本研究係探討Bacillus cereus TKU006所生產幾丁質分解酵素及其他抗氧化質之研究，探討其較適培養條件，進行酵素純化與定性為研究目的。
    TKU006以1％烏賊軟骨粉末為碳/氮源培養於100 mL含  0.1％ K2HPO4及0.05％ MgSO4．7H2O之液態培養基，於25℃搖瓶培養三天可得較高幾丁聚醣酶活性；以2％蝦頭粉末為碳/氮源於25 mL液態培養基培養二天得較高幾丁質酶活性。所得發酵上清液經硫酸銨沉澱、陰離子交換層析、膠體過濾層析步驟純化得幾丁質酶、幾丁聚醣酶。幾丁質酶分子量為39 kDa，Fe2+完全抑制酵素活性，最適反應溫度、pH分別為60℃、pH 5，熱安定性、pH安定性分別為≦50℃、pH 2-8。幾丁聚醣酶分子量為44 kDa，受到Fe2+、Cu2+完全抑制酵素活性，最適反應溫度與pH分別為60℃與pH 7，熱安定性、pH安定性分別為≦50℃與pH 3-10。
    以25 mL含1％烏賊軟骨、0.1％ K2HPO4及 0.05％ MgSO4．7H2O之液態培養基，於25℃搖瓶培養二天，可得較佳DPPH自由基清除能力。發酵上清液之自由基清除能力、亞鐵離子螯合能力、還原力分別為75％、97％與441 μg/mL CE，而酚類化合物含量為815 μg/mL GAE。總酚類化合物與自由基清除能力之相關係數為r=0.92。

The purpose of this thesis is to study the production of chitinolytic enzymes and other antioxidants from Bacillus cereus TKU006.
    The optimum culture conditions for chitosanase and other antioxidants production were composed of 1% squid pen powder, 0.1% K2HPO4, 0.05% MgSO4．7H2O shaken at 25℃ in 100 mL of liquid medium in an Erlenmeyer flask (250 mL) for 3 days and 25 mL of liquid medium for 2 days, respectively. The optimum culture carbon/nitrogen source for chitinase production was 2% shrimp head powder incubated for 2 days. A chitinase and a chitosanase were purified by chromatography procedures of ion exchange and gel filtration. The molecular mass of the chitinase and chitosanase determined by SDS-PAGE were approximately 39 kDa and 44 kDa, respectively. The chitinase was completely inactivated by Fe2+, and the chitosanase was completely inactivated by Fe2+ and Cu2+. The optimum temperature, optimum pH, thermal stability and pH stability of chitinase were 60℃, pH 5, ≦50℃, pH 2-8 . The optimum temperature, optimum pH, thermal stability and pH stability of chitosanase were 60℃, pH 7, ≦50℃, pH 3-10.
    After squid pen powder fermented, the antioxidant properties by the assays of radical scavenging, metal chelating, reducing power and total phenolic content were reached at 75%, 97%, 441 μg/mL cysteine equivalent and 815 μg/mL gallic acid equivalent, respectively. Correlation between DPPH scavenging ability and total phenolic content were r=0.92.

