Study On Module Deletion Of Surfactin Synthase And The Function Of Novel Surfactin Derivatives

Surfactin is a secondary metabolite synthesized by B. subtilis. It is composed of 13-15 carbon atoms with a series of seven amino acid sequence. It also has antimicrobial,antitumor, antiviral, and hemolytic properties.In this paper, we studied the method and producing novel Surfactin derivatives, and explored these novel Surfactin derivatives, and evaluated the physicochemical properties,yield and antibacterial activity of B. subtilis.The main results are as follows:1. Construction of novel Surfactin derivatives of B. subtilisSurfactin was used to amplify and connect the gene cluster in the database, and the sequence was amplified and connected to the D-Leu-pKS2, and the SurfactinA subunit was D-Leu, and the B. subtilis LS1 was obtained. Then the B. subtilis LS6 and B. subtilis LS9 were obtained respectively.2. Study on the properties and isolation of novel Surfactin derivatives from B. subtilisThe fermentation of a novel Surfactin derivative by B. subtilis was inocuLated with 5%concentrations of B. subtilis seed solution in the Landy fermentation medium, and the antibacterial substance was obtained. After that, Novel Surfactin derivatives were obtained by RP-HPLC separation and purification. Novel Surfactin derivatives finally by LC-MS and LC-MS / MS on the structure of analysis: the molecular weight of [△Leu3] Surfactin to 909, 923,937 m/z. The molecular weight of [△Asp5]Surfactin was 907, 921, 935 m/z. The molecular weight of [△Leu6]Surfactin was 909, 923, 937 m/z. Study on the properties of novel derivatives of Surfactin we found. [△Leu3]Surfactin,[△Asps]Surfactin,[△Leu6] Surfactin to temperature insensitive has good stability in acid and alkali, UV irradiation, the role of protease on the had little effect. [△Leu3]Surfactin and[△Leu6]Surfactin to the Fusarium moniliforme have inhibitory effect.3. B. subtilis LS-9 secretion of antimicrobial substances [ALeu6]Surfactin culture medium optimizationFirst by single factor experiment of sucrose, monosodium glutamate, potassium nitrate,magnesium suLfate, inocuLation quantity, initial pH, temperature and fermentation time the eight factors were selected, after based on selected (a),sucrose, (b) sodium glutamate and (c)potassium nitrateas the independent variable, in the [△Leu6]Surfactin yield (y) as the response value of box Behnken design, response surface analysis. Finally get to get the[△Leu6]Surfactin synthesis of optimum process parameters: in sucrose 22.43g/L, KNO36.06g/L, sodium glutamate 4.13g/L, fermentation time for 72h, 5% inocuLation amount,initial pH and temperature is 33 degrees, value is 2.33mg/L, the best prediction[△Leu6]Surfactin yield.4. The antibacterial mechanism of Fusarium moniliforme and Fusarium graminearum secreted by B. subtilis LS-9 on the antibacterial substance [△Leu6]SurfactinThe antibacterial substances [△Leu6]Surfactin on inhibition of Fusarium moniliforme,the inhibition rate reached 95.88%. And using electron microscopy of F.moniliforme and F.graminearum mycelia were observed, antibacterial substance on [△Leu6]Surfactin makes F.moniliforme and F.graminearum mycelia cell membrane formed on the surface of many of the sag, internal organelles form cannot be distinguished, the cell interior color becomes shallow, hyphae the decline of the state.5. The preliminary study on the [△Leu6]Surfactin effects on the Fusarium graminearum in protein expression by iTRAQ TechnologyThe results of the total protein iTRAQ detection for Fusarium graminearum, the number of up-regulated proteins was 103, down regulated proteins were 225. The number of total proteins was 328; accounting for the identified total protein ratio was 10.9%. Analysis of up-regulated protein function clustering, functional protein on membrane have been up-regulated, hydrolases and lyases have increased,cell growth and metabolism of protein were significantly reduced.6. The effects of B. subtilis LS-9 secretion of antimicrobial substance on[△Leu6]Surfactin on Fusarium moniliforme produced fumonisin vitamin B1 (FBI)and Fusarium graminearum produced Deoxynivalenol (DON) application in stored maize[ALeu6]Surfactin on Fusarium moniliforme growth and fumonisin vitamin B1 synthesis effect. The results show that [ALeu6]Surfactin concentration higher inhibitory effect is more obvious. The results also had similar effects on the growth of Fusarium graminearum and production of deoxynivalenol.