Using Proteomics Technology To Find The Related Proteins In The Process Of Cellulose Degradation By Arthrobotrys Sp.CX1

With the increasing of global energy demand,cellulose,as a renewable biomass energy after the four major energy sources,has been receiving great attention at home and abroad for its biotransformation issues.Cellulose has a dense and complex crystalline structure,and a single cellulase cannot degrade cellulose effectively.Therefore,the study of complex cellulose-degrading enzymes composed of multiple enzymes becomes particularly important.In the early stage of this study,a fungus strain of Arthrobotrys sp.CX1 with high cellulose degradation ability was screened out.This strain can express a large amount of cellulose degradation-related proteins under the conditions induced by cellulose as a carbon source.In this study,gel electrophoresis technology was first used to analyze the differences in extracellular secreted proteins of CX1 strains induced by different substrates,and to optimize the screening of strong binding cellulose proteins of Arthrobotrys sp.CX1.Then,the experimental samples with obvious differences were analyzed and identified by combining the liquid chromatography-mass spectrometry identification technology and bioinformatics analysis technology.Finally,some representative cellulose degradation-related proteins were screened from the obtained mass spectrometry results,and the quantitative expression changes of these proteins during the cellulose degradation process were analyzed by fluorescence quantitative PCR technology.The experimental results show that:(1)Under the conditions of filter paper induction medium and glucose medium,the extracellular proteins expressed by the CX1 strain are significantly different.With the extension of the cultivation time,the protein in the glucose medium only increased but there was no difference in protein expression,and the protein bands were relatively single.However,with the extension of culture time,the types of CX1 exocrine proteins in the filter paper induction medium changed significantly.At the fourth day of culture,a large number of differential protein bands were found in the filter paper induction medium,the molecular weight of these different protein bands was between 30 KDa and 90KDa and they were not single protein bands.In the subsequent research on the substrate in the filter paper induction medium,it was found that a large number of proteins would adsorb on the substrate and most of the adsorbed proteins had a strong binding capacity.(2)The separation and screening of the strongly binding protein on the substrate of the filter paper induction medium was carried out by the method of fractional elution.It was found that,after the eluent acts on the filter paper,the filter paper is more effective than the method of water washing combined with ultrasound.The method of 8M urea combined with ultrasound can be more effectively isolate strong binding proteins.As a surfactant,Triton X-100 can elute strong binding proteins better,but because it cannot enter the mass spectrum and is difficult to be removed,it is not suitable for the eluent of this experiment.In addition,in the optimization of the 8M urea combined with ultrasonic grading elution method,it was found that further grading and elution of the substrate after the 8M urea eluate was applied to the filter paper can more effectively separate and screen strong binding proteins.(3)The optimized fractional elution method was used to perform the mass spectrometry analysis on the experimental samples of the fractionated screening of strong binding proteins on the substrate of the filter paper medium.The method also proved that the method can effectively separate and screen the cellulose proteins with strong binding force.In the separation process,it can effectively reduce the significant impact of high-abundance proteins,so as to find more related proteins that play an important role in cellulose degradation.(4)Glucose medium was used as control group.The real-time PCR technology was used to detect the relative expression levels of eight protein genes including endoglucanase,exoglucanase,?-glucosidase,coenzyme LPMO and cellulose degradation pathways related metabolic enzymes such as glyceric acid phosphate kinase,turn aldol enzyme,CoA acetyltransferase and enolase in filter paper induction medium and microcrystalline cellulose induction medium.The research found that exoglucanase and LPMO were significantly upregulated in the two media,and the relative expression levels were higher in the microcrystalline cellulose induction medium,while endoglucanase and ?-glucosidase were significantly upregulated only in the microcrystalline cellulose induction medium.The results show that compared with microcrystalline cellulose which only contains crystalline cellulose,the filter paper with both crystalline cellulose and non-crystalline cellulose is more difficult to degrade.Therefore,the expressions of three major cellulose degrading enzymes,such as endoglucanase,exoglucanase and ?-glucosidase,were greatly induced and increased,and the relative expression of lytic polysaccharide monooxygenase,as an auxiliary enzyme for cellulose degradation,was also much higher than that of the filter paper induced medium.Except for the up-regulation of transferase in the filter paper induction medium,the other three enzymes were downregulated significantly,and the relative expression of several metabolic enzymes in the microcrystalline cellulose induction medium was lower than that in the filter paper induction medium.It indicated that glucose was more easily used by bacteria than cellulose as a carbon source,so the expression of genes related to glucose metabolism in the cellulose induction medium was lower than that in the glucose medium.The crystalline structure of microcrystalline cellulose is denser than that of filter paper,and it is also more difficult to achieve degradation.The utilization rate of monosaccharides in the culture medium of microcrystalline cellulose is lower than that of filter paper induced culture medium,so the relative expression level of several genes related to glucose metabolism is lower.This study is helpful to deeper understand the types,functions and mechanism of related proteins in the process of cellulose degradation,and to further improve the understanding of the components of Arthrobotrys sp.CX1 enzyme system.It provides a favorable basis for the follow-up research on the optimization of cellulose degrading enzyme system and the efficient biological transformation of cellulose.