封面內頁
簽名頁
授權書
中文摘要 I
英文摘要	III
誌謝	V
目錄	VI
圖目錄	IX
表目錄	X

第一章 緒論	1
第二章 文獻回顧	3
2.1  Bacillus cereus TKU006之簡介	3
2.2  幾丁質與幾丁聚醣	3
2.2.1  幾丁質類之分子構型	5
2.2.2  幾丁質類之應用	5
2.3  幾丁質酶	7
2.4  N-乙醯幾丁寡醣/幾丁寡醣	7
2.5  自由基與氧化性傷害	9
2.6  抗氧化劑作用機制	9
第三章 材料與方法	11
3.1  菌株	11
3.2  化學材料	11
3.3  實驗儀器	12
3.4  較適培養條件探討	13
3.4.1  碳/氮源之選擇	13
3.4.2  碳/氮源濃度之影響	13
3.4.3  培養體積	13
3.4.4  培養時間	14
3.5  懸浮態幾丁質之製備	14
3.6  酵素活性測定	14
3.6.1  幾丁質酶之活性測定	14
3.6.2  幾丁聚醣酶之活性測定	15
3.7  酵素之純化分離	15
3.7.1  粗酵素液之製備	15
3.7.2  陰離子交換樹脂層析	16
3.7.3  膠體過濾層析	17
3.8  酵素之特性分析	17
3.8.1  酵素之最適反應溫度與熱安定性	17
3.8.2  酵素之最適反應pH與pH安定性	18
3.8.3  金屬離子與抑制劑對酵素之影響	19
3.8.4  界面活性劑對酵素之影響	19
3.8.5  基質特異性	19
3.9  抗氧化性質分析	20
3.9.1  DPPH自由基清除能力之測定	20
3.9.2  金屬離子螯合能力之測定	20
3.9.3  還原能力之測定	21
3.9.4  酚類化合物之定量分析	22
第四章 結果與討論	23
4.1  較適培養條件探討	23
4.1.1  碳/氮源之選擇	23
4.1.2  碳/氮源濃度之影響	25
4.1.3  培養體積之影響	25
4.1.4  培養時間之影響	28
4.1.5  培養條件總結	28
4.2  酵素之純化分離	31
4.2.1  粗酵素液	31
4.2.2  陰離子交換樹脂層析	33
4.2.3  膠體過濾層析	34
4.2.4  酵素之分子量判定（CBR染色法）	34
4.2.5  綜合結果	35
4.3  酵素之特性分析	44
4.3.1  酵素之最適反應溫度與熱安定性	44
4.3.2  酵素之最適反應pH與pH安定性	44
4.3.3  金屬離子與抑制劑對酵素之影響	45
4.3.4  界面活性劑對酵素活性之影響	46
4.3.5  基質特異性	46
4.3.6  胜肽質譜鑑定	47
4.4  抗氧化性質分析	55
4.4.1  自由基清除能力	55
4.4.2  金屬離子螯合能力	55
4.4.3  還原能力	56
4.4.4  酚類化合物之定量	57
4.4.5  綜合結果	57
參考文獻	60
附錄	73

圖 目 錄
圖2.1  幾丁質、幾丁聚醣之結構	4
圖4.1  不同碳/氮源對TKU006幾丁聚醣酶生產及DPPH自由基清除能力之影響。	24
圖4.2  SPP濃度對TKU006幾丁聚醣酶生產及DPPH自由基清除能力之影響。	26
圖4.3  培養體積對TKU006幾丁質酶生產及DPPH自由基清除能力之影響。	27
圖4.4  幾丁質酶/幾丁聚醣酶生產菌之發酵上清液DPPH自由基清除能力。	30
圖4.5  TKU006幾丁質酶/幾丁聚醣酶之純化分離流程	32
圖4.6  幾丁聚醣酶之DEAE-Sepharose CL-6B層析圖譜	36
圖4.7  幾丁聚醣酶之Macro-Prep DEAE層析圖譜	37
圖4.8  幾丁質酶之Sephacryl-S100層析圖譜	38
圖4.9  幾丁聚醣酶之Sephacryl-S100層析圖譜	39
圖4.10 TKU006幾丁質酶於SDS-PAGE之分子量分析	40
圖4.11 TKU006幾丁聚醣酶於SDS-PAGE之分子量分析	41
圖4.12 幾丁質酶最適反應pH、最適反應溫度及pH安定性與熱安定性	48
圖4.13 幾丁聚醣酶最適反應pH、最適反應溫度及pH安定性與熱安定性	49
圖4.14  TKU006發酵上清液與α-生育醇之DPPH清除能力比較本	58
圖4.15 發酵上清液與EDTA之亞鐵離子螯合能力比較	59

