| Lignin, a group of aromatic polymer in nature, is built up by propane unit using stable chemical bond. Microorganisms in nature that can degrade lignin are mainly bacteria, actinomycetes and fungi, and fungi play a major role. The major enzymes involved in lignin degradation of fungi are lignin peroxidase, manganese peroxidase and laccase, which are extracellular oxidase secreted by fungi. These oxidases could degrade a variety of pollutants, including phenols, polycyclic aromatic hydrocarbons, various dyes, the PCBs and many other refractory materials. With the nature resource crisis and environmental pollution becoming serious, isolating microorganisms from natural which can biodegrade pollutants and deepening understanding lignin-degrading enzyme are the major issues of contemporary economic's and social's development, it is also the demand of science and technology.This research was done in order to isolate some strains from the environment with lignin degradation ability. First, indentified if the isolated strains can produce lignin-degrading enzyme or not. And then, for the promising strains different lignin-degrading enzyme activity in liquid medium was assayed.1. Screening lignin degrading fungi.14 pure strains were obtained from tissue culture isolation. Flat color reaction, a way for rapid screening lignin-degrading fungi, was used as the first screening method. Consided guaiacol oxidation and Azure B bleaching, the promsing strains are Ga2, S2 and W1, and they are used for research later.The strains, showed a good performance on first screening, and their lignin-degrading productivity were assayed in liquid culture as the second screening.2. Identification strain W1 and building the phylogenetic tree. Based on the preliminary results of flat color reaction, strain W1 showed a good performance, so we do more research on this strain. First, its total DNA was extracted. Then, use universal fungal rRNA primers, ITS sequences, cloned 5.8sRNA and both sides of the transcribed spacer (rDNA-ITS) of strain W1 by PCR.We found that W1 has a high sequence similarity with many strains in GenBank. To determine the evolutionary relationship among them, we did aligmant among W1 and other 8 strains in Genbank, then phylogenetic tree was built using software MEGA4.0. From the phylogenetic tree, we know that strain W1 has the closest relations to I.lacteus XSD-2.3. Optimization of lignin peroxidase and manganese peroxidase by I.lacteus W1. To optimize the culture condition for strain I.lacteus W1 to produce manganese peroxidase in liquid culture, one-factor-at-a-time, orthogonal design and response surface methodology of enzyme production was done respectively. Single factor test showed that the best carbon and nitrogen sources were glucose, yeast extract respectively, and the best culture conditions were 28℃, pH5.5,120rpm/min. Orthogonal test indicates three main factors influenceing MnP production are the inducer Tween 80, yeast powder and glucose. Optimal medium constitution are glucose 15g/L, yeast 0.2 g/L, KH2PO43.0g/L, MgSO4·7H2O 0.7g/L, Tween 80 lg/L. After optimization, MnP production was 67%higher than the orginal test. Plackeet-Burman design showed three main effects factors affecting MnP production are Tween 80, Yeast and dextose. Used Box-Beheken design, we optimized the three main effects. The Box-Beheken design predicted the best conditions for producing MnP was dextose, Yeast and Tween 80 were 17.67g/L,0.18g/L and 1.40g/L rsepectively. MnP production is 1.26 times to the original medium after optimization. Optimization of LiP production was done by single factor test. Different concentrations of glucose, ammonium tartrate, induce agents of Tween 80 and temperature,rote speed, initial medium pH were done on LiP yield, results showed that the best culture conditon were glucose 20g/L, ammonium tartrate 0.1 g/L, Tween 800.5g/L, initial pH 5.0, incubated temperature 30℃and static condition. Under the optimum culture conditions, LiP production was 31%higher than the initial test.
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