Comparative Proteomic Analysis Of Oil-effect On Lipase With Synthetic Activity Produced By Rhizopus Chinensis

Rhizopus chinesis CCTCC M201021 is an important filamentous fungi isolated from Daqu, a traditional leaven in the production of Chinese liquor. It can produce a membrane-bound lipase with high ester synthesis activity in organic phase. Research shows that olive oil can significantly improve the cell biomass and the level of the lipase fermentation in submerged culture of R. chinesis, but the regulatory mechanism is not clear. In this study, proteomics approach based on two-dimensional electrophoresis(2-DE) was used to analyze and compare the effects of different amounts of olive oil on total protein and membrane protein profiles of R. chinesis. The influence mechanisms of the lipase synthesis and cell biomass caused by olive oil were preliminary discussed, which is helpful to understanding the R. chinesis lipase production mechanism and fermentation regulation. The main results are as follows:(1) Typital samples of proteomic research was obtained by investigating the effect of different amounts of olive oil on R. chinesis liquid fermentation with the same concentration of carbon. On this basis, the two-dimensional electrophoresis system of total proteins and membrane proteins of R. chinesis were established and optimized. Using Ready Prep? Protein Extraction Kit and the detergent Triton X-100 to extract total proteins and membrane proteins from R. chinensis. p H 3–10 nonlinear IPG strip was selected. The analysis of the 2-DE maps by PDQuest 8.0.1 software showed that the protein points matching on 2-DE maps of total proteins and membrane proteins reached 87% and 94%, respectively. It can meet the requirements of proteomics.(2) PDQuest 8.0.1 software was used to analyze the 2-DE maps of typical samples. Compared with the 1018 protein spots on the 2-DE map of sample cultured with 0 g·L-1 olive oil, protein spots on the 2-DE maps of samples cultured with 1.3 g·L-1and 20 g·L-1 olive oil respectively reduces 10% and 17%. Compared with the 2-DE map of sample cultured with 0 g·L-1 olive oil, the differently express protein spots contain up and down protein spots and unique protein spots of the samples cultured with 1.3 g·L-1 and 20 g·L-1 olive oil respectively acount for 61% and 74% of the total proteins, and acount for 56% and 69% of the membrane proteins. It showns that the differential expression of proteins is significantly. The lipase protein abundance on the 2-DE maps of membrane protein increased significantly, and occurs of variety of post-translational modifications indicates that lipase may exist multiple functions. 139 differentially expressed total proteins and 32 differentially expressed membrane proteins were identified.(3) GO and KEGG pathway enrichment analysis were carried for differentially expressed proteins identified.With the addition of olive oil, the biological process such as generation of precursor metabolites and energy and small molecule metabolic process, the cellular component such as gtricarboxylic acid cycle enzyme complex, the molecular function such as unfolded protein binding, anion and cation binding, the KEGG pathway such as glycolysis/gluconeogenesis and amino acids metabolism changed significantly.With the change of olive oil amounts, the enrichment significant of thioester metabolic process, proteasome core complex and Peptidase activity changed significant. The results indicate that olive oil improve R. chinesis lipase ester synthesis activity may by multiple processes not the simply induction.(4) Differentially expressed proteins identified were classified with KOG. Adding of olive oil not only affect the expression of proteins related to carbohydrates, lipids, amino acid and energy metabolism which can affect cell growth, but also affect information storage and processing, protein folding and posttranslational modification, and protein transportion, secretion and location. Protein folding and posttranslational modification associated proteins are upregulated. Phosphoinositide signaling pathway is enhanced, GAPDH are significantly upregulated. These may promote the proper folding, transportation and location in membrane of the lipase.