土壤中篩選出之假單孢菌屬對六溴環十二烷之有氧生物轉化與其功能性酵素的鑑定

Abstract

六溴環十二烷 (Hexabromocyclododecane, HBCD)為被廣泛使用之溴化阻燃劑，因其於環境中不易被降解之特性，使HBCD累積於環境與生物內，對食物鏈各階層之生物造成毒害。雖然近年來以微生物去除汙染物的概念廣為學界倡議，但目前對細菌如何進行HBCD生物轉化的所知仍有限。本研究於土壤中共篩選三株能降解HBCD之細菌，其中兩株細菌為不同品系之Bacillus cereus，另一種細菌為Pseudomonas plecoglossicida。前人已對Bacillus cereus做過HBCD之生物降解的研究，本研究以P. plecoglossicida做為研究目標菌株。P. plecoglossicida能於35°C、pH 8.0以及HBCD濃度為0.125 ppm之條件下，於降解試驗48小時後，去除70.9%之HBCD。培養於LB medium與PBS buffer的條件下，P. plecoglossicida皆能有效去除HBCD，但觀察細菌生長的狀態時，可以發現PBS buffer之降解試驗組，細菌族群密度未能增加，顯示P. plecoglossicida可能無法僅靠HBCD做為生長所需之碳源。此外，添加葡萄糖或蔗糖時，可以發現細菌在有額外之碳源時，能增加HBCD之降解效率。透過鑑定細菌降解HBCD之副產物，我們確認P. plecoglossicida能透過兩個途徑將HBCD轉化。第一個途徑為脫溴途徑，生物轉化之副產物為五溴環十二烯 (pentabromocyclododecene)、四溴環十二烯 (tetrabromocyclododecadiene)、三溴環十二烯 (tribromocyclododecadiene)、二溴環十二烯 (dibromocyclododecadiene)、一溴環十二烯 (bromocyclododecatriene)、環十二碳三烯 (cyclododecatriene)。第二個生物轉化之途徑為同時透過對HBCD進行脫溴與羥化的反應，生物轉化之副產物為七溴環十二烷 (heptabromocyclododecane)、七溴環十二碳二醇 (heptabromocyclododecanediol)、四溴環十二醇 (tetrabromocyclododecadienol)，以及四溴環十二碳三醇 (tetrabromocyclododecenetriol)。P. plecoglossicida之粗蛋白萃取液於pH 8.0、35°C以及HBCD濃度為50.0 ppb之條件下，能降解97.8%的HBCD。蛋白酶譜之活性染色，確認粗蛋白中含有能透過脫溴途徑降解HBCD之酵素。由P. plecoglossicida全基因體序列註解資訊與鑑定與HBCD作用之蛋白質，我們推測降解HBCD之可能酵素為脫氫酶(dehydrogenase)、谷胱甘肽S-轉移酶(glutahione S-transferase)以及細胞色素c (cytochrome c oxidase)。將P. plecoglossicida之粗蛋白以電紡之方式固定於聚乙烯醇 (polyvinyl alcohol, PVA)之奈米纖維上，能有效加強蛋白之穩定性，且使蛋白具有重複使用性。這個固定化酵素的方法可提供利用P. plecoglossicida之粗蛋白去除環境之HBCD的不同應用策略。本篇研究為HBCD之細菌與酵素的生物轉化提供新的觀點。

Hexabromocyclododecane (HBCD) is widely used as a brominated flame retardant (BFR) and is identified as an emerging and persistent comtaminant. It has been detected in various environment matrix and is dangerous to human health. However, limited information is available about bacterial biotransformation of HBCD. In this study, two different Bacillus cereus strains and one Pseudomonas plecoglossicida strain were isolated from BFR comtaminated soil with good HBCD biodegradation capability in aerobical condition. We focus on P. plecoglossicida for the sparse knowledgement on the involvement of HBCD biotransformation of this species. P. plecoglossicida is able to biotransform 70.9% of HBCD at the initial concentration of 0.125 ppm at 35°C and pH 8.0 within 48 hours. This metabolic capability is observed in LB medium as well as in PBS buffer, while the cell density generally decreased in minimum salt medium. In addition, P. plecoglossicida shows a higher biotransformation rate when glucose or sucrose is supplied to minimal salt medium, indicating P. plecoglossicida could require extra carbon source than HBCD. Based on the identification of metabolites, P. plecoglossicida may biotransform HBCD by two pathways. In the first, HBCD was sequentially debrominated to pentabromocyclododecene, tetrabromocyclododecadiene, tribromocyclododecadiene, dibromocyclododecadiene, bromocyclododecatriene and cyclododecatriene. The second pathway is a debrominating and hydroxylating process to form heptabromocyclododecane, heptabromocyclododecanediol, tetrabromocyclo- dodecadienol and tetrabromocyclododecantriol. Crude protein from P. plecoglossicida is able to efficiently degrade 50.0 ppb HBCD at 35°C and pH 8.0. Zymogram assay demonstrated that the ability of dehalogenating HBCD is contributed by proteins in particular electrophoretic character. Based on the assembled genome sequence of P. plecoglossicida and proteome analysis, glutathione S-transferase, dehydrogenase and cytochrome c oxidase were proposed to be the possible enzymes involved in biodegradation of HBCD via debromination. Immobilizing crude protein into polyvinyl alcohol nanofibers via electrospinning could enhance protein stability and allow the protein to be recovered and reused. To sum up, this study provides a novel insight into microbial and enzymatic biotransformation of HBCD.