透過您的圖書館登入
IP:54.224.124.217
  • 學位論文

利用植物固醇為原料以醱酵法生產雄烯酮化合物之研究

Microbial Production of Androstenones from Phytosterol

指導教授 : 劉文雄
若您是本文的作者,可授權文章由華藝線上圖書館中協助推廣。

摘要


本研究係探討利用植物固醇為原料以微生物轉換法生產荷爾蒙中間體雄烯酮化合物AD及ADD之醱酵生產研究,其主要結果敘述如下: 分枝桿菌Mycobacterium sp. NRRL B-3683經NTG人工誘導變異後之變異株,以高濃度類固醇培養基挑選其中生長快速具植物固醇耐性之變異株(PT變異株)及具植物固醇與ADD耐性之變異株(PD變異株),經探討培養基組成份以及ADD之生產能力後,得知具植物固醇與ADD耐性之變異株PD3為最適之ADD生產菌株。利用PD3變異株以五公升醱酵槽轉換0.1%台糖植物固醇,在培養至96小時時,ADD最大蓄積量可達1.56 mM,其莫耳轉換率為70.5%。 在兩段式微生物轉換生產雄烯酮化合物方面,利用構成性膽固醇氧化酶生產原生質體融合株Arthrobacter simplex F2-20-8將植物固醇轉換成植物固酮後,供第二階段微生物轉換使用。以Hinton氏三角瓶探討以上述植物固酮醱酵液為原料生產AD、ADD之最適條件後得知,AD之最適生產菌株為具植物固醇耐性之雄烯二酮生產變異株Mycobacterium sp. AD1,最適培養基為添加1%酵母抽出物及2%葡萄糖之合成培養基,在30°C下培養96小時後, AD1菌株可轉換5.2 mM之植物固酮醱酵液並生成3.30 mM之雄烯二酮,其轉換率為63.5%。ADD之最適生產菌株為Mycobacterium sp. PD3,其最適培養基為添加0.5%酵母抽出物及2%葡萄糖之合成培養基,在30°C下培養96小時後,PD3菌株可蓄積最高量之ADD,濃度為3.86 mM,其轉換率為74.2%。在轉換過程中出現產物抑制現象,AD或ADD醱酵液中均無法蓄積超過4 mM,利用Amberlite XAD-7樹脂吸附產物AD(D),順利解除上述產物抑制效應,其最佳添加量為7.5% XAD-7。 以五公升醱酵槽檢討AD及ADD之生產條件後得知,以Mycobacterium sp. AD1為生產菌株,在含10 mM植物固酮,1% yeast extract, 2% glucose及100 g XAD-7樹脂之培養基,工作體積為2.0 L,溫度為30°C,轉速300 rpm,表面通氣量為2.0 L/min之條件下培養168小時後,回收XAD-7樹脂並以溶劑沖提後可得6.31 g粗產物,其中含有2.21 g AD,莫耳轉換率為38.6%;以Mycobacterium sp. PD3為生產菌株,在含10 mM植物固酮,0.5% yeast extract, 1% glucose及100 g XAD-7樹脂之培養基,工作體積為2.0 L,溫度為30°C,轉速300 rpm,表面通氣量為2.0 L/min之條件下培養168小時後,回收XAD-7樹脂並以溶劑沖提後可得6.34 g粗產物,其中含有3.34 g ADD,莫耳轉換率為58.8%。 巨孔性非離子交換性樹脂Amberlite XAD-7可有效以非選擇性吸附醱酵液中之雄烯酮化合物,將樹脂回收後經過乾燥程序去除水分後,再以三倍體積之乙酸乙酯沖提即可有效溶離吸附於樹脂上之粗產物,達到回收產物並減少有機溶劑使用量之目的。將粗產物AD或ADD經過兩次瞬瀉管柱層析(第一次以ethyl acetate : n-hexane = 40:60;第二次以ethyl acetate : chloroform = 20:80)後可得純度為99%之AD,其回收率為48%;ADD之純度為98%,其回收率為53%。將純化所得之AD及ADD進行熔點測定、紫外線/可見光吸收光譜、紅外線吸收光譜及氫原子核磁共振光譜等儀器分析後確認其結構即為雄烯二酮與雄二烯二酮。 將五株含膽固醇氧化酶基因之嗜甲醇酵母(P. pastoris)轉形株以0.5%(v/v)甲醇進行誘導培養72小時,篩得具有最佳之膽固醇氧化酶生產能力之菌株P. pastoris pPC-1,其酵素活性可達1.01 U/mL。利用五公升醱酵槽探討P. pastoris pPC-1轉形株生產膽固醇氧化酶之條件得知,在轉速為500 rpm,通氣量為2 vvm,溫度為30°C,接種量為10% (v/v)及操作體積為2 L之條件下,當菌體濃度(O.D. 600 nm)達300時開始進行甲醇誘導培養,甲醇濃度以儀器自動控制在0.7% (w/v),共進行誘導72小時,膽固醇氧化酶活性可達4.1 U/mL。 將經過甲醇誘導培養之醱酵液以5,000 ´ g離心後可獲得粗酵素液共2,500 mL,酵素總活性約11,500 U。以蛋白質電泳分析粗酵素液得知,膽固醇氧化酶為最主要產物,經限外過濾裝置濃縮後,可獲得比活性提高1.24倍之酵素。本酵素經內糖苷酶(endoglycosidase H, Endo H)作用後與原態蛋白做比較,得知其分子量均為55 kDa,推測膽固醇氧化酶並未被P. pastoris在N端予以糖基化。膽固醇氧化酶之最適pH值為7.5,最適溫度為50°C,pH安定範圍為5.0∼10.0,在50°C以下具有熱安定性。 在五公升醱酵槽培養以0.7% (w/v)進行甲醇誘導12小時後,以幫浦進行500 mL 4% (w/v)植物固醇之餽料共48小時,經過96小時之酵素誘導與84小時之生物轉換後,植物固酮可蓄積7.6 mM,轉換率約為61%。取一升醱酵液以等體積之乙酸乙酯萃取三次後,可得6.35 g粗植物固酮,純度約為54%,將粗植物固酮以矽膠管柱層析進行純化(沖提液為正己烷:乙酸乙酯=80:20),可得純度為95%之植物固酮共3.43 g,回收率約為54%。 利用微生物轉換法所得之植物固酮主要成份有三種,其結構相近,無法利用一般之薄層層析法分析,本研究利用高效能液相層析儀探討植物固酮之分析方法後得知,利用配備MetaChem Polaris C18-A管柱(5 mm顆粒, 500 ´ 4.6 mm直徑)之高效液相層析儀,在移動相為100%甲醇、流速為1.2 mL/min之條件下,可有效地分離三種主要植物固酮。三種主要植物固酮經結晶、熔點測定、紅外線光譜、紫外線及可見光光譜、核磁共振光譜、質譜分析等物理及儀器分析後,鑑定其結構分別為菜籽固酮(campest-4-en-3-one; II)、豆固酮(stigmasta-4,22-dien-3-one; I)及穀固酮(sitost-4-en-3-one; III)。

