Serratia marcescens TKU011係一株以蝦頭殼粉末作為唯一碳/氮源之蛋白酶及幾丁聚醣酶生產菌。本研究發現以烏賊軟骨粉末(SPP)作為碳/氮源時，TKU011生產蛋白酶及幾丁聚醣酶之較適條件為1%烏賊軟骨粉末、0.1% K2HPO4及0.05% MgSO4．7H2O，在25℃、pH 7之100 mL液態培養基振盪培養5天。延長培養基滅菌時間(在121℃下滅菌120分鐘)可得較佳之蛋白酶及幾丁聚醣酶活性。在培養基之不同滅菌時間條件下，所得發酵上清液經SDS-PAGE分析發現，酵素濃度隨滅菌時間延長而增加。自發酵物回收之烏賊粉末乾重，會隨著培養天數增加而逐漸減少，上清液可測得含有胜肽、寡糖。發酵上清液經清除DPPH自由基能力、還原力、螯合亞鐵離子能力及總酚量等測定抗氧化能力試驗，TKU011發酵SPP可生產抗氧化物質，這些抗氧化物質可能為胜肽、幾丁質及幾丁聚醣等化合物。將較適培養條件下發酵所得上清液經硫酸銨沉澱、DEAE-Sepharose及Phenyl Sepharose等層析步驟，可純化出二種幾丁質酶/幾丁聚醣酶(C1及C2)，經SDS-PAGE測得分子量分別為49 kDa及66 kDa，將進一步探討酵素生化特性分析及基質特異性。

A protease- and chitosanase-producing strain, Serratia marcescens TKU011, was cultivated by using shrimp head shell powder (SHP) as the sole carbon/nitrogen source. In this study, the optimized condition for protease and chitosanase production using squid pen powder (SPP) as the carbon/nitrogen source was found to be when the culture was shaken at 25℃ for five days in 100 mL of medium (pH 7) containing 1% SPP, 0.1 % K2HPO4, 0.05% MgSO4．7H2O. In a prolonged autoclave (at 121℃ for 120 min), the protease and chitosanase activity were measured were better than others. The culture supernatants from various time of the medium in an autoclave by SDS-PAGE analysis was found that the enzyme concentration increased with an increase in heating time. The dry weight of the recovered squid pen decreased with an increase in cultivation. The peptides and oligosaccharides in the culture supernatants was determined. The antioxidative activity of the supernatant was derermind by DPPH radical-scavenging activity, reducing activity, total phenolic, Fe2+-Chelating activity. Two chitosanase( or chitinase) (C1,C2) were purified from the culture supernatant by chromatography procedures of DEAE-Sepharose and Phenyl Sepharose. The molecular mass of C1 and C2 determined by SDS-PAGE was approximately 49 kDa and 66 kDa, respectively. The substrate specificity and biochemical characterization of the enzymes were studied.

封面內頁
簽名頁
授權頁
中文摘要I
英文摘要II
誌謝 III
目錄IV
圖目錄VII
表目錄VIII

第一章 緒論1
第二章 文獻回顧2
2.1 幾丁質2
2.1.1 幾丁質之結構2
2.1.2 幾丁質之應用3
2.2 幾丁質酶3
2.3 幾丁聚醣酶4
2.4 Serratia marcescens8
2.4.1 基本特性8
2.4.2 S. marcescens之幾丁質酶8
2.4.3 S. marcescens之蛋白酶9
2.5 抗氧化11
2.5.1 抗氧化劑作用機制11
第三章 材料與方法13
3.1 實驗材料13
3.1.1 實驗菌株13
3.1.2 實驗材料13
3.1.3 實驗儀器14
3.2 懸浮態幾丁質之製備14
3.3 蛋白酶之活性測定15
3.4 幾丁聚醣酶之活性測定15
3.5 幾丁質酶之活性測定16
3.6 不同碳/氮源之比較16
3.7 不同滅菌時間之影響16
3.8 還原糖量之測定16
3.9 回收沉澱物乾重之測定17
3.10 酵素之純化分離17
3.10.1粗酵素液之製備17
3.10.2 離子交換樹脂層析法18
3.10.3 疏水性層析18
3.10.4 蛋白質電泳分析18
3.11 抗氧化特性分析19
3.11.1 DPPH自由基清除能力之測定19
3.11.2螯合亞鐵離子能力之測定20
3.11.3還原力之測定20
3.11.4總酚類化合物之定量分析21
第四章 結果與討論22
4.1培養條件22
4.1.1 碳/氮源之選擇22
4.1.2 烏賊軟骨粉末培養基之滅菌時間24
4.2 TKU011發酵烏賊軟骨粉末之產物分析29
4.3 同一滅菌時間以不同碳氮源作為培養基之比較32
4.4幾丁質酶/幾丁聚醣酶之純化分離36
4.4.1 綜合結果41
4.5 幾丁質酶/幾丁聚醣酶之分子量測定41
4.6 以TKU011發酵SPP所得上清液之抗氧化效果45
第五章 結論47
參考文獻48

圖目錄
圖2.1幾丁聚醣、纖維素及幾丁質之化學結構5
圖4.1 TKU011培養於SHP及SPP培養基所生產蛋白酶、幾丁聚醣酶及DPPH自由基清除能力之變化情形23
圖4.2 TKU011培養於不同滅菌時間之SPP培養基所生產蛋白酶、幾丁聚醣酶之變化情形25
圖4.3 TKU011培養於不同滅菌時間之SPP培養基所生產蛋白酶、幾丁聚醣酶之變化情形26
圖4.4 TKU011培養於不同滅菌時間之SPP所得上清液之電泳分析27
圖4.5 TKU011培養於滅菌120 min之SPP培養基所生產蛋白酶、幾丁聚醣酶之變化情形28
圖4.6 (A)所得發酵上清液中測得之蛋白質含量、還原糖量及回收殘餘SPP之乾重(B)回收烘乾殘餘粉末之照片30
圖4.7 TKU011發酵SPP培養基所產幾丁質酶之變化情形31
圖4.8 TKU011發酵不同碳氮源培養基所生產蛋白酶及幾丁聚醣酶之變化情形33
圖4.9 TKU011發酵不同碳氮源培養基所生產幾丁質酶之變化情形34
圖4.10 TKU011發酵不同碳氮源培養基所得上清液中還原糖量及清除DPPH自由基能力之變化情形35
圖4.11 S. marcescens TKU011所生產酵素之純化分離流程圖38
圖4.12 幾丁聚醣酶/幾丁質酶之DEAE-Sapharose CL-6B層析圖譜39
圖4.13 幾丁聚醣酶/幾丁質酶之Phenyl-Sepharose層析圖譜40
圖4.14 幾丁質酶/幾丁聚醣酶(C1,C2)於SDS-PAGE之分子量分析42
圖4.15 TKU011發酵SPP培養基所得上清液之抗氧化試驗結果46
 
表目錄
表2.1 幾丁質與幾丁聚醣的應用6
表2.2 幾丁質酶之分類7
表2.3 S. marcescens之幾丁質酶與幾丁質結合蛋白質10
表4.1 TKU011生產蛋白酶、幾丁質酶及幾丁聚醣酶之較適條件36
表4.2 S.marcescens TKU011幾丁質酶/聚醣酶C1之純化總表43
表4.3 S.marcescens TKU011幾丁質酶/聚醣酶C2之純化總表44

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