表 目 錄
表2.1  幾丁質/幾丁聚醣之應用	6
表2.2  幾丁質分解酵素之種類	8
表4.1  TKU006生產幾丁質酶/幾丁聚醣酶與DPPH自由基清除能力之較適培養條件	29
表4.2  B. cereus TKU006幾丁質酶純化總表	42
表4.3  B. cereus TKU006 幾丁聚醣酶純化總表	43
表4.4  金屬離子和抑制劑對幾丁質酶/幾丁聚醣酶活性之影響	50
表4.5  不同界面活性劑對酵素活性之影響	51
表4.6  酵素之基質特異性	52
表4.7  TKU006幾丁質酶之胺基酸序列比對	53
表4.8  TKU006幾丁聚醣酶之胺基酸序列比對	54
表4.9  微生物發酵產物之還原力比較	60

王三郎，2000。生物技術，高立圖書有限公司。

Aiba, S., 1992. Studies on chitosan: 4. Lysozymic hydrolysis of partially N-acetylated chitosans. Int J Biol Macromol. 14, 225-8.

Allen, G. G., Peyron, M., 1995. Molecular weight manipulation of chitosan I: kinetic of depolymerization by nitrous acid. Carbohydr Res. 277, 257-72.

Arora, N., Ahmad, T., Rajagopal, R., Bhatnagar, R. K., 2003. A constitutively expressed 36 kDa exochitinase from Bacillus thuringiensis HD-1. Biochem Biophys Res Commun. 307, 620-5.

Braham, R., Dawson, B., Goodman, C., 2003. The effect of glucosamine supplementation on people experiencing regular knee pain. Br J Sports Med. 37, 45-9.
	
Chiang, C. L., Chang, C. T., Sung, H. Y. 2003. Purification and properties of chitosanase from a mutant of Bacillus subtilis IMR-NK1. Enzyme Microb Technol. 32, 260-7.

Caillet, S., Yu, H., Lessard, S., Lamoureux, G., Ajdukovic, D., Lacroix, M., 2007. Fenton reaction applied for screening natural antioxidants. Food Chem. 100, 542-52.

Cao, Z., Sun, Y., 2009. Chitosan-based rechargeable long-term antimicrobial and biofilm-controlling systems. J Biomed Mater Res. 89, 960-7.

Choi, Y. J., Kim, E. J., Piao, Z., Yun, Y. C., Shin, Y. C., 2004. Purification and characterization of chitosanase from Bacillus sp. strain KCTC 0377BP and its application for the production of chitosan oligosaccharides. Appl Environ Microbiol. 70, 4522-31.

Deshpande, M.V., 1986. Enzymatic degradation of chitin & its biological applications. J Sci Ind Res. 45, 273-81.

Ding, Y., Zhao, Y., Tao, X., Zheng, Y. Z., Chen, J. F., 2009. Assembled alginate/chitosan micro-shells for removal of organic pollutants. Polymer 50, 2841-6.

Dinis, T.C., Madeira, V.M.C., Almeida, L., 1994. Action of phenolic derivatives (acetominophen, salicylate, and 5-aminosalicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers . Arch Biochem Biophys. 315, 166-74.

Dutta, P. K., Tripathi, S., Mehrotra, G. K., Dutta, J., 2009. Perspectives for chitosan based antimicrobial films in food applications. Food Chem. 114, 1173-82.

Dziezak, J. D., 1986. Preservatives: Antioxidants. Food Technol. 40, 94-102.

Gao, X. A., Ju, W. T., Jung, W. J., Park, R. D., 2008. Purification and characterization of chitosanase from Bacillus cereus D-11. Carbohydr Polym. 72, 513-20.

