醣類之 HPLC 分析結果 ............................................................. 70~78

5. 文獻收集與研讀整理

7.1 醣類分析與測定

7.1.5 醣類之 HPLC 分析結果 ............................................................. 70~78

表D01 醣類之 HPLC 分析結果

醣類滯留時間(R.T.)(min.) 積分面積

木聚糖 (Xylan) 7.348 709051

纖維水解酵素 (Cellulase) 7.808 115266

纖維五糖 (Cellopentaose) 8.237 250168

纖維四糖 (Cellotetraose) 8.308 473141

纖維三糖 (Cellotriose) 9.063 246318

纖維二糖 (Cellobiose) 10.249 1335336

蔗糖 (Sucrose) 10.450 1706176

麥芽糖 (Maltose) 10.606 1299774

乳糖 (Lactose) 10.946 1353955

葡萄糖 (Glucose) 12.752 1478458

半乳糖 (Galactose) 13.947 1299034

木糖 (Xylose) 13.955 1366342

甘露糖 (Mannose) 14.402 1286161

果糖 (Fructose) 15.377 1506256

阿拉伯糖 (Arabinose) 15.829 1389951

核糖 (ribose) 27.219 1120409

(1).木聚糖(Xylan)

(2).纖維水解酵素(Cellulase)

(3).纖維五糖(Cellopentaose)

(4). 纖維四糖(Cellotetraose)

(5).纖維三糖(Cellotriose)

(6).纖維二糖(Cellobiose)

(7).蔗糖(Sucrose)

(8).麥芽糖(Maltose)

(9).乳糖(Lactose)

(10).葡萄糖(Glucose)

(11).半乳糖(Galactose)

(12).木糖(Xylose)

(13).甘露糖(Mannose)

(14).果糖(Fructose)

(15).阿拉伯糖(Arabinose)

(16).核糖(ribose)

7.2 酵素分析結果 7.2.1 纖維素水解酵素

y = 0.0058x R² = 0.9963

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5

0 10 20 30 40 50 60 70 80 90 100

濃度(%)

Cellulase from Trichoderma reesei ATCC 26921

圖D01 纖維素水解酵素之濃度校正圖

7.2.2 纖維二醣水解酵素

Cellobiase from Aspergillus niger

y = 0.0718x + 0.0065 R² = 0.9931

0.2 0.3 0.4 0.5 0.6 0.7 0.8

7.3 黑麴菌濃度校正曲線

Glucose 校正曲線濃度(ppm) μ(h^-1

)

0 0 10 0.7536

9 0.649 8 0.571 7 0.497 6 0.417 5 0.363 4 0.3081 3 0.191 2 0.184 1 0.087

y = 0.0718x + 0.0065 R² = 0.9931

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

0 2 4 6 8 10 12

圖D03 黑麴菌濃度校正曲線圖

8. 討論

μμ predicted

圖E01 黑麴菌比成長速率之基質濃度效應(Andrews model)

8.1.2 溫度效應

黑麴菌成長之批次動力實驗顯示；在pH = 5.1 下，基質濃度 10 ~ 100 g/L，溫度 20~27.5 ºC 時，黑麴菌比生長速率之溫度效應可以Arrhenius plot 描述(如圖 B37 所示)。由圖上斜率可以求得：黑麴菌之生長活化能為5.377 kcal/gmol，此值遠大於傳統化學反應的活化能(10 ~ 20kcal/gmol)，

顯然黑麴菌之生長速率極受溫度之影響。此外，由圖B38 可知：黑麴菌之最佳生長溫度為 27.5 ºC。

6.512 4.2665 5.8555 黑麴菌之最佳生長pH 為(k1 + k2)/2 = 5.06。

8.2 黑麴菌最佳生長條件

由圖C01 可得知：黑麴菌比成長速率之基質濃度效應可以 Andrews 模式描述，基質(葡萄糖) 之濃度在80g/L 時，即可達到最大比生長速率。

至於溫度效應方面，圖 B37 顯示黑麴菌比生長速率遵循阿忍尼亞定律(Arrhenius Law)；此外，由圖B38 可得知黑麴菌最佳生長溫度在 27.5°C。 kcal/gmole。此活化能遠低於傳統化學反應活化能的 10-20 kcal/gmole,意謂黑麴菌比生長速率受溫度影響程度不大。

8.4 纖維素水解菌株比生長最佳操作條件

由本次研究的黑麴菌、黃孢亮光伏革菌、半知菌木霉、與酵母菌等四種纖維素水解菌株比生長動力所得實驗結果，經整理列於表 E03 與表 E04。表 E03 顯示：纖維素水解菌株比生長的最佳操作條件，包括基質濃度效應、溫度效應、pH 效應、生長活化能等，可作為下一階段酵素量

產時反應器操作之研究的重要參考依據。表 E04 列出黑麴菌、半知菌木霉、黃孢亮光伏革菌、與

Aspergillus niger 31494 10864 27.5 5.06 Aerobic 5.0435 Andrews model 5.377 半知菌木霉

由表2 黑麴菌相關資料中，可以看出黑麴菌(BCRC 31494)可以比黑麴菌(BCRC 31130)分泌更多的纖維素水解酵素，因此，黑麴菌(BCRC 31494)被選為研究用菌株。

