生物界面活性劑—沙雷濕潤素之醱酵生產及相關性質分析

Production and characterization of a lipopeptide biosurfactant-serrawettin from Serratia marcescens SM△R

闞公晧(Kung-Hao Kan)

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(2006年)

摘要

沙雷氏菌 Serratia marcescens為革蘭氏陰性菌、短桿菌，能同時代謝生產紅色色素-靈菌紅素(Prodigiosin)，與脂胜肽類生物界面活性劑-沙雷溼潤素(Serrawettin)。本篇論文旨在使用Serratia marcescens經 quorum sensing 處理之突變株Serratia marcescens SMΔR，對其所代謝生產之沙雷濕潤素進行分離、純化及鑑定，確立其結構組成，並尋求其增進沙雷溼潤素最適化之發酵培養基，同時探討其相關之界面性質與物/化性質。首先針對發酵液進行分離、純化，利用1H-NMR、FAB-MS及IR等技術分析，結果證實Serratia marcescens SMΔR，所生產的沙雷濕潤素為分子量515 m/z，分類上屬於Serrawettin W1，又名Serratamolide，為一可用作抗癌之代謝產物。沙雷濕潤素發酵生產培養基部份，經碳、氮源篩選，研究顯示以24 g/L Glucose、16g/L Bacto sotyone，可得到最高Serrawettin W1產量達718 mg/L。其次利用RSM(回應曲面)法，探討微量元素最佳組成，研究證實Serratia marcescens SMΔR在2.55μM MgSO4、2.36μM MnSO4添加下，Serrawettin W1產量可達1174 mg/L，與微量元素未最適化之培養基相比，約有1.6倍提昇。在誘發Serrawettin W1產量之發酵策略方面，研究顯示使用3 %(w/v)藻酸鈣顆粒作為固體載體，能進一步提升Serrawettin W1產量至2006 mg/L，相較於未添加固體載體之控制組而言產量約有1.7倍之提升，更為碳氮比最佳化培養基之2.8倍。在界面特性分析部分，利用接觸角探討濕潤性質結果顯示，Serrawettin W1在1500 mg/L濃度下，即可使水接觸角由68度降低至32度，為一不錯之濕潤劑。透過量測界面性質亦證實，僅須添加100 mg/L的Serrawettin W1即可將水溶液與正庚烷之界面張力由40.8 mN/m降至6 mN/m。量測表面滑行現象結果顯示，添加1500 mg/L之Serrawettin W1，菌體滑行速率達0.31cm/h，為未加Serrawettin控制組之3倍。在物/化性質分析方面，研究顯示Serrawettin W1對溫度耐受性強，經100℃加熱2小時、高溫高壓滅菌釜121℃，20分鐘下，仍能維持活性表現，顯見其溫度耐受性高；在酸鹼度耐受性實驗中，發現Serrawettin處在酸性pH 1-7中，仍能維持活性表現，但在pH >7時，對其結構則會造成破壞，故培養條件應維持在酸性pH值較佳；在鹽的耐受性方面，添加鹽度1-7 %，並放置於10-25℃水浴槽中12小時，皆不影響Serrawettin W1活性表現，顯示Serrawettin W1具有應用於海上溢油控制之潛力。

關鍵字

沙雷濕潤素 ； 靈菌紅素 ； 生物界面活性劑, 臨界微胞濃度, 表面滑行

並列摘要

Being biodegradable and less toxic, biosurfactants possess great advantages over synthetic surfactants and hold potential applications in petroleum recovery, and as the emulsifiers and de-emulsifiers in pharmaceutical, cosmetics and food industries. Serrawettin W1, an important lipopeptide-type biosurfactant produced by various strains of Serratia marcescens, is the most powerful biosurfactant ever discovered. This work aimed to develop better culture medium and fermentative strategy enabling more efficient production of serrawettin W1 from S. marcescens SM△R. We shown that the optimal culture media was 24 g/L Glucose; 16g/L Bacto soytone; 2.55μM MgSO4 and 2.36μM MnSO4. Serrawettin W1 production was optimal in batch cultures when the temperature and agitation rate wre controlled at 30℃ and 200 rpm. Per above culture medium, we can be obtained the concentration of serrawettin W1 was 1174 mg/L, which was 1.6-fold over the control LB medium. Addition of a small quantity of solid porous carrier (e.g., activated carbon, sponge and alginate bead) into fermentation broth significantly increased serrawettin W1 production with Serratia macescens SM△R. Culture medium containing 3% (w/v) of alginate bead gave an optimal serrawettin W1 production yield of 2006 mg/L, which was approximately 1.7-fold higher than that obtained from carrier-free liquid culture. Serrawettin W1 in the culture broth was purified primarily via solvent extraction and column chromatography. The NMR and mass spectrometry analysis shows that the purified product is a lipopeptide biosurfacrant, called serrawettin W1. At 1500 mg/L serrawettin W1 decreases the contact angle of water from 68 degrees to 32 degrees. Meanwhile, 100 mg/L serrawettin W1 decreases interface tension and surface tension of water to 6 mN/m and 40.8 mN/m, respectively. Furthermore, we also shown that serrawettin W1 possess higher thermal, saline and acidic tolerance. However, this work was a preliminary feasibility assessment of using the Serratia macescens strains for biosurfactant (serrawettin W1) production in a commercial scale.

並列關鍵字

Serratia marcescens ； Serrawettin ； Serratamolide ； Prodigiosin ； biosurfactant

參考文獻

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