González-Aguilar, G. A., Valenzuela-Soto, E., Lizardi-Mendoza, J., Goycoolea, F., Martínez-Téllez, M. A., Villegas-Ochoa, M. A., Monroy-García, I. N., Ayala-Zavala, J. F., 2009. Effect of chitosan coating in preventing deterioration and preserving the quality of fresh-cut papaya 'Maradol' . J Sci Food Agric. 89, 15-23.

Halliwell, B., Gutteridge, J. M., 1984. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J. 219, 1-14.

Han, S., Lee, M., 2009. Crosslinking reactions of oxidized cellulose fiber. II. reactions between dialdehyde cellulose and chito-oligosaccharides on lyocell fabric. J Appl Polym Sci. 112, 709-14.

Hasegawa, M., Isogai, A., Onabe, F., 1993. Preparation of low-molecular-weight chitosan using phosphoric acid. Int J Pharm. 311, 122-9.

Imoto, T., Yagishita, K., 1971. A simple activity measurement by lysozyme. Agric Biol Chem. 35, 1154-6.

Jang, H. K., Yi, J. H., Kim, J. T., Lee, K. E., Choi, S. G., 2003. Purification, characterization, and gene cloning of chitosanase from Bacillus cereus H-1. Korean J Microbiol Biotechnol. 31, 216-23.

Jeon, Y. J., Kim, S. K., 2000. Continous production of chitooligosaccharides using a dual reactor system. Process Biochem. 35, 623-32.

Kendra, D. F., Hadwiger, L. A., 1984. Characterization of the smallest chitosan oligomer that is maximally antifungal to Fusarium solani and elicits pisatin formation in Pisum sativum. EXP Mycol. 8, 276-81.

Khan, A. J., Choudhuri, G., Husain, Q., Parmar, D., 2009. Polymorphism in glutathione-S-transferases: A risk factor in alcoholic liver cirrhosis. Drug Alcohol Depend. 101, 183-90.

Klevan, A., Tourasse, N. J., Stabell, F. B., Kolstø, A. B., Økstad, O. A., 2007. Exploring the evolution of the Bacillus cereus group repeat element bcr1 by comparative genome analysis of closely related strains. Microbiol. 153, 3894-908.

Knorr, D., 1984. Use of chitinous polymer in food-A challenge for food research and developent. Food Technol. 38, 85-97.

Koga, D., Sueshige, N., Orhono, K., Utsumi, T., Tanaka, S., Yamada, Y., Ide, A., 1988. Efficiency of chitinolytic enzyme in the formation of Tricholoma matsutake protoplasts. Agric Biol Chem. 52, 2091-3.

Koga D., Tsuyoshi, T., Sueshige, N., Utsumi, T., Ide, A. 1989. Kinetics of chitinase from yam, Dioscorea opposita THUNB. Agric Biol Chem. 53, 3121-6.

Kumar, 2000. A review of chitin and chitosan applications. React Funct Polym. 46, 1-27.

Lee, Y. S., Park, I. H., Yoo, J. S., Chung, S. Y., Lee, Y. C., Cho, Y. S., Ahn, S. C., Kim, C. M., Choi, Y. L., 2007. Cloning, purification, and characterization of chitinase from Bacillus sp. DAU101. Biores Tech. 98, 2734-41.

Lee, Y. S., Yoo, J. S., Chung, S. Y., Lee, Y. C., Cho, Y. S., Choi, Y. L., 2006. Cloning, purification, and characterization of chitosanase from Bacillus sp. DAU101. Appl Microbiol Biotechnol. 73, 113-21.

Li, F. H., Zhang, B. Y., Li, X. F., Xi, M., Zhang, N., 2009a. Preparation and characterization of thermoplastic biomaterial chitosan oligo-saccharide-g-polycaprolactone-g-cholesterol. J Funct Mater. 40, 677-80.

Li, Y. C., Sun, X. J., BI, Y., GE, Y. H., Wang, Y., 2009b. Antifungal activity of chitosan on Fsarium sulphureum in relation to dry rot of potato tuber . Agric Sci China. 8, 597-604.

