嗜高溫放線菌木聚糖酶基因及人類芳香酶基因在嗜甲醇酵母菌中之表現

Studies on the expression of xylanase gene from thermophilic actinomycete and human aromatase gene in Pichia pastoris

鄭伊芳(Yi-Fang Cheng)

指導教授 ： 劉文雄

國立臺灣大學/生命科學院/微生物與生化學研究所/博士(2006年)

https://doi.org/10.6342/NTU.2006.01744

摘要

本研究之目的可分為兩部分，第一部分為耐熱性木聚醣酶之生產，第二部分則為以生物轉換法生產雌激素，其主要結果分述如下：
將嗜高溫放線菌Thermobifida fusca NTU22之木聚糖酶基因(xyl11)，藉由表現載體pPICZαA於酵母菌Pichia pastoris KM71H中進行外泌性表現。基因重組酵母菌P. pastoris (pPICαXYL6) 在50 mL Hinton氏搖瓶培養中，經由甲醇誘導96小時之後，其胞外木聚醣酶活性可達88.29 U/mL。於5 公升醱酵槽中，以工作體積為2.0公升、通氣量3 L/min、攪拌速率500 rpm、培養溫度30℃及酸鹼值5.0之條件下，以basal salt medium及甘油饋料進行菌體生長，經過144小時的甲醇誘導可於胞外測得最高木聚糖酶活性為324.2 U/mL。探討P. pastoris (pPICαXYL6) 木聚糖酶酵素性質，得知其最適作用pH值及最適作用溫度分別為6.0-7.0及70℃，而酵素活性可於pH值6.0-9.0之間維持穩定，此外，酵素於70℃作用3小時仍維持70% 酵素活性。醱酵液經由限外過濾濃縮、Sepharose CL-6B管柱之層析純化，其純化倍率提高6.9倍，回收率為23%。純化之木聚糖酶經由酵素去醣基化分析，得知其具有多重醣基化之現象。
由一人類胎盤cDNA library中選殖出芳香酶基因，並藉由胞內表現載體pPICZB於酵母菌P. pastoris KM71H中進行表現。基因重組酵母菌P. pastoris (pAro3)之粗酵素液經離心區分並進行免疫染色的結果，確認人類芳香酶表現於胞內微粒體膜上，且經由酵素去醣基化分析結果，得知表現於酵母菌中之人類芳香酶大部分帶有醣基。於50 mL Hinton氏搖瓶培養中，不論以雄烯二酮或是睪固酮為基質，經P. pastoris (pAro3) 轉換所得之唯一雌激素產物經薄層層析、高效液相層析光電二極體陣列分析儀及氣相層析質譜分析儀確認為雌一酮。
P. pastoris (pAro3) 於500 mL Hinton氏搖瓶中，以甘油為碳源培養16小時後，分別以睪固酮與雌二醇為反應基質，於胞內粗酵素液中可測得17β-羥基類固醇去氫酶之活性為61.54 U/g及 56.41 U/g。經離心區分得知17β-羥基類固醇去氫酶為可溶性細胞質蛋白。粗酵素液以還原態聚丙烯醯胺電泳分析之後，分別以睪固酮及雌二醇為反應基質進行活性染色，兩反應基質之活性染色結果均呈現單一、且其相對位置一致之色帶
於50 mL Hinton氏搖瓶中，檢討P. pastoris (pAro3)轉換生產雌激素之最適條件，得知以50 mg/L雄烯二酮為基質、酸鹼值為6及1.0 %甲醇持續誘導120小時後，雌一酮的生成量可達0.060 mM，其轉換率為34.56%。探討以兩階段之生物轉換生產雌激素之研究中，先以Mycobacterium NRRL B-3805為生產菌株，於含1 g/L 膽固醇之培養基中，於30℃下進行第一階段之生物轉換96小時後，可得雄烯二酮及睪固酮之最高生成量分別為1.970 mM及0.066 mM。上述培養液經由高溫蒸氣滅菌處理後，以P. pastoris (pAro3) 及雌激素最適轉換生產條件進行第二階段之生物轉換。經由甲醇誘導120小時後之雌一酮生成量為0.054 mM，其轉換率為30.73%。
於5 公升醱酵槽中，以工作體積2.0公升、通氣量3 L/min、攪拌速率500 rpm及培養溫度30℃之條件下，以basal salt medium及甘油饋料進行菌體生長。進入甲醇誘導階段後調降通氣量為1 L/min，且基質雄烯二酮隨著甲醇饋料添加於培養基中，使其終濃度達300 mg/L。經由甲醇誘導168小時後，可得最高雌一酮及雌二醇生成量分別為0.690 mM及0.019 mM，雌激素之總轉換率為67.76%。
以P. pastoris (pAro3)建立芳香酶抑制劑之篩選模式。於24孔培養
皿之篩選模式中，以甲醇誘導後72小時後，添加濃度為1 mM之7-methoxyflavanone及formestane之芳香酶抑制率分別可達100%及63.38%。至於naringenin 及DL-aminoglutethimide均無法偵測到明顯的芳香酶抑制性。

