富馬酸生產菌米根霉的高效選育

生物工程論文編號:SW160  論文字數:10028,頁數:30

目  錄
中文摘要 I
英文摘要 Ⅱ
目錄 Ⅲ
1. 緒論 1
 1.1  富馬酸的理化性質 1
 1.2　富馬酸的應用 2 
 1.3　富馬酸的生產進展 2
 1.4　米根霉發酵生成富馬酸的代謝途徑 3
 1.5　誘變育種 4
 1.6  本論文主要研究內容 7
2. 實驗部分 8
 2.1  實驗材料 8
 2.2  實驗方法 9
 2.3  分析方法 9
 2.4  主要試劑 10
 2.5  主要實驗設備與儀器 11
 2.6  富馬酸高產菌株的選育 11
3. 結果與討論 13
 3.1   獲得單菌落 13
 3.2　指示劑的選擇 13
 3.3　KMnO4滴定結果 17
 3.4　出發菌株產富馬酸測定 18
 3.5　誘變劑的劑量選擇 18
 3.6　米根霉在搖瓶中發酵 19
 3.7　篩選方法評估 19
 3.8　誘變菌株的產酸量測定 19
 3.9　小結 21
4．總結與展望 22
 4.1  結論與創新點 22
 4.2  展望 23
致謝 24
參考文獻 25

 摘 要：本文以實驗室自行保存的富馬酸生產菌米根霉ZJU作為初始菌株，通過傳統的紫外誘變，結合簡單有效的平板篩選，輔以培養基的優化，大幅度提高了富馬酸生產菌的產酸能力。
 建立了適合于產酸絲狀真菌誘變選育的變色圈平板初篩法，大大降低了誘變育種后高產菌株選育的工作量。以實驗室儲藏的米根霉作為出發菌株，初始富馬酸產量為29.8g/l，經UV反復誘變處理后，應用上述方法篩選得到了一株富馬酸高產菌株－米根霉，在出發菌株的最佳發酵條件下富馬酸產量為50.9g/l,比原始菌株提高了70.8％。對突變菌株的培養基進行了優化，發現突變菌株和原始菌株最佳培養基組成相似，最佳培養溫度為35℃，初始搖床轉速100rpm，到大量產酸時轉速升至200rpm。培養基中需要加入0.6g碳酸鈣以控制pH值。
關鍵詞：富馬酸；米根霉；紫外誘變；制霉菌素

Abstract: A systematic investigation was carried out to improve the productivity of fumaric acid producing strain by UV irritation and bioprocess optimization. During the selection procedure, a simple way of screening for high-yield fumaric acid producing strain was developed and eva luated, in which, area ratio of area of colored ring to that of colony was designed as an indicator.
 Strain improvement has been conventionally achieved through mutation and selection. A fumaric acid over-producing strain mutant, Rhizopus oryzae , was isolated by mutagenizing the parent strain by UV mutagenesis. Starting with an optimized concentration of 29.8 g/l glucose, the mutant Rhizopus oryzae ZJU11 produced 50.9 g/l fumaric acid in shaking-flask fermentation , which were 70.8% higher than that of the parent strain produced respectively.
 The influences of nitrogen sources, carbon sources, concentration of CaCO3 and its addition method CaCO3 in culture medium on fumaric acid production by the mutant Rhizopus oryae ZJU11were investigated.
Keywords：Fumaric acid; Rhizopus oryzae; UV irritation; optimization; Nystatin