以乳酸桿菌Lactobacillus reuteri Pg4表達內酯水解酶Zhd101以降解玉米烯酮毒素

Expression of lactonohydrolase Zhd101 for detoxifying zearalenone by Lactobacillus reuteri Pg4

楊雯鈞(Wen-Chun Yang)

指導教授 ： 鄭光成

共同指導教授 ： 劉嚞睿

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.10579

摘要

玉米烯酮（zearalenone ; ZEN）為Fusarium產生的一種類雌激素黴菌毒素，利用酵素水解玉米烯酮毒素可有效減少毒素含量，由Clonostachys rosea中選殖到的zhd101即為一可表現具有分解玉米烯酮的内酯水解酶（lactonohydrolase）的基因。應用益生菌做為表現宿主除了其安全性高、耐酸、耐膽鹽和腸道吸附性以外，也具很高的便利性，本實驗中所使用的乳酸桿菌為由肉雞腸道中分離出的Lactobacillus reuteri Pg4，已證實具有益生菌之特性。將zhd101基因構築於乳酸菌-大腸桿菌穿梭載體pNZ3004，並利用電穿孔轉形獲得L. reuteri pNZ-zhd101轉形株，在DNA、RNA及蛋白質層次可證實重組菌株具表達Zhd101之能力；以高效能液相層析儀進行酵素活性檢測，由於乳酸菌生長代謝產生酸會使Zhd101酵素失活，因此藉由發酵槽控制pH值，結果顯示重組乳酸菌之生長相較於L. reuteri Pg4慢，但在pH 7的環境下具有降解ZEN的活性，質體及基因的存在率在發酵14小時後分別為91%及100%。本實驗成功利用重組乳酸桿菌L. reuteri pNZ-zhd101表達玉米烯酮內酯水解酶Zhd101，利用發酵槽放大培養的轉形株具有降解玉米烯酮的能力而且表現高度穩定度。

關鍵字

玉米烯酮 ； 內酯水解酶 ； 乳酸桿菌

並列摘要

Mycotoxins are fungal secondary metabolites and contaminate agricultural products during crops growing, harvest, transportation, processing, or storage. Zearalenone （ZEN） is a nonsteriod estrogenic mycotoxin, produced by Fusarium species, and has been frequently implicated in reproductive disorders of farm animals and occasionally in hyperoestrogenic syndromes in humans. Biological, chemical, and physical detoxification procedures are trying to figure out mycotoxin contaminations by absorption or biotransformation, and enzymatic detoxification could be an efficient method for ZEN detoxification. ZEN-detoxifying gene, zhd101, is isolated from Clonostachys rosea IFO 7063. Zhd101 converts ZEN to the nonestrogenic product. Maximal activity of Zhd101 toward ZEN was measured at pH 10.5 and 37 to 45°C. Lactobacillus reuteri Pg4 is a lactic acid bacteria isolated from the gastrointestinal tract of broilers and possesses probiotic characteristics, so it has great potential for application. This study is aimed to construct expression vector with zhd101 and electroporation into L. reuteri Pg4 to express Zhd101. The zhd101 gene was cloned into Escherichia coli-Lactobacillus shuttle vector, pNZ3004, and the plasmid pNZ-zhd101 was electoporotion into L. reuteri Pg4. Zhd101 was successfully expressed by recomninant L. reuteri pNZ-zhd101 at DNA, mRNA, and protein levels. L. reuteri pNZ-zhd101 transformants showed ZEN degradation activity at pH 7.0 and highly plasmid stability.

並列關鍵字

zearalenone ； lactonohydrolase ； Lactobacillus

參考文獻

廖建華。2013。應用乳酸桿菌表達家禽流行性感冒病毒之血球凝集素並評估其益生菌特性及對雞隻之疫苗功效。碩士論文。國立台灣大學動物科學技術學系。

Abid-Essefi, S.; Baudrimont, I.; Hassen, W.; Ouanes, Z.; Mobio, T. A.; Anane, R.; Creppy, E. E.; Bacha, H. DNA fragmentation, apoptosis and cell cycle arrest induced by zearalenone in cultured DOK, Vero and Caco-2 cells: prevention by Vitamin E. Toxicology. 2003, 192, 237-248.

Ahamed, S.; Foster, J. S.; Bukovsky, A.; Wimalasena, J. Signal transduction through the Ras/Erk pathway is essential for the mycoestrogen zearalenone-induced cell-cycle progression in MCF-7 cells. Mol. Carcinog. 2001, 30, 88-98.

Alvarez, M. A.; Herrero, M.; Suárez, J. E. The site-specific recombination system of the Lactobacillus species bacteriophage A2 integrates in gram-positive and gram-negative bacteria. Virology. 1998, 250, 185-193.

Anandharaj, M.; Sivasankari, B.; Parveen R. R. Effects of probiotics, prebiotics, and synbiotics on hypercholesterolemia: a review. Chinese J. Biol. 2014, 2014.