脂胜肽生物界面活性劑-豐原素之醱酵生產及其相關特性分析

Production and characterization of a lipopeptide biosurfactant – fengycin from Bacillus subtilis F29-3

王莉娟(Li-Chuan Wang)

指導教授 ： 魏毓宏

元智大學/工程學院/生物科技暨生物資訊研究所/碩士(2007年)

摘要

Bacillus subtilis F29-3經研究證實具合成抗真菌抗生素及生物界面活性劑—豐原素(fengycin)之能力。本研究主要是著重在開發Bacillus subtilis F29-3醱酵代謝合成豐原素之培養基組成、醱酵製程、分離純化及其結構鑑定分析。本研究證實：Bacillus subtilis F29-3醱酵產物，在結合酒精(ethanol)破微胞、超過濾(ultrafiltration)及串聯奈過濾(nanofiltration)等分離純化製程，可得純化之豐原素。純化之豐原素，經以FAB-MS、UV-Vis、FT-IR、13C-NMR及MALDI-TOF之定性分析，證實Bacillus subtilis F29-3醱酵合成之產物為豐原素。其次，利用HPLC系統配合MALDI-TOF/MASS證實本研究所開發的HPLC系統可有效分離及分析豐原素之多種異構物。
另外，在豐原素之醱酵製程方面，本研究也探討不同的因素，如溫度、轉速、酸鹼值、接菌量及不同碳氮源對於Bacillus subtilis F29-3代謝豐原素之影響，並以反應曲面實驗設計法(Response Surface Methodology, RSM)來探討碳源、氮源及微量元素之間的交互關係和最適化濃度。研究結果證實：培養Bacillus subtilis F29-3代謝豐原素之最適化培養基組成為：mannitol：26.2 g/L、soybean meal : 21.9 g/L、NaNO3 : 3.1 g/L、FeCl2．4H2O : 0.55 g/L、MgSO4．7H2O : 3.0 g/L、MnSO4．H2O : 0.15 g/L、Na2MoO4．2H2O : 0.055 g/L。Bacillus subtilis F29-3在上述最適化培養基培養下，可得豐原素最大產量約為3.5 g/L，是一般文獻值之2.4倍。
最後，本研究也經由搖瓶的重複批次饋料實驗證實：將置換出來
之醱酵液，加入2.6 % mannitol及2.2 % soybean meal並重新接入1 % 的菌液後，其豐原素產量可由原醱酵液之3 g/L提升至5.2 g/L，約為文獻值之3.6倍。而由醱酵槽實驗證實：於四個時間點各添加0.65 % mannitol及0.55 % soybean meal，可將豐原素之產量由批次醱酵之3 g/L提升至4.7 g/L，約為文獻值之3.2倍。由此可推論，適時適量添加營養源，有助於菌體生長及豐原素之代謝合成。

關鍵字

Bacillus subtilis F29-3 ； 生物界面活性劑 ； 豐原素 ； 抗生素 ； 反應曲面法

並列摘要

Fengycin is an antifungal lipopeptide produced by Bacillus subtilis F29-3. It inhibits filamentous fungi but is ineffective against yeast and bacteria. Fengycin was isolated and purified from the broth and was characterized by FAB-MS, FT-IR, UV-Vis, 13C-NMR and MALDI-TOF /MASS. The results of characteristic analysis show that the molecular weight and molecular structure of the fermentative metabolite resembled those of fengycin . Fengycin consists of two main components differing by one amino acid exchange. Fengycin A is composed of 1 D-Ala, 1 L-Ile, 1 L-Pro, 1 D-allo-Thr, 3 L-Glx, 1 D-Tyr, 1 L-Tyr, 1 D-Orn, whereas in fengyicn B the D-Ala is replaced by D-Val.
Fengycin production from the B. subtilis F29-3 was affected by temperature and agitation rate, as 30 ℃ and 200 rpm agitation were favorable for fengycin production. Furthermore, statistical experimental design (Response surface methodology, RSM)) was applied for the purpose of identifying optimal formulation in the medium. For five-level four-factor central composite design, the production was significantly affected by Soybean meal, NaNO3, MnSO4, mannitol-mannitol, soybean meal-mannitol, soybean meal-soybean meal, NaNO3-NaNO3 and MnSO4-MnSO4. The model had a coefficient of determination (R2) of 0.9043, which can explain 90.43 % variability of the data. The results of steepest ascent and central composite design revealed that 26.2 g/L mannitol, 21.9 g/L soybean meal, 3.1 g/L NaNO3 and 0.2 g/L MnSO4 were optimum for the production of fengycin. The optimization strategy led to the enhancement of fengycin from 1.45 g/L to 3.5 g/L.
The results of repeated fed-batch cultures in fermentor revealed that the optimum feeding nutrient is 2.6 % mannitol and 2.2 % soybean meal as carbon and nitrogen source, and the optimum feeding timing is 12, 24, 36 and 48 hr. The fengycin production was boosted to 4.7 g/L. Meanwhile, the results of repeated fed-batch cultures in shaking flask revealed that obtain the supernatant, add 1 % inoculum and culture for 96 hr, fengycin production could not inhibit by fengycin or toxic in the supernatant. This strategy led to the enhancement of fengycin from 3.0 g/L to 5.2 g/L.

並列關鍵字

Fengycin ； Bacillus subtilis F29-3 ； Response surface methodology (RSM) ； Lipopeptide ； Media optimization

參考文獻

Adinarayana, K., K.V.V.S.N. B. Raju, and P. Ellaiah. 2004. Investions on alkaline protease production with Bacillus subtilis PE-11 immobilized in calcium alginate gel beads. Process Biochem., 39 : 1331-9

Ahimou, F., M. Paquot, P. Jacques, P. Thonart, and P. G. Rouxhet. 2001. Influence of electrical properties on the evaluation of the surface hydrophobicity of Bacillus subtilis. J. Microbiol. Methods., 45: 119-26

Akpa, E., P Jacques, B. Wathelet, M. Paquot, R. Fuchs, H. Budzikiewicz and P. Thonart. 2001. Influence of Culture Conditions on Lipopeptide Production by Bacillus subtilis. Appl. Biochem. Biotechnol., 91-93: 551-61

Bais, H. P., R. Fall, and J. M. Vivanco. 2004. Biocontrol of Bacillus subtilis against Infection of Arabidopsis Roots by Pseudomonas syringae Is Facilitated by Biofilm Fromation and Surfactin Production. Plant Physiol., 134: 307-19

Bodour, A. A., C. G. Barajas, B. V. Jiorle, M. E. Malcomson, A. K. Paull, A. Somogyi, L. N. Trinh, R. B. Bates, and R. M. Maier. 2004. Structure and Characterization of Flavolipids, a Novel Class of Biosurfactants Produced by Flavobacterium sp. Strain MTN11. Appl. Environ. Microbiol., 70: 114-20