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圖一、Yen 在 C-10 固體培養基上，在室溫下培養2天之菌落形態 Fig. 1. Colony morphology of Yen, followed by incubation 2 days at RT
in C-10 plate.
圖二、Yen 在 C-10 固體培養基上，在室溫下培養2天後，利用碘 液來偵測洋菜酶活性
Fig. 2. Assay of agarase activity by iodine reagent, followed by incubation 2 days at RT in C-10 plate.
CTGGCTCAGATTGAACGCTAGCGGGATGCCTTACACATGCAAGTCGAACG Fig. 3. 16S rDNA sequence of Yen
圖四、Yen 之洋菜酶 CBR 染色
Fig. 4. SDS-PAGE CBR staining of agarase fromYen.
( Yen 之洋菜酶，每個 Lane 加 30 µL 粗酵素液，以 120 V 電壓 進行 4~20 % SDS-PAGE 電泳，以 Coomassie btilliant blue R-250 染劑染色 1 天，再以褪染液褪染 4 hr 並與蛋白質標準品(注入量 為 10 µL )比對，M:mark；Lane 1:Sephadex G-100 膠體管柱過濾純 化)
A B
圖五、Yen 之洋菜酶 Native-PAGE 活性染
Fig. 5. Native-PAGE activity staining of agarase fromYen.
( Yen 之粗洋菜酶，每個 Lane 加 30 µL 粗酵素液，以 120 V 電 壓進行 4~20 % SDS-PAGE 及 Native page電泳，電泳膠片以緩衝 液洗過後，在膠片上倒上 5% soft agar，35 ℃反應3 小時後以碘 液進行活性染。上圖為 SDS-PAGE CBR 染色；圖A，M:
standards；Lane A、B:Sephadex G-100 膠體管柱過濾純化；圖B，
M: standards；Lane 1~:0~20 %；Lane 1~:0~20 %；Lane 2~:20~40 %；
Lane 3~:40~60 %；Lane 4~:60~80 %；Lane 5~:80~100 % 硫酸銨分 化；Lane 6、7:Sephadex G-100 膠體管柱過濾純化)
0
Citrate-Na2HPO4 buffer
Tris-HCl buffer Sodium phosphate buffer Phosphate buffer Glycine-NaO
圖六、Yen-agarases 於不同緩衝液中的最適作用 pH 值。
Fig. 6. Optimum pH of Yen-agarases in various buffer solutions.
(分別取 5U/mL 之酵素加入含不同 pH 値緩衝溶液之基質中，
50 mM citrate-Na2HPO4 buffer, pH 3.0-5.0; 50 mM Tris-HCl buffer, pH 6.0-7.5; 50 mM Sodium phosphate buffer pH 7.5-8.5; 50 mM phosphate buffer, pH 8.0-8.5; 50 mM glycine-NaOH buffer, pH 9.0-10.0.，在35℃ 反應下 30 min後，依據還原糖酵素活性測定 之方法測還原糖變化量。)
Each point represents the average of three experiments.
Activity at zero time was taken as 100% activity.
0 20 40 60 80 100
2 3 4 5 6 7 8 9 10 11
pH
Relative acctivity (%)
圖七、pH 值對 Yen-agarases 之安定性。
Fig. 7. Effect of pH on the stability of Yen-agarases.
(分別取 5U/mL 之各酵素與 50 mM 不同 pH 值
citrate-Na2HPO4 buffer, pH 3.0-5.0; Tris-HCl buffer, pH 6.0-7.5;
Sodium phosphate buffer pH 7.5-8.0; phosphate buffer, pH 8.5;
glycine-NaOH buffer, pH 9.0-10.0 溶液混合均勻，於 4℃ 下儲存 24 hr 後，取各 pH 值的酵素液與 0.2% 基質在 35℃ 反應下 30 min 後，依還原糖酵素活性測定之方法測酵素活性。) Each point represents the average of three experiments.
Activity at zero time was taken as 100% activity.
0 20 40 60 80 100
0 10 20 30 40 50 60 7
Temparature (℃)
Relative activity (%)
0
Low agarose High agarose
圖八、Yen-agarases 的最適反應溫度。
Fig. 8. Optimum temperature of Yen-agarases.
(將 0.2% 的 low-melting-point agarose 和 high-melting-point agarose，分別置於各反應溫度 20℃、25℃、30℃、37℃、45℃、
50℃、55℃、60℃中，再加入 5U/mL 酵素液混合均勻，反應 30 min 後，依還原糖酵素活性測定之方法測酵素活性。)
Each point represents the average of three experiments.
Activity at zero time was taken as 100% activity.
0 20 40 60 80 100
20 30 40 50 60 70
Temperature (℃)
Relative activity (%)
圖九、Yen-agarases 對溫度穩定性分析 Fig. 9. Thermostability of Yen-agarases.
(將 Yen-agarases 粗洋菜酶在不同溫度下處理 30min 之酵素殘 存活性，把 Yen-agarases 粗洋菜酶濃度調整成約 5U/mL，在溫 度 20、25、30、35、40、45、50、55、60℃ 下處理 30 min，然 後依還原糖酵素活性測定之方法測酵素殘存活性。)
Each point represents the average of three experiments.
Activity at zero time was taken as 100% activity.
0 20 40 60 80 100 120
0 20 40 60 80 100 120
CaCl2(mM)
Relative activity (%)
Yen-agarase
圖十、氯化鈣濃度對洋菜酶活性之影響。
Fig. 10. Effect of CaCl2 concentration to Yen-agarases on their activities.
(不同鈣離子濃度0-100 mM分別加入 0.2% 基質中混合均勻後，
加入各酵素 5U/mL 與基質在 35℃ 反應下 30 min 後，依還原 糖酵素活性測定之方法測酵素活性。)
Each point represents the average of three experiments.
Activity at zero time was taken as 100% activity.
0 20 40 60 80 100
0 200 400 600 800 1000
Na Cl ( mM )
Relative activity (%)