本研究以蝦蟹殼粉（SCSP）為 Pseudomonas sp.TKU008 生產蛋白酶與幾丁質酶之唯一碳/氮源，於含有 1% SCSP、0.05% MgSO4．7H2O、0.1% K2HPO4（pH7）之液態培養基搖瓶培養 4 天可得較高酵素活性，將所得發酵上清液進行酵素之純化分離及定性。首先將所得發酵上清液經硫酸銨沉澱濃縮、透析去除鹽類，再經過 DEAE-Sepharose、Phenyl Sepharose 及 Sephacryl S-100 管柱層析等分離步驟，純化出一種幾丁質酶，經 SDS-PAGE 測其分子量為 40 kDa。純化後幾丁質酶之活性回收率為 9%，純化倍數為 24.1倍，比活性為 0.175 U/mg。以懸浮態幾丁質為基質測得幾丁質酶最適pH值、最適反應溫度、pH安定性及熱安定性分別為 pH7、50℃、pH6～8 及 <50℃；幾丁質酶活性受 Cu2+、Mn2+所抑制。
由幾丁質酶的液相層析串連式質譜分析得到胺基酸序列片段，序列比對有 26％ 序列接近 Bacillus cereus 的 chitinase B（GenBank accession number gi 10944304）。

The protease and chitinase producing strain, Pseudomonas sp.TKU008, was isolated from the soil in Taiwan. The shrimp and crab shell powder was used as sole carbon / nitrogen source. The optimized condition for protease and chitinase production were found when the culture was shaken at 30℃ for 4 days in 100mL of medium contain 1% shrimp and crab shell powder （SCSP）, 0.1% K2HPO4 and 0.05% MgSO4．7H2O （pH7）.One chitinase was purified by chromatography procedures of DEAE-sepharose CL-6B, Phenyl Sepharose and Sephacryl S-100. The molecular weight of the chitinase was 40k Da estimated by SDS–PAGE and gel-filtration, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of the chitinase were pH7, 50°C, pH6～8, and <50°C, respectively. The chitinase was inhibited completely by Mn2+, and Cu2+.
The results of peptide mass mapping showed that three tryptic peptides of the chitinase were identical to a chitinase B from Bacillus cereus（GenBank accession number gi 10944304） with 26% sequence coverage.

目錄

簽名頁	
授權書	
中文摘要	i
英文摘要	ii
誌謝	iii
目錄	iv
圖目錄	viii
表目錄	ix

第一章 緒論	1
第二章 文獻回顧	4
2.1 菌種之簡介	4
2.2 幾丁質之簡介	4
2.2.1 幾丁質的結構	4
2.2.2 幾丁質的特性	5
2.2.3 幾丁質的天然分佈	5
2.2.4 幾丁質的水解產物與水解方法	6
2.2.5 幾丁質水解產物之應用	8
2.3 幾丁質酶之簡介	10
2.3.1 幾丁質酶的天然分佈與功能	10
2.3.2 幾丁質酶的分類方式	10
2.3.3 幾丁質酶的結構	12
2.3.4.幾丁質酶之應用	12
第三章 材料與方法	15
3.1 實驗材料	15
3.1.1 實驗菌株	15
3.1 2 藥品與試劑	15
3.1.3 實驗儀器	16
3.2 懸浮態幾丁質之製備	16
3.3 幾丁質酶之活性測定	17
3.4 蛋白酶之活性測定	17
3.5 酵素之分離純化	18
3.5.1 粗酵素液之製備	18
3.5.2 陰離子交換層析	18
3.5.3 疏水性層析	19
3.5.4 膠體過濾層析	19
3.6 蛋白質電泳分析	20
3.7 胜肽質譜鑑定	20
3.8 蛋白酶活性電泳	20
3.9 幾丁質酶活性電泳	21
3.10 酵素之生化特性分析	21
3.10.1 酵素最適反應溫度	21
3.10.2 酵素之熱安定性	21
3.10.3 酵素最適反應pH	22
3.10.4 酵素之pH安定性	22
3.10.5 金屬離子與抑制劑對酵素活性之影響	22
3.10.6 界面活性劑對酵素活性之影響	23
第四章 結果與討論	24
4.1 菌株之特性與培養條件	24
4.2 幾丁質酶之分離純化	26
4.2.1 粗酵素液製備	27
4.2.2 陰離子交換層析	27
4.2.3 疏水性層析	29
4.2.4 膠體過濾層析	31
4.2.5 綜合結果	32
4.3 幾丁質酶之分子量測定	33
4.4 酵素之活性染色	34
4.4.1 幾丁質酶之活性染色	34
4.4.2 蛋白酶之活性染色	35
4.5 幾丁質酶胜肽質譜鑑定	36
4.6 純化後幾丁質酶之生化特性分析	37
4.6.1 幾丁質酶之基質特異性	37
4.6.2 幾丁質酶之最適反應溫度及熱安定性	38
4.6.3 幾丁質酶之最適反應 pH 及 pH 安定性	40
4.6.4 金屬離子對幾丁質酶之影響	42
4.6.5 界面活性劑對幾丁質酶之影響	43
第五章 結論	44
第六章 參考文獻	46
 
圖目錄

圖 2.1 幾丁質及幾丁聚醣之構造	14
圖 4.1 Pseudomonas sp.TKU008 16S rDNA 部分序列	25
圖 4.2 幾丁質酶之分離純化流程圖	26
圖 4.3a、4.3b 幾丁質酶之 DEAE-Sepharose CL-6B 層析圖譜	28
圖 4.4a、4.4b 幾丁質酶之 Phenyl Sepharose 層析圖譜	30
圖 4.5 幾丁質酶之 Sephacryl S-100 層析圖譜	31
圖 4.6 幾丁質酶之 SDS-PAGE 電泳分析圖	33
圖 4.7 幾丁質酶活性染色	34
圖 4.8 蛋白酶活性染色	35
圖 4.9 幾丁質酶之最適溫度及熱安定性	39
圖 4.10 幾丁質酶之最適 pH 及 pH 安定性	41

 表目錄

表 1.1 幾丁質與幾丁聚醣的應用	3
表 4.1 TKU008 幾丁質酶純化總表	32
表 4.2 TKU008 幾丁質酶胜肽質譜鑑定結果	36
表 4.3 TKU008 幾丁質酶基質特異性	37
表 4.4 金屬離子對幾丁質酶活性之影響	42
表 4.5 界面活性劑對幾丁質酶之影響	43
表 4.6 微生物來源之幾丁質酶特性比較	45

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