關鍵字

木聚醣酶芳香酶雌激素 ； 嗜甲醇酵母菌

並列摘要

This study was focused on two purposes, the first was to produce thermophilic xylanase, and the second was to produce estrogen via bioconversion. The results were as follows:
A xylanase gene (xyl11) from thermophilic actinomycete Thermobifida fusca NTU22 was secretively expressed in Pichia pastoris KM71H with pPICZαA., The extracellular xylanase activity of a recombinant yeast P. pastoris (pPICαXYL6) was 88.29 U/mL after methanol induction for 96 h in a 50 mL Hinton’s shaking flask. The cultivation of P. pastoris (pPICαXYL6) in a 5-L fermentor loaded 2-L basal salt medium was performed under the conditions as follows: aeration rate, 3 L/min; agitation speed, 500 rpm and pH 5.0 at 30℃. After glycerol feeding and methanol induction for 144 h, the maximum extracellular xylanase activity was 324.2 U/mL in the fermentation broth. The optimum pH and temperature for the xylanase produced by P. pastoris (pPICαXYL6) was 6.0-7.0 and 70℃, respectively. The xylanase was stable at pH 6.0 - 9.0, and 70% of original activity was remained after treated at 70℃ for 30 min. The xylanase of P. pastoris (pPICαXYL6) in fermentation broth was purified 6.9-fold through ultrafiltration concentration and Sepharose CL-6B gel-filtration chromatography. The overall yield of the purified xylanase was 23%. The result of deglycosylation analysis revealed that the xylanase expressed in P. pastoris (pPICαXYL6) was glycosylated in various degrees.
An aromatase gene cloned from a human placenta cDNA library was intracellularly expressed in P. pastoris KM71H with pPICZB. After the crude enzyme of P. pastoris (pAro3) was examined through centrifugation fractionation and immuno-stain assay, the localization of aromatase was identified as microsomal membrane. The result of deglycosylation analysis revealed that most aromatase was glycosylated. No matter androstenedione or testosterone were used as substrate, the only estrogenic product converted by P. pastoris (pAro3) was identified as estrone by TLC, HPLC-PDA and GC-Mass.
By using glycerol as carbon source, P. pastoris (pAro3) was cultivated in a 500 mL Hinton’s shaking flask for 16 h. The 17β-hydroxysteroids dehydrogenase activity of crude extract was determined as 61.54 U/g and 56.41 U/g while using testosterone and estradiol as substrate, respectively. The 17β-hydroxysteroids dehydrogenase was identified as a soluble cytoplasmic protein after centrifugation fractionation. The crude extract was examined on Native-PAGE, and only one band located at the same position was detected by activity stain when testosterone or estradiol was used as substrate.
The optimum culture conditions for production of estrogen by P. pastoris (pAro3) were investigated in a 50 mL Hinton’s shaking flask. It was found that the production of estrone was 0.060 mM with conversion rate of 34.56% using 50 mg/L androstenedione as substrate under pH 6 and 1% methanol induction for 120 h. In the production of estrogen by two-step bioconversion, Mycobacterium NRRL B-3805 was used firstly to convert 1 g/L cholesterol into 1.970 mM androstenedione and 0.066 mM testosterone at 30℃ for 96 h. In the second-step, P. pastoris (pAro3) was used to convert the sterilized Mycobacterium culture broth under the optimum conditions for production of estrogen, and 0.054 mM estrone was produced with conversion rate of 30.73% after methanol induction for 120 h.
The cultivation of P. pastoris (pAro3) in a 5-L fermentor loaded 2-L basal salt medium was performed under the conditions as follows:aeration rate, 3 L/min; agitation speed, 500 rpm and pH 5.0 at 30℃. When the methanol induction phase was initiated, aeration speed was decreased to 1 L/min and androstenedione was added with methanol feeding to a final concentration of 300 mg/L. The maximum production of estrone and estradiol was 0.690 mM and 0.019 mM, respectively. The overall conversion rate of estrogen was 67.79%.
An aromatase inhibitor screening model was built based on P. pastoris (pAro3). In a 24-well plate model, the aromatase inhibition of 1 mM 7-methoxyflavanone and 1 mM formestane was estimated as 100% and 63.38%, respectively, after methanol induction for 72 h. However, no aromatase inhibition of 1 mM naringenin and 1 mM DL-aminoglutethimide was detected.

並列關鍵字

xylanase ； aromatase ； estrogen ； Pichia pastoris

參考文獻

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