have the same function:causing white rot of woodiness.Relying on its capacity of degrading lignocellulose selectively, Its hypha can penetrate woodiness,invade the cell cavity of woodiness and release enzymes which can degrade lignin and other components of woodiness,leading to woodiness rot into light spongiose gobbet-white rot. Phanerochaete Chrysosporium is the type strain for the white rot fungi research and it is the representative strain of the whole fungi biotechnology system as well.White rot fungi can produce special enzyme system under nutrient-limitation conditions such as N-limitation,C-limtitation and S-limitation.The enzymes include LiP,MnP and Lac and they can degrade lignin effectively. White rot fungi are the only microbes that can degrade lignin independently and completely.But the environment mostly belongs to eutrophic environment which restricts the application of Phanerochaete Chrysosporium.At the same time, in the incubation,its metabolic activities such as the production of lignin degradative enzymes and the enzymatic activity are largely influenced by cultivation factors.Furthermore,the formation of degradation enzymes system of white rot fungi needs a kind of cosubstrate and it can't take lignin or other degradation substrate as the only carbon source.If Phanerochaete Chrysosporium can take lignin or other degradation substrate as the only carbon source, undoubtedly,it will have a broad application prospect.Meanwhile,it is hard to improve the enzyme output substantially because the cell growth isn't synchronized with the enzymatic synthesis.Based on a large amount of experiment,by ultraviolet radiation predecessors have selected and bred the mutant strains PC5324,PC5326 and PC5305 which can grow quickly and synthesize lignin degradation enzymes system efficiently as well under N-S condition. This project aims to carry out systematic research on these mutant strains, including lignin isozyme types and lignin isozyme production mechanism of different strains and ligin biodegradation mechanism of mutant strains.The results are as follows:(1) Through analysis of Peroxidase Isoenzyme produced by the original strain and the mutant strains respectively under N-L and N-S conditions,we find that isoenzyme bands are 1-5. The activity of enzyme bands can assessed according to the shades and width of the colors of the enzyme bands in a certain degree. Peroxidase Isoenzyme of different strains show differences in a certain degree in the respects of quality(quantity of enzymes bands and relative mobility) and quantity(enzyme activity),but the arrangement of part of enzyme bands have certain regularity. In general,the enzyme bands of the mutant strain PC5305 represents the darkest color and the widest bands and the enzyme bands of the mutant strain PC5324 shows dark bands while the bands of the mutant strain PC5326 shows pale dark bands.The enzyme bands of the original strain PC553 shows the lightest color and the narrowest band width.(2)Through the research on the relationship between the cell growth and enzyme production of the original strain and the mutant strains,it is found that the original strain PC553 belongs to nutritional-repression strains,namely,the growth isn't synchronized with the enzymatic synthesis;but the mutant strains break this rule and the amount of growth and the enzyme output can reach the peak at the same time.Furthermore,the amount of growth and enzyme activity of the mutant strains are much higher than those of the original strain.Through the research on N consumption curve and C consumption curve of the original strain and the mutant strains under N-L and N-S conditions,we find N and C concentrations critical points of these different strains in the secondary growth stage. Under N-L conditions, N concentrations critical points of PC553?PC5324?PC5326?PC5305 are 0.10g/L,0.11 g/L,0.14 g/L and 0.16 g/L.C concentrations critical points are 12.0 g/L,13.0 g/L,17.0g/L and 18.0 g/L.Under N-S conditions,N concentrations critical points are 1.2 g/L,1.3 g/L,1.6 g/L and 1.9 g/L.C concentrations critical points are 14.0 g/L,15.2 g/L,14.6 g/L and 16.9 g/L.We also research the nutrition mechanisms of the original strain and the mutant strains:the effect of different nutrient conditions on enzyme production,the change regularity and the reason.Under N-L condition, the optimal concentration for PC553:Zn2+ 0.8g/L,Mn2+ 0.2g/L, oxalic acid 0.3g/L, phenylalanine 0.2g/L;Under N-S condition,the optimal concentration:Zn2+ 0.6g/L, Mn2+ 0.4g/L, oxalic acid 0.3g/L, phenylalanine 0.4g/L.(3)Through the research on lignin degradation mechanism of the original strain and the mutant strains,it is found that the original strain can't use lignin as the only carbon source while the mutant strain PC5305 breaks this rule and can take lignin as the only carbon source.Without the culture medium,PC5305 can degrade 25% of lignin.Through the research on lignin degradation by PC5305 under different conditions,we try to look for the change regularity and the reason and find that different factors have different effects on the degradation rate of calcium lignosulphonate by PC5305.They represent the following sequence:temperature> the concentration of benzil alcohol>pH> the concentration of tween 80.The optimal culture condition in the orthogonal experiment of calcium lignosulphonate degradation rate by PC5305 is as follows:temperature 37?,pH 4.5, benzil alcohol 5.2mmol/L, tween 80 l.Og/L.