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研究生:范宜琮
研究生(外文):Van Yi Tsong
論文名稱:以苔蘚桿菌生產聚麩胺酸之研究
論文名稱(外文):Study of production of γ-Poly glutamic acid by Bacillus licheniformis
指導教授:施英隆施英隆引用關係
指導教授(外文):Shih-Ing-Lung
學位類別:碩士
校院名稱:大葉大學
系所名稱:環境工程研究所
學門:工程學門
學類:環境工程學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:97
中文關鍵詞:聚麩胺酸回應曲面法苔蘚桿菌
外文關鍵詞:γ-Poly(glutamic acid)Response surface MethodologyBacillus licheniformis
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中文摘要
培養基E(麩胺酸20g/L,檸檬酸12g/L,甘油80g/L,NH4Cl 7g/L,MgSO4 7H2O 0.5g/L,FeCl3 6H2O 0.04g/L,K2HPO4 0.5g/L,CaCl2 2H2O 0.15g/L,MnSO4 H2O 0.04g/L)為多株桿菌生產聚麩胺酸之最適培養基。本研究發現苔蘚桿菌(Bacillus licheniformis) CCRC12826在培養基E中可生產聚麩胺酸5.23g/L,此與其他桿菌株於相同培養基所生產之聚麩胺酸產量相差甚距。當探討以其他碳氮源或他們不同組合取代培養基E之碳氮源結果發現麩胺酸、檸檬酸、甘油與氯化銨仍是Baccillus licheniformis CCRC12826生產聚麩胺酸之最佳碳氮源。因此本研究選定麩胺酸,檸檬酸甘油與pH等四個自變因子,並以回應曲面法探討此四因子對Bacillus licheniformis CCRC12826生產聚麩胺酸之影響,經由一階回應區面實驗設計結果,我們發現在培養基中麩胺酸、檸檬酸、甘油對聚麩胺酸產量有顯著的影響性而pH值則無影響性,就單因子對聚麩胺酸的影響以甘油最為顯著(當濃度增加),其次是麩胺酸及檸檬酸,在因子間之交互影響性對聚麩胺酸產量,以甘油與麩胺酸交互影響最為顯著(當濃度增加),由一階回應曲面設計法所得之結果,再以一階實驗之中心點(原點)加上實際爬升距離進行陡升路徑實驗。陡升路徑之最高點為檸檬酸17.9g/L、麩胺酸55.4g/L、甘油148.4g/L、因此以此為新原點進行中心混成實驗設計由中心混成實驗之數據進行二階模式的迴歸分析。此一模式對於描述實驗數據之適切程度,可由變異數分析加以檢驗,由Fisher’s F檢驗之具有顯著性(p<0.05),且檢定係數R2值達0.9078,誤差項中模式契合不良(Lack of fit)項不顯著(p>0.05),皆表示所迴歸得到的二次多項式模式適切甚佳。
本研究先以培養基E探討Bacillus licheniformisCCRC12826生產聚麩胺酸,進而以回應曲面法探討最適產量,從一階回應曲面實驗設計、陡升路經實驗設計到二階回應曲面實驗設計(中心混成實驗設計),聚麩胺酸產量從5.27g/L(Medium E)增至最終之21g/L(二階),產量增加了300%,而麩胺酸、檸檬酸、甘油三碳源之最佳培養基為麩胺酸(65g/L)、檸檬酸(22g/L)、甘油(170g/L);以回應曲面法探討聚麩胺酸生產之最適培養基,確實為有效的方法。
Abstract
Medium E(L-glutamic acid 20g/L,citric acid 12g/L,glycerol 80g/L, NH4Cl 7g/L, MgSO4 7H2O 0.5g/L, FeCl3 6H2O 0.04 g/L, K2HPO4 0.5 g/L, CaCl2.2H2O 0.15 g/L, and MnSO4 H2O 0.04 g/L) is a medium of choice for many researchers investigating production of poly-γ-glutamic acid(γ-PGA)by Bacillus species. When Bacillus licheniformis CCRC12826 was cultivated in medium E, the amount of γ-PGA produced was only 5.27g/L, which is much less than that produced by other Bacillus species in the medium. When glutamic acid、citric acid、glycerol and ammonium chloride were substituted by other carbon and nitrogen sources, it was found that the production of γ-PGA by B.lciheniformis CCRC12826 decreased significantly. It was concluded that glutamic acid、citric acid、glycerol and ammonium chloride were still the most suitable carbon and nitrogen sources for the production of γ-PGA by Bacillus licheniformis CCRC12826, Response surface methodology(RSM) was employed to study the effect of glutamic acid, citric acid, glycerol and initial medium pH on the production of γ-PGA by Bacillus licheniformis CCRC12826 shaken cultures.