並列摘要


The purpose of this study was to investigate suitable microbial transformation conditions for the production of androstenones from phytosterol. The main results were stated as follows: Phytosterol-tolerant mutants were initially isolated from parental strain Mycobacterium sp. NRRL B-3683 via the NTG-inducted mutagenesis and tested for productivity of androstenones. The optimum ADD-producing mutant was strain PD3 after investigation of the medium composition and productivities of the phytosterol-tolerant mutants. When cultivation was carried out in a 5-L fermentor with a medium supplemented with 1% glucose, 0.1% TSC phytosterol, the maximum ADD yield (1.56 mM) could be obtained at 96-h cultivation, the molar conversion rate was 70.5%. The phytosterol was converted into phytostenones by a constitutive cholesterol oxidase producing protoplast-fusant Arthrobacter simplex F2-20-8. Phytostenones in fermentation broth were used for the second step microbial transformation by Mycobacterium sp. When a shaking culture was carried out to investigate the optimum conditions of production of AD and ADD with the phytostenones broth mentioned above, it was found that the optimum medium for production of AD was a synthetic medium supplemented with 1% yeast extract and 2% glucose. A maximum accumulation of AD (3.30 mM) from phytostenones broth (5.2 mM) could be obtained at 96-h cultivation by phytosterol-tolerant and AD-producing mutant AD1 at 30°C. The optimum medium for production of ADD was a synthetic medium supplemented with 0.5% yeast extract and 2% glucose. A maximum accumulation of ADD (3.86 mM) from phytostenones broth (5.2 mM) could be obtained at 96-h cultivation by phytosterol-tolerant and ADD-producing mutant PD3 at 30°C. The molar conversion rate was 74.2%. The maximum accumulation of AD or ADD was less than 4 mM due to the product inhibition effect. By using Amberlite XAD-7 as absorption material, we successfully disabled the product inhibition effect and the optimum quantity of XAD-7 was 7.5% (w/v). The optimum operating conditions for the production of AD and ADD with a 5-L microprocessor controlled fermentor were investigated. When a cultivation was carried out with a medium supplemented with 1% yeast extract, 2% glucose, 100 g of XAD-7, 10 mM phytostenones and the operating conditions were 300 rpm of agitation speed, 2.0 L/min of surface-aeration rate for 168-h cultivation with mutant AD1. XAD-7 resin was recovered and eluted by solvent, 6.31 g of crude product containing 2.21 g of AD was obtained. The molar conversion rate was 38.6%. Under the same operating conditions mentioned above except 0.5% yeast extract, 1% glucose and mutant PD3, 6.34 g of crude product containing 3.34 g of ADD was recovered. The molar conversion rate was 58.8 %. Androstenones in fermentation broth were absorbed by Amberlite XAD-7. The XAD-7 resins were recovered, washed by clean water and then dried in the oven. The crude androstenones in XAD-7 resins were efficiently washed out by 3 times of volume of ethyl acetate. The crude AD or ADD was subjected on a flash column chromatography twice (solvent system were ethyl acetate : n-hexane=40:60 and ethyl acetate : chloroform=20:80, respectively). AD with the purity of 99% and ADD with the purity of 98% were recovered; the rate of recoveries were 48% and 53%, respectively. The molecular structure of AD and ADD were confirmed after chemicophysical and instrumental analyses including melting point, UV/Vis spectroscopy, IR