| Biosurfactants are surface-active compounds synthesized by a number of microorganisms. Biosurfactants show several advantages over chemical surfactants for their biodegradablity, low toxicity, eco-compatibility and biotcompatibility.. Therefore, surface-active compounds produced by microorganisms are attracting more attention in recent years and will replace chemical surfactants in some industrial fields in the near future.Sophorolipid, as an important glycolipid surfactant, are attracting more and more attention. Sophorolipids consist of2-O-β-D-glucopyranosyl-D-glucopyranose units linked β-glycosidically to17-L-hydroxyoctadecanoic or17-L-hydroxy-9-octadecenoic acids and the sugar moieties could be partly acetylated. The wild type nonpathogenic yeast (Wickerhamiella domericqiae Y2A) could produce sophorolipids up to120g/L and possesses great comercial application. The thesis conducted research on the production of SLs with fatty acids of medium-chain length so that the type of sophorolipids could be enriched and the novel sophorolipids with novel characteristics will also enlarge the application of sophorolipids.The main results of the research are as follows:l.Separation, purification and identification of sophorolipids with medium-chain length.The fermention medium for sophorolipids production by W. domericqiae was supplemented with glucose and an alkane (C8～14), respectively, the foure selected alkanes were n-octane, n-decane, n-dodecane and n-tetradecane. The control medium was added only glucose and no alkane.. The HPLC-MS method was applied to analysis the structureof the sophorolipids and the GC-MS method was used to detect the fatty acid moieties. When the medium was supplemented with octane, both HPLC-MS and GC-MS methods would not detect the sophorolipids and fatty acids with medium-chain length. While decane was added, the chain length of the acidic sophorolipid was identified as14by HPLC-MS method. When dodecane was added, the acidic sophorolipids with12and14chain length were identified by both methods. While the substrate was tetradecane, the sophorolipid with C14chain length was detected. Otherwise, the chain length of the main sophorolipids is16or18. Overall, when dodecane and tetradecane were added, large amounts of sophorolipids with12and14chain length were produced.2.Transcription analysis of the genes referred to the fatty acid elongation and metabolism by Real-Time PCRThe transcription analysis of four genes about fatty acid metabolism and β-oxidation and one gene responsible for the fatty acid elongation in mitochondria were conducted when W. domericqiae was cultured on the medium with medium-chain length alkane (octane, decane, dodecane, tetradecane supplemented with glucose). Compared with octane/decane, the genes about fatty acid metabolism and P-oxidation were regulated at transcriptional level when the substrate was tetradecane/dodecane. It was concluded that the genes about fatty metabolism and β-oxidation were regulated when the chain length of alkane was longer.3.The deletion of the gene encoding fatty acid synthase a subunit and the identification of sophorolipids produced by the ΔF strainBased on the gene recombinant principle and the double-joint PCR method, the6157bp ura3gene knockout cassette was constructed and the uracil auxotrophic strain was obtained by the electrotransformation method. Then the fatty acid synthase α subunit gene knockout cassette with ura3gene was transfered to the uracil auxotrophic strain and then the knockout strain (ΔF) was obtained. The sophorlipids produced by the ΔF strain were identified by HPLC-MS and GC-MS methods when the dodecane was added to the medium. It can be concluded that the main sophorolipids synthesized by ΔF strain was acidic and the lipophilic group was oleic acid. |
PDF Full Text Request