纤维素分解酶与聚木醣分解酶基因转型乳酸菌.DOC

纖維素分解酶與聚木醣分解酶基因轉型乳酸菌 發酵植物殘渣生產乳酸之研究 Fermentation of plant debris for lactic acid production by xylanase and cellulase genes transformed lactic acid bacteria 研究生：齊大鈞 Chi, Da-Jiun 指導教授：陳又嘉 Chen, Yo-Chia 【摘要】 乳酸是一種應用廣泛的有機酸，估計全世界每年對乳酸的需求約130,000～150,000公噸，因此如何利用生資源生產乳酸將是重要的方向。本研究利用再生資源-植物殘渣中的聚木糖及纖維素為碳源，分別以聚木醣分解酶及纖維素分解酶基因轉型乳酸菌來進行發酵生產乳酸。本實驗建構出聚木醣分解酶基因轉型株 (Lactococcus lactis R8、Lactbacillus brevis R8) 及纖維素分解酶基因轉型株 (Lb. plantarum CD)，並以剛果紅染色確認其具有分泌出聚木醣分解酶及纖維素分解酶之能力。將L. lactis R8及Lb. brevis R8培養在燕麥聚木醣培養液中，轉型株都可分泌出聚木醣分解酶及β-木糖苷酶，並分解聚木醣產生木醣，轉型株都可利用其產生出相較於野生株更多的菌數，但L. lactis R8無法有效利用木醣產生乳酸，而Lb. brevis R8則可利用木醣產生0.71 g/L乳酸。再將Lb. brevis R8培養在玉米穗軸培養液中，轉型株雖然可分泌出聚木醣分解酶，但無法有效分解玉米穗軸產生還原醣，因此無法利用其產生乳酸。將Lb. plantarum CD培養在葡萄糖及纖維二醣培養液中，發現轉型株代謝葡萄糖及纖維二醣產生乳酸之能力相較於野生株是較差的。將Lb. plantarum CD培養在羧甲基纖維素及甘蔗渣培養液中，轉型株能分泌出活性較差的纖維素分解酶，也可分解羧甲基纖維素及甘蔗渣產生還原醣，轉型株雖然可利用纖維寡醣產生更高之菌數，但卻無法利用其產生乳酸。 關鍵字：纖維素分解酶、乳酸、植物殘渣、聚木醣分解酶 【Abstract】 Lactic acid is one of widely used organic acids. The worldwide demand for lactic acid was estimated to be 130,000 to 150,000 tones per year. For the development of bioresource, it would be an important way to produce lactic acid by using recyclable bioresources as material. In this study, the lignocellulosic materials including xylan and cellulose were used in lactic acid fermentation of xylanase and cellulase gene transformed lactic acid bacteria, respectively. The xylanase gene and cellulase gene were respectively introduced to lactic acid bacteria and obtained their transformants, Lactococcus lactis R8, Lactobacillus brevis R8 and Lb. plantarum CD. The xylanolytic or cellulolytic activity of transformants were confirmed by Congo red staining.L. lactis R8 and Lb. brevis R8 were able to grow in the medium using oat spelts xylan as sole carbon source, the xylose and the activities of xylanase and β-xylosidase would be detected in the broth. The cell density were also much higher than wild type under the same condition, these indicate that xylanase gene would be helpful to lact