本研究利用烏賊軟骨作為Bacillus sp. TKU004生產幾丁聚醣酶及生物活性物質之唯一碳/氮源。
    Bacillus sp. TKU004生產幾丁聚醣酶之較適培養條件為：含有3%烏賊軟骨粉、0.1% K2HPO4及0.05% MgSO4．7H2O之100 mL液態培養基(pH 7)於250 mL三角錐形瓶，經滅菌45分鐘，於30℃搖瓶培養二天。發酵所得離心上清液，經硫酸銨沈澱、DEAE-Sepharose、Macro-Prep DEAE、Sephacryl S-100等層析步驟，純化出一種幾丁聚醣酶，其活性回收率為20%，比活性為2.8 U/mg；以SDS-PAGE及膠體過濾層析測定分子量分別為29 kDa及25 kDa；TKU004幾丁聚醣酶胜肽質譜鑑定結果，與B. subtilis subsp. subtilis str. 168之幾丁聚醣酶(GenBank編號為 gi16079742)相似度為23%。幾丁聚醣酶之最適反應溫度、最適反應pH、熱安定性、pH安定性分別為37℃、pH 7、＜40℃、pH 4~7；其活性會受5 mM之Cu2+及Fe2+所抑制，而在2% Tween 20、2% Tween 40、2% Triton X-100及2 mM SDS存在下，分別還維持原活性94%、96%、93%、95%。
    此外，以烏賊軟骨粉及蝦頭粉作為不同碳/氮源，利用TKU004培養1~6天，結果發現，發酵3%烏賊軟骨粉所得上清液第3天具較高DPPH自由基清除能力，第5天有較佳的總酚含量、還原力、螯合亞鐵離子能力。

This study focused on the utilization of squid pen as the sole carbon/nitrogen source by Bacillus sp. TKU004 to produce chitosanase and bioactive materials. 
    The optimized culture condition for chitosanase production was composed of 3% squid pen powder (SPP), 0.1% K2HPO4, 0.05% MgSO4．7H2O (pH 7), with autoclave treatment for 45 min, afterward, TKU004 was incubated in 100 mL of above liquid medium in an Erlenmeyer flask (250 mL) and kept shaking at 30℃ for two days. The chitosanase was purified from the culture supernatant by using ammonium sulfate precipitation, chromatography procedures of DEAE-Sepharose, Macro-Prep DEAE, and Sephacryl S-100. The overall activity yield of the purified chitosanase was 20%, with specific chitosanase activity of 2.8 U/mg. The molecular mass of TKU004 chitosanase determined by SDS-PAGE and gel filtration was approximately 29 kDa and 25 kDa, respectively. The results of peptide mass mapping indicated that TKU004 chitosanase matched to chitosanase from B. subtilis subsp. subtilis str. 168 (GenBank accession number gi16079742) with 23% sequence coverage. The optimum temperature, optimum pH, thermal stability, and pH stability of TKU004 chitosanase were 37℃, pH 7, ＜40℃, and pH 4~7, respectively. The chitosanase was inhibited by 5 mM Cu2+ and Fe2+, but retained 94%, 96%, 93%, 95% of its original activity in the presence of 2% Tween 20, 2% Tween 40, 2% Triton X-100, and 2 mM SDS, respectively.
    Additionally, TKU004 was cultivated for 1~6 days by using squid pen powder and shrimp head powder as the different carbon/nitrogen sources. The results indicated that culture supernatant (3% SPP) had higher DPPH free radical scavenging effect at the third day and better total phenolic contents, reducing activity, and Fe2+ chelating ability at the fifth day.

