香葉草醇10-羥基酶之異源表現之研究

本研究的目標為建構一個微生物表現系統進行香葉草醇10-羥基酶 (G10H) 的異源表現。柚皮素 (naringenin) 可經G10H轉化成具有抗氧化以及抗腫瘤增殖活性的藥物聖草酚 (eriodictyol) 因而提高其經濟價值，使得大量表現G10H酵素具有工業生產的潛力。本研究中嗜甲醇酵母菌P. pastoris以及大腸桿菌E. coli 被選作表現的宿主，其分別使用pPICZaA以及pQE-30 Xa作為表現質體進行表現。在嗜甲醇酵母菌X33以及SMD1168品系表現G10H的研究結果顯示，無法以SDS- PAGE測得G10H。由於當初建構表現質體時為了顧及酵素折疊構形的正確性以及確保酵素的完整活性，沒有將6 x His-tag接於G10H上。現階段的結果顯示所建構的轉殖嗜甲醇酵母菌沒有達成表現G10H的目的。進一步重新建構表現質體以使用像是組胺酸標定 (His-tag) 的抗體偵測系統能較敏感和專一性地測定目標蛋白。而轉殖宿主能進一步以抗生素梯度篩選以取得高拷貝數的基因轉殖宿主。以大腸桿菌作為原核表現系統也於本研究中進行探討。大腸桿菌JM109以及M15轉殖株中均可以抗體標定偵測到外源蛋白的表現。宿主有效調控噬菌體T5啟動子是影響G10H的誘導表現量的重要因子。M15轉殖株儘管過量表現lacI蛋白，噬菌體T5啟動子於未添加誘導劑的情況下仍然會表現下游基因，因而持續性表現外源蛋白。這現象對於宿主細胞造成代謝上的負擔。大量表現外源蛋白只能在M15品系的轉殖株破菌後的不可溶蛋白劃分中測得。高效能液相層析儀分析G10H酵素活性的研究發現，目前的分析系統無法在嗜甲醇酵母菌或是大腸桿菌的轉殖株破菌抽出物中成功將柚皮素轉化生成聖草酚。本研究目前找到的大腸桿菌M15轉殖株的最適生長以及誘導的溫度為25oC，而最適合的誘導條件為以0.01 mM IPTG 進行誘導9個小時。

關鍵字

大腸桿菌；嗜甲醇酵母菌；香葉草醇10-羥基酶柚皮素；聖草酚

並列摘要

The aim of this research was to establish microbial hosts expression of economically valuable protein geraniol 10-hydroxylase (G10H). Naringenin was one of the substrates of G10H, which could be catalyzed into eriodictyol. Eriodictyol served as an antioxidant and was found to have antiproliferation activity against tumor cells. Pichia pastoris and Escherichia coli were chosen as expression hosts of G10H by using pPICZaA and pQE-30 Xa as expression vector respectively. During expression of G10H in P. pastoris host strains X33 and SMD1168, the expression level could not reach a level to be confirmed by SDS-PAGE. An antibiotic gradient during transformant selection to obtain high copy number transgenic strains and a more sensitive and specific detection system such as using His-tag detection were needed. Procaryotic expression system using E. coli stains JM109 and M15 were also examined. A tight regulation of the phage T5 promoter was an important factor for high induction expression of G10H. M15 transgenic strains had a leakage in phage T5 promoter inhibition even when lacI was overexpressing. The constitutive expression of foreign protein was presumed to cause a metabolic burden. A high level accumulation of foreign proteins could only be achieved in insoluble protein fractions of M15 transformants. High performance liquid chromatography analysis of G10H enzyme activity in vitro failed to detect eriodictyol production for all P. pastoris and E. coli transfomants. The results so far indicated that the optimum incubation and induction temperature for M15 transformants was 25oC, and 0.01 mM IPTG induction for 9 h was the most adequent condition for G10H expression.

並列關鍵字

Escherichia coli ； Pichia pastoris ； geraniol 10-hydroxylase ； naringenin ； , eriodictyol

參考文獻

鄭伊芳，2006。嗜高溫放線菌木聚糖酶基因以及人類芳香酶基因在嗜甲醇酵母菌中之表現。國立台灣大學微生物與生化學研究所博士論文。

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香葉草醇10-羥基酶之異源表現之研究

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