The result of first order RSM showed that the liner terms of glutamic acid, citric acid and glycerol had significant positive effect, but the initial medium pH exhibited no significant effect on γ-PGA. The effects decreased in the order of glycerol, glutamic acid and citric acid. In addition, the interaction term of glutamic acid-glycerol exhibited a significant positive effect. Based on the results of the first-order RSM, the path of steepest ascent was determined to bring the factors level to more close to their optimum level. The results of the path of steepest ascent showed that the highest production response was 14.35 g/L when the concentration of citric acid, glutamic acid and glycerol was 17.9g/L, 55.4g/L, 148.4 g/L respectively. After having climbed up to response surface to regions of higher production response, the optimum composition was then investigated by using a central composite design (CCD). The experimental results of CCD were fitted with a second-order polynomial equation by a multiple regression analysis. The goodness of fit of the model was checked by several criteria. The determination coefficient R2=0.9078 indicated that 9% of the total variance is not explained by the model。 The Fisher F-test is significant at upper 5% level,and the lack of fit is insignificant at 5% level。 All of these indicate a good adequacy of the second-order polynomial model。
The results of this study have show that there is significant 300%, from 5.27g/L to 21g/L, improvement of γ-PGA production by Bacillus licheniformis CCRC12826 through RSM. The maximal γ-PGA yield(21g/L) appeared at the regions where respective concentrations of glutamic acid、citric acid and glycerol were around 65 g/L、22 g/L、170 g/L respectively。
目 錄
頁次
封面內頁
簽名頁
博碩士論文電子檔上網授權書iii
大葉大學碩士論文全文授權書iv
中文摘要 v
英文摘要……………………………………………………………….vii
誌謝ix
目錄x
圖目錄………………………………………………………………xiii
表目錄……………………………………………………………….xv
第一章 緒論1
第二章 文獻回顧4
2.1 聚麩胺酸之生合成4
2.2 需使用L-麩胺酸之菌株7
2.2.2 Bacillus licheniformis ATCC9945a7
2.2.3 Bacillus subtilis IFO3335 ...13
2.3 不需用L-麩胺酸之菌株…………………………………...16
2.3.1 Bacillus subtilis TAM-4...16
2.3.2 Bacillus lichenifromis A35...17
2.4 聚麩胺酸之應用……………………………………………18
2.4.1 聚麩胺酸在生醫材料(Biomaterial)上應用……………...19
2.4.2 聚麩胺酸在抗癌藥物(Antitumor drug)的應用………….20
2.4.3 聚麩胺酸在環境工程上的應用………………………….22
2.4.4 聚麩胺酸處理廢水中重金屬…………………………….22
2.4.5 聚麩胺酸在食品上的應用……………………………….23
2.4.6 聚麩胺酸在化妝品上的應用…………………………….23
2.4.7 回應區面法……………………………………………….24
2.4.8 二水準因子設計實……………………………………….25
2.4.9 陡升路徑法..……………………………………………...26
2.4.10中心混成設計..…………………………………………..26
2.4.11 變異數分析法………………………..………………….27
第三章 利用苔顯桿菌生產聚麩胺酸之搖瓶培養探討…………...31
3.1 前言…………………………………………………………31
3.2 實驗材料……………………………………………………31
3.2.1 器材……………………………………………………….31
3.2.2 藥品……………………………………………………….32
3.2.3 菌株……………………………………………………….32
3.2.4 實驗步驟………………………………………………….33
3.2.4.1 預培養…………………………………………………..33
3.2.4.2聚麩胺酸產量探討……………………………………...33
3.2.5 聚麩胺酸純化…………………………………………….34
