臺灣博碩士論文加值系統

本研究之目的在分離人類糞便中具幾丁質和 (或) 幾丁聚醣水解能力的細菌並探討其產生幾丁質和 (或) 幾丁聚醣水解酵素之培養條件並純化此酵素。由10-8糞便稀釋液在含膠體幾丁質之BHIA (37oC, 2天) 上可發現三個具透明環菌落，鑑定後該三個菌落皆為Bacteroides fragilis。因此挑選其中一株分離株與參考株 B. fragilis CCRC 10620 一起進行酵素活性試驗。不論分離株或參考株，均不需誘導即有相當高之幾丁質酶活性 (培養一天活性即可達30 unit/ml以上)。但N-乙醯葡萄糖胺酶活性皆相當低。兩菌皆以 BHI 培養液、添加 0.5% (w/v) yeast extract，在37 oC培養五天為最佳之幾丁質酶生產條件。分離株所產粗酵素液經由超過濾膜濃縮後比活性僅有未濃縮前的1/8，再經硫銨沈澱後，以hight Q cartridge 層析後蛋白質量很少。經吸附試驗發現膠體幾丁質 (CC) 及幾丁質粉末 (CP) 等會吸附本分離株之幾丁質酶，因此以此為酵素快速純化之依據。粗酵素液經硫酸銨沈澱後以 1% CP 吸附並去除未吸附之蛋白質，於50oC水解24小時後，經native-PAGE分析後以CBR-250及4-MU-(GlcNAc)2活性染色後可見單一的蛋白質區帶；經SDS-PAGE判斷，此幾丁質酶為多個分子量為 31.8 kDa 的次單元所構成，比活性為433.96 unit/ml，純化倍率為2.57。

目 錄
頁次
中文摘要 I
英文摘要 II
第一章 前言 1
第二章 文獻整理 3
一、腸道微生物 3
1. 腸道菌相 4
2. 腸內菌對宿主健康的影響 4
3. Bacteroides fragilis 的特性 4
二、幾丁質及其衍生物的結構與應用 5
1. 幾丁質、幾丁聚醣的分子結構 5
2. N-乙醯幾丁寡醣及幾丁寡醣的分子結構 6
3. 幾丁質、幾丁聚醣的應用 6
4. 幾丁質及幾丁聚醣水解物的應用 6
4-1. 抑菌方面 6
4-2. 其他方面 7
三、幾丁質酶系統 8
1. 幾丁質酶的分佈、種類及水解幾丁質模式 8
1-1. 幾丁質酶的分佈 (動物、植物、微生物) 8
1-2. 幾丁質酶的種類 8
1-3. 水解幾丁質的模式 9
2. 影響微生物產生幾丁質酶的因子 10
3. 幾丁質酶的結構分析 10
3-1. 催化區 (catalytic domain) 10
3-2. 幾丁質結合區 (chitin-binding domains, ChBDs) 11
3-3. 纖維網蛋白第三功能部位 (Fibronectin type III-like domain) 12
3-4. Cadherin-like domain 12
四、動物消化幾丁質、幾丁聚醣及幾丁寡醣的情形 12
1. 幾丁質、幾丁聚醣在動物消化道的變化 13
2. 動物體消化道中幾丁質酶的分佈 13
3. 腸道微生物的消化作用 14
五、幾丁質酶活性測定及純化方法 16
1. 細菌幾丁質酶的純化 16
2. 幾丁質酶活性測定的探討 17
2-1. 傳統檢測法 17
2-2. 快速檢測法 18
第三章實驗方法與步驟 20
一、材料 20
1. 菌株來源 20
2. 糞便來源 20
3. 化學試藥 20
3-1. 有機溶劑 20
3-2. N-乙醯幾丁寡醣標準品 20
3-3. 酵素基質 21
3-4. 電泳試劑 21
3-5. 其他 21
4. 設備與器材 22
4-1. 厭氧菌培養 22
4-2. HPLC 之設備及管柱規格 22
4-3. FPLC 之設備 23
4-4. 蛋白質電泳 23
4-5. 其他 23
二、步驟 24
1. 糞便中幾丁質、幾丁聚醣水解菌之分離 24
1-1. 糞便取樣 24
1-2. 菌株分離 24
1-3. 菌落計數與菌株單離 25
1-4. 菌株鑑定 25
1-5. 菌株保存 25
1-6. 接種源的製備 25
2. B. fragilisg生產幾丁質酶最適培養條件 26
2-1. 不同培養基對幾丁質酶及N-乙醯葡萄糖胺酶活性之影響 26
2-2. 膠體幾丁質濃度對幾丁質酶活性及菌數之影響 26
2-3. 在BHI、37 oC 培養下幾丁質酶活性、菌數、pH的變化 26
2-4. 培養基中yeast extract濃度對幾丁質酶活性之影響 27
2-5. 不同培養溫度對幾丁質酶活性之影響 27
3. 幾丁質酶與其受質的吸附作用 27
3-1. 吸附作用對酵素活性之影響 27
3-1. 幾丁質粉末吸附幾丁質酶後水解產物分析 27
4. 分離株硫酸銨沈澱酵素幾丁質酶最適反應條件 28
4-1 最適反應pH的測定 28
4-2 最適反應溫度的測定 28
4-3 酵素熱穩定性的測定 29
5. 幾丁質酶的純化 29
5-1. 粗酵素液 (CES) 之製備 29
5-2. 超過濾膜濃縮粗酵素 29
5-3. 酵素之硫銨區分 29
5-4. Econo-Pac high Q Cartridge 層析法 30
6. 幾丁質酶的快速純化 30
三、方法 30
1. 幾丁聚醣的製備 30
