以蛋白質體學探討雙份A降解菌Rhodococcus pyridinivorans(PDB9)之蛋白調控與降解機制

Abstract

雙酚 A（BPA）據之前研究指出，對生物具有雌激素活性和毒性。本篇論文中,篩選出具有降解雙酚 A的降解菌株Rhodococcus pyridinivorans (PDB9)。生物降解菌株生長在具基本鹽類的培養液中,內含有雙酚 A（50毫克/升）作為唯一碳源的培養基，菌株48小時內降解90％的雙酚 A。藉由高壓液相層析儀(High-performance liquid chromatography)及液相層析串聯質譜儀(Liquid Chromatography/Mass Spectrometry)，鑑定降解後的中間產物，其中鑑定出四個中間產物。藉由二維電泳（Two Dimensional Electrophoresis），膠體內水解(In-gel digestion)，質譜分析，並利用2D-ImagesMaster分析軟體，鑑定出與對造組有差異的蛋白質與酵素，其中包括五個差異蛋白質，分別為N-二甲基亞硝胺甲醇去氫酶(NDMA-dependent methanol dehydrogenase)，延長因子Tu (Elongation factor Tu)，半胱氨酸合酶(Cysteine synthase)，電子轉移黃素蛋白β/α亞基(Electron transfer flavoprotein beta/alpha subunit)，ATP依賴蛋白降解酶(ATP-dependent Clp protease proteolytic subunit)。我們已經分離了一株雙酚A降解菌Rhodococcus pyridinivorans，這幾乎可以在48小時內完全降解50mg/L BPA。此外，使用液相質譜，我們發現了一種新的降解途徑。藉由以上實驗技術，可以運用在之後生物催化與生物降解等探討。

Bisphenol A (BPA) has been reported has an estrogenic activity and toxicity to aquatic organisms. This study was conducted to show the isolation and property of Rhodococcus pyridinivorans(PDB9) having BPA biodegradability. The bacterial strain was able to grow in a basal mineral salt medium containing BPA (50 mg/L) as the sole carbon source, and to degrade 90% BPA within 48 h. The effects of BPA on the biology of Rhodococcus pyridinivorans(PDB9) were elucidated using 2D proteomic. The cytoplasmic proteins isolated from these BPA –treated and -untreated cells after 24, 48hour were carried out for proteomic analysis. Four intermediated compounds were detected using Liquid Chromatography/Mass Spectrometry when BPA was degraded by Rhodococcus pyridinivorans via a novel pathway. There were five differential protein, respectively, as compared to their corresponding control (without BPA addition), at the indicated incubation times when 40% in 24h and 90% 48h of BPA had been removed. The five protein ID were identified by LTQ-FT-MS/MS. Proteins identified included NDMA-dependent methanol dehydrogenase, Elongation factor Tu, Cysteine synthase, Electron transfer flavoprotein beta/alpha subunit, ATP-dependent Clp protease proteolytic subunit. We had isolated a potential bacteria Rhodococcus pyridinivorans which could almost completely degrade 50 mg/L BPA within 48 hours. Moreover, using LC-Mass, we discovered a novel pathway to degrade BPA by microbe. Characterizing the bacterial response to BPA at the biochemical level identifies proteins that can be used by biocatalysts for biodegradation deserving further investigations.