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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/30294

Title: Alginate Lyase 之基因轉殖至Lactococcus lactis NZ3900表現及Alginate Lyase所產寡醣產物分析和用於酵母菌發酵生產乙醇之探討
Study on Cloning and Expression of Pseudomonas vesicularis MA103 Alginate Lyase Gene on Lactococcus lactis NZ3900, and the Analysis of Oligosaccharides Produced by Alginate Lyases, and Their Use on Ethanol Fermentation by Yeasts
Authors: Yi-Shan Yang
楊依珊
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 轉殖;褐藻膠裂解酶;乳酸菌;乙醇
Transformation;Alginate lyase;Lactic acid bacteria;Ethanol
Date: 2011
Issue Date: 2011-11-25T07:38:12Z
Abstract: 本研究目的在探討alginate lyase rpalIII 之 palIII基因構築至乳酸菌(nisin controlled expression system, NICE) 表現系統之 pNZ8149載體上,再以電穿孔法轉殖至 Lactococcus lactis NZ3900 中,並測試轉殖株 Lc. lactis NZ3900 rpalIII 之質體穩定性及以nisin 誘導下其所產 alginate lyase 之酵素活性變化。並探討菌株 MA103 之crude alginate lyases 降解褐藻膠所得的寡醣產物,以及用P. vesicularis MA103粗酵素液、纖維素酶、和酸水解等製程製備馬尾藻熱萃多醣水解液用於酵母菌發酵生產乙醇產率之變化。 乳酸菌之轉殖在目標 pal III基因引子對之兩端分別加入 SpeI 和 XbaI 限制酶切位 (alg-pNZ-for 和 alg-pNZ-rev2),以 alginate lyase rpal III 質體為模板經 PCR擴增。成功擴增之 palIII基因以 TA-cloning 方式與 pGEM-T vector 組合之重組載體I轉殖入 E. coli DH5,將此重組載體純化後和乳酸菌載體pNZ8149以SpeI 、 XbaI 限制酶剪切,並透過接合反應形成乳酸菌之重組載體II後,電穿孔轉殖至 Lc. lactis NZ300 中,在 BCP-lactose 洋菜平板上培養基上篩選出呈現 lac+ (菌落周圍為黃色) 之成功之轉殖株。將轉殖成功之重組 palII 基因定序,經比對確認其長度 (1,041 bp) 及核苷酸序列無誤,並命名為Lc. lactis NZ300 rpalIII。測試 Lc. lactis NZ300 rpalIII 於含有 0.5% lactose 之 M17 broth 中連續培養 5 天後,其質體保有率仍可維持在 90% 以上。且以 1 ng/mL 濃度之 nisin 在 30oC 下誘導 3 hr 時呈現較佳的 alginate lyase 胞內酵素活性 (0.095 U/mg protein),經 SDS-PAGE 分析結果顯示約在 40 kDa 處有被誘導出之蛋白質條帶的產生。 使用經 0.3% 市售褐藻膠誘導菌株 MA103 之crude alginate lyases 水解褐藻膠,所得寡醣水解物 (< 3 kDa) 經Sephadex G25 膠體過濾層析 (gel permeation chromatography, GPC),可收集到 5 個寡醣產物 (peaks A1、A5、A6、A7、與 A8)。以市售 alginate lyase 經相同處理程序後,可得到 3 個寡醣產物 (peaks B3、B4、與B5)。區分之寡醣經薄層層析 (thin layer chromatography, TLC) 以及高效能液相層析 (high performance liquid chromatography, HPLC) 分析後推測兩種水解酵素皆可產生聚合度 (degree of polymerization, dp) 為3 (A6 與 B5) 和 4 (A5 與 B4) 之褐藻膠衍生寡醣。測試寡醣產物之抗氧化活性發現 A7 之寡醣具有最佳的清除 DPPH 自由基率 (62.16%) 和螯合亞鐵離子能力 (86.19%)。 最後探討以0.3% 褐藻膠誘導菌株 MA103之crude alginate lyases (6.75-6.83 U/mL) 、 3% 纖維素酶 (88 U/g) 以及0.4 M HCl 降解 5% 馬尾藻熱萃多醣液,所得之酵素水解液用於酵母菌發酵生產乙醇試驗,獲得乙醇最佳產率為13.33%,發酵前後還原醣利用率高達 51.07%。
The aim of this study is using palIII gene from alginate lyase rpalIII construct on LAB nisin controlled expression (NICE) system vector, pNZ8149, then cloned into Lactococcus lactis NZ3900 by electroporating; and testing the vector stability of Lc. lactis NZ3900 rpalIII and its alginate lyase activity induced by nisin. And investigate the lytic products of alginate from crude alginate lyases of Pseudomonas vesiculars MA103. Also, strain MA103 crude enzyme, cellulase, and acid were used to produce hydrolyzates from polysaccharide by hot water extraction from Sargassum, then using these hydrolyzates to evaluate the yield of ethanol fermentation by yeasts. The palIII forward primer encode SpeI restriction site and the reverse primer encode XbaI restriction site. Then using the gene of alginate lyase rpalIII as a template for PCR reaction, and this PCR amplified product as ligated into pGEM-T vector, and then transformed into the competent cells of E. coli DH5α. Then pGEM-T vector, which carrying the cloned palIII gene, was digested with SpeI and XbaI, and ligated into the LAB vector pNZ8149. The recombined plasmid (pNZ8149-palIII) was electroporated into the Lc. lactis NZ3900. The yellow colonies were selected on the BCP lactose agar plates. It was proved that the sequence of the cloned DNA fragments was completely identical to the DNA sequence of the palIII gene (1,041 bp), and the succeed clone was designated as Lc. lactis NZ300 rpalIII. After alignment, Lc. lactis NZ300 rpalIII was tested for the stability of transformed gene by continuously transferring in M17L and M17G broth for 5 days. The results showed that this clone could maintain over 90% cells still bearing the cloned recombinant plasmid. The clones induced by 1 ng/mL nisin at 30oC for 3 hr could obtain higher alginate lyase activity (0.095 U/mg protein), which revealed a 40 kDa protein band by SDS-PAGE. While 0.3% alginate was used to induced crude alginate lyases from strain MA103 to digested alginate, and the lytic oligosaccharides (< 3 kDa) were separated by Sephadex G25 gel filtration to obtain peaks A1, A5, A6, A7, and A8. Besides, commercial alginate lyase could get 3 lytic oligosaccharides (peaks B3, B4, and B5) by the same procedures. Both of these two degradation could produce degree of polymerization (dp) 3 (A6 and B5) and dp 4 (A5 and B4) lytic oligosaccharides, which analyzed and determined by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The experiment on antioxidant activity of lytic oligosaccharides revealed peak A7 have better DPPH scavenging effect (62.16%) and Fe2+ chelating effect (86.19%). While using the induced crude alginate lyases (6.75-6.83 U/mL) of strain MA103, 3% cellulase (88 U/g), and 0.4 M HCl to hydrolyze 5% hot water polysaccharide extracts of Sargassum, the obtained hydrolyzates were utilized as substrates for ethanol fermentation by yeasts. In this Sargassum bioethanol production experiment, the best ethanol yield rate was 13.33% and the reducing sugar utilization rate was 51.07%.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M98320049
http://ntour.ntou.edu.tw/handle/987654321/30294
Appears in Collections:[食品科學系] 博碩士論文

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