授權書
簽名頁
誌謝…I
中文摘要…II
英文摘要…III
目錄…V
圖目錄…X
表目錄…XII

第一章 緒論…1
第二章 文獻回顧…2
     2.1 Bacillus sp. TKU004之簡介…2
     2.2 幾丁質與幾丁聚醣…2
        2.2.1 幾丁質與幾丁聚醣之分布及構造…2
        2.2.2 幾丁質與幾丁聚醣之應用…3
     2.3 幾丁質酶與幾丁聚醣酶…3
     2.4 N-乙醯幾丁寡醣與幾丁寡醣…4
     2.5 抗氧化…7
        2.5.1 自由基與活性氧	…7
        2.5.2 自由基與氧化性傷害…7
        2.5.3 抗氧化劑作用之原理與機制…8
第三章 材料與方法…10
     3.1 實驗菌株…10
     3.2 實驗材料…10
     3.3 實驗儀器…11
     3.4 幾丁聚醣酶之活性測定…11
     3.5 蛋白酶之活性測定…12
     3.6 幾丁聚醣酶較適生產條件探討…12
        3.6.1 碳/氮源…12
        3.6.2 培養基滅菌時間	…13
        3.6.3 培養溫度…13
        3.6.4 培養基酸鹼值(pH)…13
        3.6.5 培養體積…13
        3.6.6 培養時間…14
     3.7 幾丁聚醣酶之分離純化…14
        3.7.1 粗酵素液之製備	…14
        3.7.2 陰離子交換層析…14
        3.7.3 膠體過濾層析…15
     3.8 幾丁聚醣酶之分子量測定…16
        3.8.1 蛋白質電泳分析…16
        3.8.2 膠體過濾層析…16
     3.9 幾丁聚醣酶之特性分析…17
        3.9.1 幾丁聚醣酶最適反應溫度…17
        3.9.2 幾丁聚醣酶熱安定性…17
        3.9.3 幾丁聚醣酶最適反應pH…17
        3.9.4 幾丁聚醣酶pH安定性…18
        3.9.5 化學藥品對幾丁聚醣酶活性之影響…18
        3.9.6 界面活性劑對幾丁聚醣酶活性之影響…18
        3.9.7 幾丁聚醣酶之基質特異性…19
     3.10 粗酵素液之蛋白酶及幾丁聚醣酶活性關係…19
     3.11 還原醣量之測定…19
     3.12 抗氧化分析…20
        3.12.1 總酚類化合物含量之測定…20
        3.12.2 DPPH自由基清除能力之測定…20
        3.12.3 還原力之測定…21
        3.12.4 螯合亞鐵離子能力之測定…21
第四章 結果與討論…23
     4.1 幾丁聚醣酶較適生產條件探討…23
        4.1.1 碳/氮源…23
        4.1.2 培養基滅菌時間	…24
        4.1.3 培養溫度…24
        4.1.4 培養基酸鹼值(pH)…24
        4.1.5 培養體積…25
        4.1.6 較適培養條件探討結果…25
     4.2 幾丁聚醣酶之分離純化…37
        4.2.1 粗酵素液之製備…37
        4.2.2 陰離子交換層析…37
        4.2.3 膠體過濾層析…38
        4.2.4 綜合結果…38
     4.3 幾丁聚醣酶之分子量測定…45
        4.3.1 SDS-PAGE…45
        4.3.2 膠體過濾層析…45
        4.3.3 綜合結果…45
        4.3.4 幾丁聚醣酶胜肽質譜鑑定…46
     4.4 幾丁聚醣酶之特性分析…50
        4.4.1 幾丁聚醣酶之最適反應溫度及熱安定性…50
        4.4.2 幾丁聚醣酶之最適反應pH及pH安定性…50
        4.4.3 化學藥品對幾丁聚醣酶活性之影響…50
        4.4.4 界面活性劑對幾丁聚醣酶活性之影響…51
        4.4.5 幾丁聚醣酶之基質特異性…51
     4.5 還原醣量與培養基烏賊軟骨粉剩餘重量(%)之變化情形
         …59
     4.6 抗氧化分析…59
        4.6.1 總酚類含量…59
        4.6.2 DPPH自由基清除能力…60
        4.6.3 還原力…60
        4.6.4 螯合亞鐵離子能力…61
        4.6.5 綜合結果…61
第五章 結論…68
參考文獻…69

圖目錄
頁次
圖2.1 幾丁質、幾丁聚醣及纖維素之結構…5
圖4.1 不同碳/氮源對TKU004幾丁聚醣酶生產之影響…27
圖4.2 SPP濃度對TKU004幾丁聚醣酶生產之影響…28
圖4.3 培養基滅菌時間對TKU004幾丁聚醣酶生產之影響…29
圖4.4 溫度對TKU004幾丁聚醣酶生產之影響…30
圖4.5 pH對TKU004幾丁聚醣酶生產之影響…31
圖4.6 培養體積對TKU004幾丁聚醣酶生產之影響…32
圖4.7 Bacillus sp. TKU004於SPP培養基所生產幾丁聚醣酶及蛋白
      酶之生長曲線圖…33
圖4.8 B. subtilis TKU007於SHP培養基所產蛋白酶及幾丁聚醣酶之
      生長曲線圖…33
圖4.9 TKU004幾丁聚醣酶與蛋白酶活性關係…35
圖4.10 TKU004幾丁聚醣酶與蛋白酶於37℃下反應後之SDS-PAGE…36
圖4.11 Bacillus sp. TKU004所生產幾丁聚醣酶之純化分離流程
       圖…40
圖4.12 TKU004幾丁聚醣酶之DEAE-Sepharose CL-6B層析圖譜…41
圖4.13 TKU004幾丁聚醣酶之Macro-Prep DEAE層析圖譜…42
圖4.14 TKU004幾丁聚醣酶之Macro-Prep DEAE再層析圖譜…43
圖4.15 TKU004幾丁聚醣酶之Sephacryl S-100層析圖譜…44
圖4.16 TKU004幾丁聚醣酶SDS-PAGE之分子量分析…48
圖4.17 分子量標準品和幾丁聚醣酶於Sephacryl S-100之層析圖譜	…49
圖4.18 幾丁聚醣酶之最適反應溫度及熱安定性…53
圖4.19 幾丁聚醣酶之最適反應pH及pH安定性…54
圖4.20 發酵上清液還原醣量、幾丁聚醣酶活性與培養基烏賊軟骨粉
       剩餘重量之變化情形…63
圖4.21 不同碳/氮源對(a)酚類含量生產及(b) DPPH自由基清除能力
       之影響…64
圖4.22 不同碳/氮源對(a)還原力及(b)螯合亞鐵離子能力之影響
       …65
圖4.23 TKU004發酵3天上清液與α-生育醇不同濃度之DPPH自由基
       清除能力…66
圖4.24 TKU004發酵5天上清液與EDTA不同濃度之螯合亞鐵離子能 
       力	…67

表目錄
頁次
表2.1 幾丁質與幾丁聚醣之應用…6
表4.1 TKU004幾丁聚醣酶之較適生產條件…34
表4.2 Bacillus sp. TKU004、B. subtilis TKU007、Pseudomonas
      sp. TKU015幾丁聚醣酶較適生產條件之比較…34
表4.3 Bacillus sp. TKU004幾丁聚醣酶之純化總表…39
表4.4 TKU004幾丁聚醣酶胜肽質譜鑑定結果…47
表4.5 化學藥品對幾丁聚醣酶活性之影響…55
表4.6 界面活性劑對幾丁聚醣酶活性之影響…55
表4.7 TKU004幾丁聚醣酶之基質特異性…56
表4.8 Bacillus spp.幾丁聚醣酶特性比較…57

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