由表4 可分泌多種纖維水解酵素的菌株與其分泌酵素種類中，可以看出：可以分泌內切型與外切型纖維素水解酵素、纖維二醣水解酵素、與木糖異構酶等四種酵素的菌株，只有半知菌木霉 (Trichoderma reesei)，能夠分泌三種酵素的菌株，包括黑麴菌(Aspergillus niger)、卷枝毛黴(Mucor

circinelloides) 等，能夠分泌兩種酵素的菌株，包括綠木黴 (Trichoderma viride) 、康寧木腐黴

(Trichoderma koningii)、風乾菌(Scierotium rolfsii UV-8)、酵母菌(Saccharomyces cerevisiae)等，而黃 孢亮光伏革菌(Phanerochaete chrysosporium)只能分泌一種酵素--外切型纖維素水解酵素；但是由於黃孢亮光伏革菌是一種白腐菌，也能分泌木質素水解酵素，因此，可以分解木材，所以對於木質纖維素的水解，仍有賴黃孢亮光伏革菌。

因此，菌株的研究優先順序如下：半知菌木霉 > 黑麴菌, 卷枝毛黴 > 酵母菌, 綠木黴, 康寧木腐黴, 風乾菌 > 黃孢亮光伏革菌。

8.6 未來纖維素水解酵素生產的研究方向

列於表5 中所選出的纖維水解菌株中，除了本次研究的黑麴菌、黃孢亮光伏革菌、半知菌木霉、

與酵母菌等四種纖維素水解菌株外，尚待進一步探討的菌株，包括綠木黴(Trichoderma virid

e

)、康寧木腐黴(Trichoderma koningii)、風乾菌(Scierotium rolfsii UV-8)、卷枝毛黴(Mucor circinelloides)等四種纖維素水解菌株的比生長速率動力實驗，其次，進行纖維素水解、半纖維素水解、與木質素水解的菌株篩選；還有菌株生產酵素的生化分離技術探討。至於纖維素水解酵素與基質、酵素與酵素間的抑制作用，有待從文獻中整理與動力探討，以供酒精醱酵之製程設計參考。

9.結論綜合以上的實驗觀察結果，可以歸納以下結論

1. 黑麴菌（BCRC 31494）之比生長動力模式，在基質為葡萄糖濃度範圍 10~100g/L 之間，基質濃度效應可以Andrews 模式描述， Andrews 模式參數為 = 97 h^-1, KS = 90000 g/L, KI = 0.07 g/L,

= 0.01 h^-1，最大黑麴菌比成長速率之基質濃度為79.37 g/L。

^μ μc

2. 黑麴菌比生長速率之溫度效應，在 pH = 5.1，基質濃度 10 ~ 100 g/L 及溫度 20~27.5 ºC 時，黑麴菌比生長速率之溫度效應可以Arrhenius plot 描述，黑麴菌之生長活化能為 5.377 kcal/gmol；黑麴菌之最佳生長溫度為27.5 ºC。

3. 黑麴菌比生長速率之 pH 效應，在溫度 30°C, 40g/L 葡萄糖下，可以 Michaelis pH 函數描述，

Michaelis pH 函數之參數如下：δ= 6.512 h^-1, k1 = 4.2665, k2 = 5.8555, 黑麴菌之最佳生長 pH 為 5.06。

4. 纖維水解菌株之比生長速率的 pH 效應，可以得知最適於菌株生長的 pH 值如下：黑麴菌 5.06，

半知菌木霉3.95，黃孢亮光伏革菌 5.00，酵母菌 4.33。

5. 纖維水解菌株之比生長速率的 pH 效應，可以得知 Michaelis pH 函數模式參數( ,k₁, k₂)值分別如下：黑麴菌(6.512, 4.2665, 5.8555)，半知菌木霉(0.0069, 2.9322, 4.9648)，黃孢亮光伏革菌(0.0618, 3.2639, 6.7258)，酵母菌(1.1133, 3.9158, 4.7398)。

6. 黑麴菌、半知菌木霉、黃孢亮光伏革菌、酵母菌等菌株的比生長速率皆符合 Arrhenius Law 模式所描述之結果。最佳比生長速率之溫度值分別為：黑麴菌(27.5 ºC)、半知菌木霉(30 ºC)、黃孢亮光伏革菌(30 ºC)、臺灣本土根瘤菌(35 ºC)、與酵母菌(28 ºC)等結果。

7. 纖維素水解產物⎯纖維寡醣之 HPLC 分析結果，各成分之滯留時間(min.)順序中，除了纖維四糖(8.308)與纖維五糖(8.237)、纖維二糖(10.249)與蔗糖(10.450)與麥芽糖(10.606)、半乳糖(13.947) 與木糖(13.955)外，其餘皆無分析上的解析問題，甚至於兩種酵素：纖維素水解酵素(4.041)、纖維二醣水解酵素(3.337)，也可精確地分析。標定用纖維二醣水解酵素為黑麴菌(Aspergillus niger) 產製，商品名Nomozyme 188, 纖維素水解酵素為半知菌木霉(Trichoderma reesei, ATCC 26921) 所產製。

8. 菌株濃度可以菌株的乾細胞重(dry cell weight, DCW)量測。

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在文檔中纖維水解菌株篩選、水解酵素分離純化、與酵素生產生質酒精製程最適化探討 (頁 79-0)