2. 膠體幾丁質 (colloidal chitin；CC) 的製備 31
3. 膠體幾丁聚醣 (colloidal chitosan；CS) 的製備 31
4. 培養基的配製 31
4-1. Brain heart infusion broth (BHI) 31
4-2. Basal medium (PY broth-Peptone Yeast Extract) 32
4-3. 含幾丁質、幾丁聚醣培養基 (BCCA及BCSA) 的製備 33
5. 厭氧稀釋液 (Anaerobic dilution blanks, ADB) 33
6. 幾丁質酶活性分析 34
6-1. 反應試劑的配製 34
6-2. 酵素的反應步驟 34
7. Native 和 SDS-PAGE 電泳分析 35
7-1. 膠體的製備 35
7-2. Native-PAGE 分析 38
7-3. SDS-PAGE 分析 37
7-4. 膠片的保存 38
8. 蛋白質的定量 38
9. 統計分析 39
第四章 結果與討論 40
一、糞便中幾丁質、幾丁聚醣水解菌之分離 38
二、B. fragilisg生產幾丁質酶之最適培養條件 41
1. 不同培養基對幾丁質水解酵素活性的影響 41
2.添加不同濃度之CC對幾丁質酶活性及菌株生長的影響 42
3. 培養溫度對幾丁質酶活性的影響 42
4. Yeast extract添加量對幾丁質酶活性的影響 42
5. 兩菌株在BHI、37 oC 培養下幾丁質水解酵素活性、菌數、pH的變化 43
三、分離株幾丁質酶的純化 43
1 粗酵素液經濃縮、硫銨沈澱等粗步純化的結果 43
2. High Q-cartrige層析 44
3. 幾丁質酶與其受質的吸附作用 44
3-1. 分離株幾丁質酶與其受質吸附特性的測試 45
3-1-1. CC、CP對粗酵素液幾丁質酶活性之影響 45
3-1-2. CC、CP吸附幾丁質酶後水解產物分析 45
3-2. 其他多醣類的吸附作用 46
•α-cellulose及 cellulose對粗酵素液活性之影響 46
4. 分離株硫酸銨沈澱酵素幾丁質酶之最適反應條件 46
4-1. 硫酸銨沈澱酵素之最適反應pH及溫度 46
4-2. 硫銨沈澱酵素之熱穩定性 47
5. 幾丁質酶的快速純化 47
第五章 結論 71
第六章 參考文獻 72
圖目錄
頁次
圖一 在含 0.3% (W/V) CC 之 BHIA 平板上，B. fragilis CCRC 10620 (A) 及一號分離株 (B) 的菌落形態 (37oC，2天)。 51
圖二 不同培養液中一號分離株 (A) 及 B. fragilis CCRC 10620 (B) 的幾丁質酶的活性。 52
圖三 不同培養液中一號分離株 (A) 及 B. fragilis CCRC 10620 (B) 的 GlcNAcase 的活性。 53
圖四 培養液中不同膠體幾丁質濃度對一號分離株 (A) 及 B. fragilis CCRC 10620 (B) 幾丁質酶活性之影響。 54
圖五 在培養液中不同膠體幾丁質濃度對一號分離株 (A) 及 B. fragilis CCRC 10620 (B) 生長之影響。 55
圖六 不同培養溫度對一號分離株 (A) 及B. fragilis CCRC 10620 (A) 幾丁質酶活性之影響。 56
圖七 在培養液中不同yeast extract濃度對一號分離株 (A) 及B. fragilis CCRC 10620 (B) 幾丁質酶活性之影響。 57
圖八 一號分離株 (A) 及 B. fragilis CCRC 10620 (B) 於BHI中培養，菌數 (A1、B1)、pH (A2、B2) 及幾丁質酶活性 (A3、B3) 之變化情形。 58
圖九 一號分離株生產之粗酵素液經超過濾濃縮、硫酸銨沈澱後在High Q cartridge 管柱層析圖。 60
圖十 一號分離株生產之粗酵素液以 1% CP 吸附後 (4 oC, 靜置1小時) 於42.5oC 水解之幾丁質水解產物 HPLC 分析圖譜。 62
圖十一 pH對硫銨沈澱酵素幾丁質酶活性之影響。 64
圖十二 溫度對硫銨沈澱酵素幾丁質酶活性之影響。 65
圖十三 硫銨沈澱酵素幾丁質酶之熱穩定性。 66
圖十四 一號分離菌所產幾丁質酶經快速純化之SDS-PAGE電泳圖。 67
圖十五 硫銨沈澱的酵素及快速純化的幾丁質酶在native-PAGE之活性染色。 68
圖十六 以SDS-PAGE檢測分子量之檢量曲線。 69
表目錄
表一 分離株以 API 20 A 套組鑑定之結果。 49
表二 分離株以 API 32 A 套組鑑定之結果。 50
表三 硫酸銨沈澱酵素活性與蛋白質區分表。 59
表四 一號分離株生產之粗酵素液以 CC 及 CP 在不同條件進行吸附試驗後之酵素活性。 61
表五 一號分離株生產之粗酵素液以 cellulose 及 α-cellulose 在不同條件進行吸附試驗後之酵素活性。 63
表六 分離株幾丁質酶之快速純化摘要。 70

第六章 參考文獻
