The Protein Secretomics Analysis Of Filamentous Fungi Rhizopus Chinesis

For fermentation industry, filamentous fungi is the important strain to produce some kinds of enzymes. But there has a compact relationship among the culture patterns, filamentous fungi's morphology and the formation of target products. Rhizopus chinesis CCTCC M201021 is an important filamentous fungi isolated by our laboratory from the traditional leaven to produce Chinese liquor- Daqu, which can produce some enzymes, including lipases and so on, that have high industrial application value. But the production and activity of these enzymes could be influenced by the mycelia morphology and the culture patterns greatly.Therefore, in this study, haploid strain already isolated was chosen as the experimental strains, proteomics technologies such as two-dimensional gels electrophoresis(2-DE) and others were chosen to analyze the differential proteins formed under different morphologies and different culture patterns, respectively. And then reasons leading to the differential morphologies of R. chinesis, the mechanism of how mycelia morphology affects lipase and the reason resulting in the formation of differential secreted proteins under different culture patterns were discussed.The main research results are as follows:(1) Differential secretomics research and analysis were conducted to the extracellular proteins of R. chinensis under solid-state and submerged cunture:The analysis of the secretomics data indicates that, there do exist significant difference of the extracellular proteins of R. chinensis no matter using wheat bran or the extracting solution as substrate or using synthetic medium to conduct the R. chinensis cultivation under solid-state and submerged fermentation. About 70% of these differential proteins are the exclusively secreted under each condition, and of most of these differential proteins' expression quality have a great difference from each other. After identification by Mass Spectrum(MS), it could be discovered that these two group's extracellular proteins' species and expression quality can be influenced by the culture patterns greatly. Most of them are hydrolases and more than 60% of these hydrolases participate the protein degradation.The different substrates' impact to extracellular proteins of R. chinensis under the same culture patterns was underwent, and the result indicated that not only the culture patterns but also the composition of substrates could influence the expression of extracellular proteins of filamentous fungi greatly. Some proteins can only be formed under the circumstance of the existence of certain substrate and culture patterns. And some proteins may experience posttranslational modification after secretion. All these can influence the fermentation performance of R. chinensis greatly. The results prompt that the inconsistency of the filtration and production of extracellular enzymes should be noticed during the culture of filamentous fungi under solid-state and submerged cultivation. A single filtration method and the simple fermentation patterns' substitution during industrial production may not be appropriate.(2) Differential secretomics research and analysis were conducted to the extracellular and membrane-bound proteins of R. chinensis of different morphologies under submerged fermentation:According to the 2-DE maps, there do exist significant differences in the extracellular and membrane-bound proteins of different morphologies. Among those 28 extracellular proteins and 70 membrane proteins identified successfully, more than 85% of them was exclusively secreted under each condition. And the protein identification result indicates that about 42.9% of them are hydrolases and about 50% of these hydrolases take part in the protein degradation. The lipase secreted by R. chinensis was mainly located on the cell membrane, and the different morphology may influence the lipase' expression quality, not the secretion, which is not the same as the lipase secreted by Rhizopus oryzae. While for membrane-bound proteins, they were classified according to its Eukaryotic Orthologous Groups of Proteins(KOG) function. And the differential proteins are mainly exist in two categories, which are energy production and conversion(18.8%) and carbohydrate transport and metabolism(15.6%). These results show that the difference of strain morphology maybe the reason to result cell differentiation. And for protein transportation and secretion, there may exist some complicated physiological and metabolic mechanism.