具去除黃麴毒素B1能力之 Chryseobacterium timonianum 菌株的篩選與特性分析

Abstract

黃麴毒素B1（aflatoxin B1, AFB1）因其在呿喃環雙鍵形成環氧化物而具有強烈肝致癌性，是食品與飼料中危害性最高的黴菌毒素之一。相較於物理或化學解毒方法，微生物細胞外且有耐熱成分的生物解毒方式更具有應用潛力。本研究首先分離並鑑定具有AFB1去除能力的菌株，並進一步篩選出其中一株菌株Chryseobacterium timonianum c4a，其胞外產物具有耐熱性且有AFB1降解活性。 本研究利用香豆素選擇性培養基，從雞盲腸內容物與富含羽毛之土壤樣本篩選具AFB1降解能力菌株。在篩選出的菌株中，c4a具有最佳AFB1活性降解效果。c4a在營養瓊脂上的菌落呈亮黃色、flexirubin反應陽性、表面光滑且常帶有淡淡果香、為革蘭氏陰性、不產孢子的短桿菌，且不具運動性。API 生化試驗顯示其吲哚酶、尿素酶、氧化酶、esculin 與明膠水解均為陽性，並能同化葡萄糖、甘露糖、麥芽糖、檸檬酸、苦杏仁苷、海藻糖、澱粉、肝醣及 gentiobiose 等多種碳源。16S rRNA之初步親緣分析及利用rpoB、gyrB、rpoD家族基因進行的串聯 MLSA 分析皆顯示c4a歸屬於Chryseobacterium timonianum之近緣支系。 c4a之AFB1降解活性在其胞外培養上清液中，胞內與細胞壁幾乎不具活性。在明膠蛋白胨與牛肉萃取物比例為4:1的營養培養基中，添加1 ppm AFB1，於 60°C、pH 8 並補充 Mn²⁺ 或 Mg²⁺ 的條件下，c4a上清液於72小時內可降低約93%的AFB1活性。c4a上清液降解AFB1活性能力可耐受高壓滅菌，但在加入蛋白酶混合物或 SDS 後即完全喪失，顯示其為蛋白質。UPLCESI MS/MS 解析含 c4a 上清液與AFB1的反應混合物，偵測到一種水加成於呿喃環雙鍵之水合代謝物（m/z 331.08），其細胞毒性遠低於AFB1。 利用c4a全基因體定序資料進行分析，並根據抗AFB1活性基因篩選，其中一個漆酶（laccase）與三個醛酮還原酶（aldoketo reductase, AKR）為目標基因。c4a 細胞亦可脫色 Remazol brilliant blue，顯示其可能具有氧化還原相關酶。所有目標基因皆成功以 pET32 Xa/LIC 進行克隆，但於 Rosettagami 2(DE3)pLysS 的表現中，即使補充銅離子，漆酶仍無法完整表現。其中 AKR 15_78 能順利表現與純化，偏好 NADPH作為輔酶，其最適活性條件為 30°C、pH 7。然而，AKR 15_78 對 AFB1、玉米赤黴烯酮（zearalenone）及去氧雪腐鐵鏽菌烯醇（deoxynivalenol）皆無活性，但可還原體積較大的酮類化合物。由 AKR 15_78 催化反應所得的手性產物，多數不適合作為藥物前驅物，惟乙基苯甲醯乙酸為例外。 總而言之，C. timonianum c4a具構成性、胞外且耐熱的AFB1生物轉化能力，但其中包含的基因與酵素之分析，需利用如Pichia pastoris或Kluyveromyces marxianus等真核表現系統進一步研究。同時，AKR 15_78催化形成的手性醇，仍需藉由不同手性色譜管柱與標準品進一步分析其立體選擇性與應用潛力。

Aflatoxin B1 (AFB1) ranks among the most hazardous mycotoxins in food and feed chains, owing to its potent hepatocarcinogenicity arising from epoxide formation at the furan ring double bond. Microbial detoxification strategies, especially those involving extracellular and thermostable components, provide practical benefits over physical or chemical alternatives. This thesis detailed the isolation and characterization of bacterial strains for AFB1 removal, with further characterization of the AFB1-degrading activity of the Chryseobacterium timonianum strain c4a, which was found to be thermostable. A coumarin-selective medium was used to screen AFB1-degrading strains from chicken cecal digesta and feather-enriched soil samples. Out of the selected AFB1-degrading strains, c4a degraded AFB1 most effectively. Colonies of the c4a strain on nutrient agar were bright yellow, flexirubin-positive, smooth, and often had a faint fruity odor. Cells stained Gram-negative, appeared as non-motile rods lacking endospores. Biochemical tests via API systems were positive for indole, urease, oxidase, esculin, and gelatin hydrolysis, with assimilation of glucose, mannose, maltose, citrate, amygdalin, trehalose, starch, glycogen, and gentiobiose, among others. Initial 16S rRNA phylogenetic analysis, as well as concatenated MLSA analysis of housekeeping genes (rpoB, gyrB, rpoD), revealed that the c4a strain is closely related to Chryseobacterium timonianum. AFB1 activity was localized to the extracellular supernatant, while the intracellular and cell wall fractions showed negligible activity. With nutrient broth at a 4:1 gelatin peptone: beef extract ratio, 1 ppm AFB1 at 60°C, pH 8, plus Mn²⁺ or Mg²⁺, the c4a supernatant showed ~93% loss in 72 h. This activity survived autoclaving yet was abolished by protease cocktails or SDS, implying its proteinic nature. UPLC-ESI MS/MS analysis of the reaction mixture containing c4a supernatant and AFB1 showed a hydrated metabolite (m/z 331.08) from water addition across the furan double bond, which is reported to be significantly low in cytotoxicity compared to the original AFB1. Gene mining using whole genome sequencing data of the c4a strain revealed one laccase and three aldo-keto reductase (AKR) target genes, which are reported to be active against AFB1. Strain c4a cells also decolorized Remazol brilliant blue. pET-32 Xa/LIC cloning of all target genes succeeded, but Rosetta-gami 2(DE3)pLysS expression was incomplete in the case of laccase, despite copper supplementation. AKR 15_78, which was expressed and purified, preferred NADPH, with optimal activity at 30°C and pH 7, and was inactive against mycotoxins AFB1, zearalenone, and deoxynivalenol. However, it reduced bulky ketones. Chiral products formed after enzymatic reactions with AKR 15_78 were undesirable as pharmaceutical precursors, except for the case of the substrate ethyl benzoyl acetate. Overall, C. timonianum strain c4a exhibited constitutive, extracellular, and thermostable AFB1 biotransformation, yet pinpointing the underlying genes and enzymes will likely require eukaryotic expression (e.g., Pichia pastoris / Kluyveromyces marxianus); in parallel, the putative AKR 15_78 mediated chiral alcohol transformation requires further investigation using alternative chiral HPLC columns and authentic reference standards.