黃珊萍。2001。不同去乙醯度幾丁聚醣對腸內細菌生長之影響。國立台灣海洋大學食品科學研究所碩士論文。
周聰麟。2000。Aspergillus clavatus NTU-FC-7 之幾丁聚醣酶純化與酵素特性之探討。國立台灣大學農業化學研究所碩士論文。
康建智。2001。細菌幾丁質酶基因的誘導表現及生產N-乙醯幾丁寡醣最適化條件的研究。國立台灣海洋大學食品科學研究所碩士論文。
蔣馥蕾。2000。產黃纖維單胞桿菌幾丁質酶基因的選殖與酵素的快速純化。國立台灣海洋大學食品科學研究所碩士論文。
Alaten, D. M. F., Synstad, B., Brurberg, M. B., Hough, E., Riise, B. W., Eijsink, V. G. H. and Wierenga, R. K. 2000. Structure of a two-domain chitotriosidase from Serratia marcescens at 1.9-A resolution. Proc. Nat. Acad. Sci. USA 97(11):5842-5847.
Bradford, M. M. 1976. A rapid and sensitive method for the quantitative of microgram quantites of protein using the principle of protein-dye binding. Anal. Biochem. 72:248-254.
Chamoy, L., Nicolai, M., Ravaux, J., Quennedey, B., Gaill, F. and Delachambre, J. 2001. A novel chitin-binding protein from the vestimentiferan Riftia pachyptila interacts specially with β-chitin. J. Biol. Chem. 276(11):8051-8058.
Chen, H. C., Chang, C. C., Mau, W. J. and Yen, L. S. 2002. Evaluaation of N-acetylchitooligosaccharides as the main carbon sources for the growth of intestinal bacteria. FEMS Microbial.Lett. 209:53-56.
Chen, K. S., Lee, K. K. and Chen, H. C. 1994. A rapid method for detection of N-acetylglucosaminidase-type chitinase activity in crossed immunoelectrophoresis and sodium dodecyl sulfate—polyacrylamide gel electrophoresis gels using 4-methylumbelliferyl-N-acetyl- D-glucosaminide as substrate. Electrophoresis. 51:662-665.
Chen, S. J. and Chang, M. C. 1994. Immobilization of chitinase on a reversibly soluble-insoluble polymer for chitin hydrolysis. J. Chem. Technol. 60:133-140.
Chen, S. W., and Chen, H. C. 1999. Effect of oral administration of Cellulomonas flavigena NTOU 1-degraded chitin hydrolysate on physiological changes in rates. Food Sci. Agric. Chem. 1:186-189.
Chernin, L. S., Fuente, L. D. L., Sobolev, V., Haran, S., Vorgias, C. E., Oppenheim, A. B. and Chet, I. 1997. Molecular cloning, structural analysis, and expression in Escherichia coli of chitinase gene from Enterobacter agglomerans. Appl. Environ. Microbiol. 63(3):834-839.
Cirano, J. U. and John, F. P. 1991. Regulation of chitinase synthesis in trichoderma harzianum. J. Gen. Microbiol. 137:2163-2169.