spectroscopy, 1H NMR spectroscopy. An optimum cholesterol oxidase-producing recombinant yeast, P. pastoris pPC-1, was selected from five P. pastoris KM71 transformants containing cholesterol oxidase structure gene from A. simplex F2-20-8 after 72-h of methanol induction (0.5%, v/v). The accumulated enzyme activity was 1.01 U/mL. P. pastoris pPC-1 was cultured in a 5 L fermentor under the following conditions: 30°C; agitation speed, 500 rpm; aeration rate, 2 vvm; inoculums, 10% (v/v) and working volume, 2 L. When the optimum cell density (O.D. 600 nm=300) was reached, 0.7% (w/v) methanol was added for the induction of cholesterol oxidase production. The maximum cholesterol oxidase activity was 4.1 U/mL after the methanol induction at 72 h. Fermentation broth was centrifuged with 5000 ´ g after the methanol induction, 2,500 mL of crude cholesterol oxidase with 11,500 U of total enzyme activity was recovered. It was found that cholesterol oxidase was the main product in the fermentation broth by analyzing the crude enzyme with SDS-PAGE. Concentrated enzymes with 1.24 times of specific activity were obtained by ultrafiltration (Pellicon, 10 kDa). The cholesterol oxidase was then digested by Endo-H. It was found that the molecular weight of both digested and native cholesterol oxidase were the same, 55 kDa. It was suggested that the enzymes did not be glycosylated at N-terminal by Pichia yeast. Some properties of cholesterol oxidase from P. pastoris pPC-1 were analyzed and listed as follows: optimal pH, 7.5; optimal temperature, 50°C; pH stability, 5.0–10.0; thermo stability, below 50°C. Bioconversion of phytosterol to phytostenones by P. pastoris pPC-1 was carried out in a 5-L fermentor. P. pastoris pPC-1 was induced by 0.7% methanol for 12 h. Then, phytosterol (4%, w/v, 500 mL) was pumped into the fermentor in the next 48 h. After 96 h enzyme induction and 84 h bioconversion, the accumulation of phytostenones was 7.6 mM with the bioconversion of 61%. The culture broth (1 L) was extracted with equal volume of ethyl acetate for three times, and 6.35 g crude phytostenones was recovered with the purity of 54%. After silica gel column chromatography with the elution system: n-hexan:ethyl acetate=80:20, 3.43 g of purified phytostenones was recovered with the purity of 95%, and the rate of recovery was 54%. There were three main molecules of phytostenones. The three main molecules could not be separated by TLC method due to the structural resemblance. To separate the mixture of the three major phytostenones from the fermentation broth, a high performance liquid chromatography (HPLC) method was investigated. The analytic conditions were as follows: column, MetaChem Polaris C18-A (5 μm particles, 500 ´ 4.6 mm I.D.); mobile phase, 100% methanol, and flow rate, 1.2 mL/min. According to the physical and instrumental analyses including crystallization, melting point, ESI-MS, IR, UV-Vis and 1H-NMR spectrometries, the three major phytostenones present in the fermentation broth were campest-4-en-3-one (II), stigmasta-4,22-dien-3-one (I) and sitost-4-en-3-one (III), respectively.

參考文獻


台灣省政府農林廳。1996。農產貿易與供需。台灣省農業統計要覽。270-271頁。
王騰旭。2000。由大豆油脫臭蒸餾物分離植物固醇之研究。國立臺灣大學農業化學研究所碩士論文。
楊祐銘。2004年。Thermobifida fusca NTU22 β-葡萄糖苷酶基因在大腸桿菌中之表現。國立台灣大學微生物與生化學研究所碩士論文。
羅之綱。1998。利用分枝桿菌由穀固醇生產男性荷爾蒙睪固酮之研究。國立臺灣大學農業化學研究所博士論文。
Achenbach H. and H. Hemrich. 1991. Alkaloids, flavonoids and phenylpropanoids of the West African plant Oxymitra velutina. Phytochem., 30: 1265-1267.

延伸閱讀