3.3 培養基中碳源消耗分析……………………………………36
3.3.1 檢量線製備……………………………………………….36
3.3.2 碳源之HPLC分析條…………………………………… 36
3.3.3 培養基中麩胺酸、檸檬酸濃度計算…………………….36
3.4 結果與討論…………………………………………………41
3.4.1 培養基中之最適碳氮源研究…………………………….41
3.4.2 搖瓶培養基中碳源酸鹼值黏度隨時間變化之情形…….41
第四章以回應區面法生產聚麩胺酸之碳源探討………………….48
4.1 前言………………………………………………………...48
4.2 材料與方法…………………………………………………...49
4.3 實驗材料……………………………………………………...49
4.3.1 培養方法……………………………………………………49
4.4 回應區面設計………………………………………………...50
4.4.1 一階因子實驗設計…………………………………………50
4.5 陡升路徑之實驗設計………………………………………...51
4.6 中心混成設計之實驗………………………………………...51
4.7 回應區面之模式設計適切性之統計檢驗…………………...51
4.8 結果與討論…………………………………………………...55
4.8.1 部分因子實驗設計…………………………………………55
4.8.2 一階回應區面模式適切性之統計檢驗……………………56
4.8.3 陡升實驗設計結果…………………………………………56
4.8.4 中心混成實驗設計結果……………………………………57
4.9 聚麩胺酸特性分析…………………………………………...80
第五章結論與建議……………………………………………………87
參考文獻………………………………………………………………90
圖 目 錄
頁次
圖2.1 聚麩胺酸結構4
圖2.2 TCA循環 12
圖2.3 Bacillus subtilis IFO3335合成γ-PGA可能生合成路………15
圖2.4 回應區面圖29
圖2.5 回應區面進行步驟流程圖…………………………………….30
圖3.1 麩胺酸檢量線………………………………………………….37
圖3.2 檸檬酸檢量線.…………………………………………………38
圖3.3 高效液相層析儀分析麩胺酸、檸檬酸標準圖譜…………….39
圖3.4 培養112小時後,以高效液相層析儀分析培養基之圖譜……40
圖3.5 培養基中碳源消耗曲線……………………………………….45
圖3.6 γ-PGA產量與黏度對培養時間之關係圖….………………..46
圖3.7 培養基之pH變化 ……………………………………………47
圖4.1 延著最陡上升路徑各步階之γ-PGA產量 ………………….64
圖4.2 檸檬與麩胺酸對生產聚麩胺酸產量之一階回應曲面圖與
等高線圖…………………………………………………….69
圖4.3 檸檬與甘油對生產聚麩胺酸產量之一階回應曲面圖與等
高線圖……………………………………………………….70
圖4.4 檸檬與pH對生產聚麩胺酸產量之一階回應曲面圖與等
高線圖…………………………………………………….…71
圖4.5 麩氨酸與甘油對生產聚麩胺酸產量之一階回應曲面圖與
等高線圖…………………………………………………….72
圖4.6 麩氨酸與pH對生產聚麩胺酸產量之一階回應曲面圖與
等高線圖………………...…………………………………..73
圖4.7 甘油與pH對生產聚麩胺酸產量之一階回應曲面圖與等
高線圖……………………………………………………….74
圖4.8 一階RSM實驗聚麩胺酸的產量觀測值與實際值之比較..75
圖4.9 一階RSM實驗之聚麩胺酸產率之期望值與殘差值之常態
機率圖……………………………………………………….75
圖4.10 麩胺酸與檸檬酸對生產聚麩胺酸產量之二階回應區面圖
與等高線圖………………………………………………...76
圖4.11 檸檬酸與甘油對生產聚麩胺酸產量之二階回應區面圖與
等高線圖…………………………………………………...77
圖4.11 麩胺酸與甘油對生產聚麩胺酸產量之二階回應區面圖與
等高線圖…………….…………………………………….78
圖4.12二階回應曲面實驗聚麩胺酸實際產率與預測值之比較...79
圖4.13 二階回應曲面實驗聚麩胺酸產率之期望值與殘差值之常
態機率圖…………………………………………………..79
圖4.15 Dextran經由膠體穿透層析儀分析之圖譜………………..81
圖4.16 聚麩胺酸經由膠體穿透層析儀分析之圖譜……………...82
圖4.17 聚麩胺酸水解氨基酸鑑定圖譜…………………………...83
圖4.18 高效液相層析儀分析聚麩胺酸之標準品………………...84
圖4.17高效液相層析儀分析聚麩胺酸純化透析後之圖譜.………85
圖4.20照片(一)為聚麩胺酸之水溶液,照片二為純化後之聚麩胺
酸.……………………………………………………………86
表 目 錄
頁次
表2.1 目前生產聚麩胺酸之菌株及碳、氮源之比較6
表2.2 Bacillus licheniformis ATCC9945a之培養基組成8
表2.3 聚麩胺酸可能應用之領域18
表3.1 碳源對以Bacillus licheniformis CCRC12826生產聚麩氨酸之
影響.……………………………………………………………35
表3.2 氮源對以Bacillus licheniformis CCRC12826生產聚麩氨酸之
影響…….………………………………………………………35
表3.3 搖瓶培養基中碳源酸鹼值黏度隨時間變化之情形………….44
表4.1 24實驗設計之變數及層階53
表4.2 陡升路徑實驗設計54
表4.3 中心混成實驗設計之變數及其層階………………………….55
表4.4 一階回應曲面設計之結果60
表4.5 24因子設計結果之複迴歸分析表61
表4.6 24因子設計結果之變異數分析表(ANOVA table)62
表4.7 一階回應曲面實驗設計經由迴歸分析觀測值與殘差值之比
較……………………………………………………………….63
表4.8 陡升路徑實驗設計之結果64
表4.9 中心混成實驗設計之結果65
表4.10中心混成實驗之複迴歸分析表……………………………….66
表4.11中心混成實驗之變異數分析表67
表4.12 二階回應曲面實驗設計之實驗值與經由迴歸分析殘差值
之比較……………………………………………..………….68
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