Liang, T. W., Chen, Y. J., Yen, Y. H., Wang, S. L., 2007. The antitumor activity of the hydrolysates of chitinous materials hydrolyzed by crude enzyme from Bacillus amyloliquefaciens V656. Process Biochem. 42, 527-34.

Lin, S. B., Chen, S. H., Peng, K. C., 2009. Preparation of antibacterial chito-oligosaccharide by altering the degree of deacetylation of β-chitosan in a Trichoderma harzianum chitinase-hydrolysing process. J Sci Food Agric. 89, 238-44.

Liu, B. L., Kao, P. M., Tzeng, Y. M., Feng, K. C., 2003. Production of chitinase from Verticillium lecanii F091 using submerged fermentation. Enzyme Microb Technol. 33, 410-5.

Martinez-Garcia, G., Johnson, A. C., Bachmann, R. T., Williams, C. J.,   Burgoyne, A., Edyvean, R. G. J., 2008. Anaerobic treatment of olive mill wastewater and piggery effluents fermented with Candida tropicalis. J Hazard Mater. 164, 1398-405.

Minke, R., Blackwell, J., 1987. The Structure of α-chitin. J Mol Biol. 120, 167-81.

Miyakawa, H., Mason, R. P., Jiang, J., Kadiiska, M. B., 2009. Lipid-derived free radical production in superantigen-induced interstitial pneumonia. Free Radical Biol Med. In press.

Morales De La Vega, L., Barboza-Corona, J.　E., Aguilar-Uscanga, M.　G., Ramírez-Lepe, M.., 2006. Purification and characterization of an exochitinase from Bacillus thuringiensis subsp. aizawai and its action against phytopathogenic fungi. Can J Microbiol. 52, 651-7.

Nagahama, H., Kashiki, T., Nwe, N., Jayakumar, R., Furuike, T., Tamura, H., 2008. Preparation of biodegradable chitin/gelatin membranes with GlcNAc for tissue engineering applications. Carbohydr Polym. 73, 456-63.

Ohtakara, A., Matsunaga, H., Mitsutomi, M., 1990. Action pattern of Streptomyces griseus chitinase on partially N-acetylated chitosan. Agric Biol Chem. 54, 3191-9.

Overdijk, B., Van Steijn, G. J., 1994. Human serum contains a chitinase: identification of an enzyme, formerly described as 4-methylumbelliferyl-tetra-N-acetylchitotetraoside hydrolase (MU-TACT hydrolase). Glycobiol. 4, 797-803.

Oyaizu, M., 1986, Antioxidative activities of browning reaction prepared from glucosamine. Jpn J Nutr. 44, 307-15.

Rajapakse, N., Mendis, E., Jung, W. K., Je, J.-Y., Kim, S. K., 2005. Purification of a radical scavenging peptide from fermented mussel sauce and its antioxidant properties. Food Res Int. 38, 175-82.

Robert, W. K., Selitrennidkoff, C. P., 1988. Plant and bacterial chitinases differ in antifungal activity. J Biol Chem. 134, 169-71.

Sakai, K., Yokota, A., Kurokawa, H., Wakayama, M., Moriguchi, M., 1998. Purification and characterization of three thermostable endochitinases of a noble Bacillus strain, MH-1, isolated from chitin-containing compost. Appl Environ Microbiol. 64, 3397-402.

Shimada, K., Fujikawa, K., Yahara, K., Nakamura, T., 1992. Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. J Agric Food Chem. 40, 945-8.

Steenkamp, G. C., Keizer, K., Neomagus, H. W. J. P., Krieg, H. M., 2002. Copper(II) removal from polluted water with alumina/chitosan composite membranes. J Membr Sci. 197, 147-56.

