以大腸桿菌建立紫色桿菌素生產系統與功能性細菌纖維素製備

Abstract

紫色桿菌素 (violacein) 為一天然由紫色桿菌 (Chromobacterium violaceum) 所產之紫色色素，具有抗菌性、抗真菌、抗癌等生理活性，可望應用於多項產業，諸如食品業 (作為著色劑)、製藥業等，然而受限於產率供不應求。細菌纖維素 (Bacterial Cellulose, BC) 是一種由 Komagataeibacter xylinus 所產之聚合物，具有高水分含量、優良機械性質與生物相容性。近年來科學家發展了許多提升 violacein 的技術，而本篇研究期望能夠將 violacein 合成途經之基因 (violacein biosynthesis related gene cluster, vio gene) 轉殖進大腸桿菌 (Escherichia coli, E. coli) 質體 (plasmid) 進行異源基因表達並將產出之 violacein 以後位修飾法 (ex-situ modification) 修飾自 Komagataeibacter xylinus ATCC 700178 培養取得之 BC，以期製備含有 violacein 之功效性 BC 並附加 BC 抗菌性之價值。結果顯示，含有 vio gene 的 E. coli BL21(DE3) pSV004 可產生約 3.53 ± 1.58 mg/mL crude violacein。以孔洞擴散法測試 (agar diffusion method) 與紙錠擴散試驗 (disc-diffusion method) 測試其抗菌性，對於 Staphylococcus aureus (S. aureus) BCRC 10451 可分別產生 10.67、12.33 (濾紙)、12.17 (BC) mm 的抑菌環直徑。將 BC 浸泡於 crude violacein 並凍乾製備成 violacein/BC (VBC)，VBC 在色澤分析中顯著地降低 b 值，顯示 violacein 使 VBCs 呈現藍色。在水含量上，VBC2 (75.8 %) 較低於一般 BC (96.5 %)。表面型態顯示 VBCs 除了相似的網狀纖維束之外，還含有短桿狀細菌顆粒。此外，VBCs 的熱穩定度較低於一般 BC，最大降解溫度 (tpeak) 由 325.95 降至 305.96oC。最後，於抗菌試驗中顯示顯示 VBCs 對 S. aureus 無顯著抗菌性。未來期望能夠提升 violacein 於 VBCs 中的含量，並結合抑制 Gram (-) 之機能性成分以獲得廣域抗菌性，藉以應用於食品包材或是傷口敷材。

Violacein is a natural pigment produced by Chromobacterium violaceum. It has the potential to apply in many fields due to its biological properties, such as antibacterial, antifungal, anticancer, and so on. However, the low productivity of violacein limits its application in industry. Bacterial cellulose (BC) is a polymer produced by Komagataeibacter xylinus that provides many material properties such as high-water content, great mechanical property and great biocompatibility. Scientists have tried a lots to improve its productivity and this study’s purpose is to clone the violacein-synthesis gene cluster into Escherichia coli (E. coli) to increase violacein productivity. The BC was combined with violacein utilizing ex-situ modification and produce the violacein/BC (VBC) that contained antimicrobial properties. The result showed that E. coli BL21/pSV004 presented the violacein production that reached 3.53 mg/mL. Crude violacein could produce 10.67 - 12.33 mm diameters of inhibition zone toward Staphylococcus aureus in agar diffusion method or disc-diffusion method. The VBCs significantly reduced the b value in color analysis from -2.81 to -15.27 after immersed BC into crude violacein and freeze-dried. Besides that, the VBCs exhibited lower water content compared to BC (75.8 % v.s. 96.5 %). In the result of morphology, VBCs showed a similar network structure with BC and contained bacterial cell particles. Violacein lower the thermal stabilities of VBCs which resulted in a lower tpeak from 325.95oC to 305.96oC. The further study could increase the violacein content and combine with other Gram (-) inhibitors to produce an antimicrobial material for food packaging or wound dressing.