Construction Of Engineering Strains Containing The Genes Of Ligninolytic Enzyme System And Degradation Of Lignin Of Plant

The resources of plant are inexhaustible since it’s renewable through photosynthesis. Its cellulose and hemicellulose are the excellent ingredients for making of ethanol and producing of biogas. But the former is inlayed by lignin which is so solid that it’s hard to be degraded to become the bottleneck problem of the industrial development on cellulosic ethanol and cellulose biogas. This paper studies on constructing and applying of the engineering strains containing of the genes of lignin peroxidase\manganese peroxidase and laccase to degrade of lignin of plant.The genes of lignin peroxidase and manganese peroxidase were amplified by reverse transcription PCR from white-rot fungi. Using DNA operations, the expression vectors of containing genes of lignin peroxidase, manganese peroxidase and laccase were constructed respectively. Then, the expressive vectors were transformed into P. pastoris, Bacillus subtilis and Saccharomyces cerevisiae with the technique of electroporation to obtain three kinds of engineering Pichia pastoris which are containing the genes of lignin peroxidase, of manganese peroxidase and of laccase, three of engineering Bacillus subtilis containing the genes of lignin peroxidase, of manganese peroxidase and of laccase and three of engineering Saccharomyces cerevisiae containing the genes of lignin peroxidase, of manganese peroxidase and of laccase.All of the engineering strains were fermented in shaking flask and the fermentation broths were used to determinate of enzyme activity. The fermentation broths from those strains of containing the gene of lignin peroxidase are all shown lignin peroxidase activity, which are 97U/ml from the engineering Pichia pastoris,57.4U/ml the engineering Bacillus subtilis and 43.4 the engineering Saccharomyces cerevisiae. The fermentation broths from those strains of containing the gene of manganese peroxidase are all shown manganese peroxidase activity, which are 0.933U/ml from the engineering Pichia pastoris,1.870U/ml the engineering Bacillus subtilis and 0.580U/ml the engineering Saccharomyces cerevisiae. The fermentation broths from those strains of containing the gene of laccase are all shown laccase activity, which are 68.7U/ml from the engineering Pichia pastoris,119.3U/ml the engineering Bacillus subtilis and 80U/ml the engineering Saccharomyces cerevisiae.A four days pretreatment of rice straws were progressed by eight situations which are three kinds of the engineering Pichia pastoris, three kinds of the engineering Bacillus subtilis, three kinds of the engineering Saccharomyces cerevisiae, White rot fungi containing lignin enzyme system, ammonia water, Bacillus subtilis, Pichia pastoris and Saccharomyces cerevisiae, The results under the scanning electron microscope indicated that degradation of lignin of the engineering Bacillus subtilis and engineering Pichia pastoris is obvious stronger than other including the White rot fungi and ammonia water. While fermentation biogas with those pretreated rice straw for 7 days, the best output of biogas is from the treated of by the engineering Bacillus subtilises and next is by the engineering Pichia pastorises. The results from statistical analysis software indicated that the output of biogas from the pretreatment by the engineering Bacillus subtilises is not only extremely significantly (P<0.001) more than those of Saccharomyces cerevisiae, Pichia pastoris and Bacillus subtilis, but also that of ammonia water White rot fungi, engineering Saccharomyces cerevisiae and engineering Pichia pastoris. And the the output from the engineering Pichia pastoris is extremely significantly (P<0.001) more than that of ammonia water, White rot fungi, Saccharomyces cerevisiae, engineering Saccharomyces cerevisiae and Pichia pastoris.Our work had successful constructed of lignin degrading enzyme genes containing engineering Bacillus subtilises, engineering Pichia pastorises and engineering Saccharomyces cerevisiaes, implemented that the lignin degrading enzyme genes expression in those engineering strains, and attested the mixing engjneering Bacillus subtilises and the mixing engineering Pichia pastorises, which including the ligninolytic enzyme system, behave the function on high efficient degradation of lignin of plant. This study benefits on provide a new way on pretreatment plant for producing of biogas and ethanol. It would promote the development of the industrials of utilizing plant to produce of biogas and ethanol.