Takayanagi, T., Ajisaka, K., Takiguchi, Y., Shimahara, K., 1991. Isolation and characterization of thermostable chitinases from Bacillus licheniformis X-7u. Biochim Biophys Acta, 1078, 404-10.

Wang, S. L., Chao, C. H., Liang, T. W., Chen, C. C., 2009a. Purification and characterization of protease and chitinase from Bacillus cereus TKU006 and conversion of marine wastes by these enzymes. Mar Biotechnol. 11, 334-44.

Wang, S. L., Chen, S. J., Wang, C. L., 2008a. Purification and characterization of chitinases and chitosanases from a new species strain Pseudomonas sp. TKU015 using shrimp shells as a substrate. Carbohydr Res. 343, 1171-9.

Wang, S. L., Lin, H. T., Liang, T. W., Chen, Y. J., Yen, Y. H., Guo, S. P., 2008b. Reclamation of chitinous materials by bromelain for the preparation of antitumor and antifungal materials. Biores Tech. 99, 4386-93.

Wang, S. L., Lin, T. Y., Yen, Y. H., Liao, H. F., Chen, Y. J., 2006a. Bioconversion of shellfish chitin wastes for the production of Bacillus subtilis W-118 chitinase. Carbohydr Res. 341, 2507-15.

Wang, S. L., Liou, J. Y., Liang, T. W., Liu, K. C., 2009b. Conversion of squid pen by using Serratia sp. TKU020 fermentation for the production of enzymes, antioxidants, and N-acetyl chitooligosaccharides. Process Biochem. 44, 854-61.

Wang, S. L., Peng, J. H., Liang, T. W., Liu, K. C., 2008c. Purification and characterization of a chitosanase from Serratia marcescens TKU011. Carbohydr Res. 343, 1316-23.

Wang, S. L., Wu, P. C., Liang T. W., 2009c. Utilization of squid pen for the efficient production of chitosanase and antioxidants through prolonged autoclave treatment. Carbohydr Res. 344, 979-84.

Wang, S. L., Yeh, P. Y., 2006b. Production of a surfactant- and solvent-stable alkaliphilic protease by bioconversion of shrimp shell wastes fermented by Bacillus subtilis TKU007. Process Biochem. 41, 1545-52.

Wang, S. L., Yeh, P. Y., 2008d. Purification and characterization of a chitosanase from a nattokinase producing strain Bacillus subtilis TKU007. Process Biochem. 43, 132-8.

Wang, Y. C., Yu, R. C., Chou, C. C., 2006. Antioxidative activities of soymilk fermented with lactic acid bacteria and bifidobacteria. Food Microbiol. 23, 128-35.

Wang, T., Jónsdóttir, R., Ólafsdóttir, G., 2009. Total phenolic compounds, radical scavenging and metal chelation of extracts from Icelandic seaweeds. Food Chem. 116, 240-8.

Wu, Y., Wang, Y., Luo, G., Dai, Y., 2009. In situ preparation of magnetic Fe3O4-chitosan nanoparticles for lipase immobilization by cross-linking and oxidation in aqueous solution. Biores Tech. 100, 3459-64.

Yen, G. C., Hsieh, C. L., 1998. Antioxidant activity of extracts from du- zhong (Eucommia ulmoides) toward various lipid peroxidation models in vitro. J Agric Food Chem. 46, 3952-7.

Yue, W. F., Li, G. L., Liu, J. M., Sun, J. T., Sun, H. X., Li, X. H., Wu, X. F., Miao, Y. G., 2007. Anti-oxidation and immune responses in mice upon exposure to manganese superoxide dismutase expressed in silkworm larvae, Bombyx mori L. Cell Biol Int. 31, 974-8.

Zhu, Y. P., Fan, J. F., Cheng, Y. Q., Li, L. T., 2008. Improvement of the antioxidant activity of Chinese traditional fermented okara (Meitauza) using Bacillus subtilis B2. Food Control. 19, 654-61.