Clark, J., Quayle, K. A., Macdonald, N. L., and Stark, J. R. 1988. Metabolism in marine flatfish-V. chitinolytic activities in dover sole, Solea Solea (L.). Comp. Biochem. Physiol. 90B:379-384.
Cohen-kupiec, R. and Chet, I. 1998. The molecular biology of chitin digestion. Curr. Opin. Biotech. 9:270-277.
Collinge, D. B., Kragh, K. M., Mikkslesen, J. D., Nielsen, K. K., Rasmussen, U., and Vad, K. 1993. Plant chitinase. J. Plant. 3:31-40.
Danielli, A., Loukeris, T. G., Lagueeux, M., Müller, H. M., Richman, A. and Kafatos, F. C. 2000. A modular chitin-binding protease associated with hemocytes and hemolymph in the mosquito Anopheles gambiae. Proc. Natl. Acad. Sci. USA 97(13):7136-7141
Danulat, E. 1986. Role of bacteria with regard to chitin degradation in the digestive tract of the cod Gadus morhua. Marine Biol. 90:335-343.
Escott, G. M., Hearn, V. M. and Adams, D.J. 1998. Inducible chitinolytic system of Aspergillus fumigatus. Microbiol. 144:1575-1581.
Finegold, S. M., Sutter, V. L., and Mathisen, G. E. 1983. Normal indigenous intestinal flora. In: human intestinal microflora in health and disease (Hentges, D. J., ed), pp. 3-31. Academic Press, London, U. K.
Frandeberg, E. and Schnurer, J. 1998. Antifungal activity of chitinolytic bacteria isolated from airtight stored cereal grain. Can. J. Microbiol. 44:121-127.
Frandberg, E. and Schnurer, J.1994. Evaluation of a chromogenic chiot-oligosaccharide analogue, p-nitrophenyl-β-D-N,N’ -diacetylchitobiose, for the measurement of the chitinolytic activity of bacteria. J. Appl. Bacteriol. 76:259-263.
Fukasawa, S., Arai, M., Wada, T., Shima, H. and Kurata, M. 1992. Properties of a chitinase from a marine luminous bacterium, Vibrio fischeri strain COT-A136. Chem. Pharm. Bull. 40:1631-1633.
Goncharova, G. I., Dorofeichuk, V. G., Smolianskaia, A. Z., and Sokolova, K. Ia. 1989. Microbial ecology of the intestines in the health and in pathology. Antibiot. Khimioter, 34: 462-466.
Gooday, G. 1990. The ecology of chitin degradation. In Advances in Microbial ecology. Edited by K. C. Marshall. Plenum Press, New York. p. 387-430.
Gupta, R. G., Saxena, R. K., Chaturvedl, P. and Virdi, J. S. 1995. Chitinase production by Streptomyces viridificans: its potential in fungal cell wall lysis. J. App. Bacteriol. 78:378-383.
Hashimoto, M., Ikegami, T., Seino, S., Ohuchi, N., Fukada, H., Sugiyama, J., Shirakawa, M. and Watanabe, T. 2000. Expression and characterization of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12. J. Biol. Chem. 182(11):3045-3054.
Hirano, S.,Itakura, C., Seino, H., Akiyama, Y., Nonaka, I., Kanbara, N., and Kawakami, T.1990.Chitosan as an ingredient for domestic animal feeds. Journal of Agricultural and Food Chemistry. 38:1214-1217.
Holdman, L. V., Cato, E. P., and Moore, W. E. (Eds.) 1997. Anaerobe Laboratory Manual, 4th edn. Virginia Polytechnic Institute Anaerobe Laboratory, Blacksburg, VA.
Hood, M. A. 1991. Comparison of four methods for measuring chitinase activity and the application of the 4-MUF assay in aquatic environments. J. Microbiol. Methods. 13:151-160.
HSU., S.C. and Lockwood, J. L. 1975. Powdered chitin agar as a selective medium for enumeration of actinomycetes in water and soil. Appl. Microbiol. 29(3):422-426.
Ikegami, T., Okada, T., Hashimoto, M., Seino, S., Watanabe, T. and Shrirakawa, M. 2000. Solution structure of the chitin-binding domain of Bacillus circulans WL-12 chitinase A1. J. Biol. Chem. 275(18):13654-13661.
Imoto, T. and Yagishita, K. 1971. A simple activity measurement of lysozyme. Argic. Biol. Chem. 35(7):1154-1156.
Iseli, B., S. Armand, T. Boller, J. M. Neuhaus and B. Henrissant. 1996. Plant chitinase use two different hydrolytic mechanisms. FEBS Lett. 382:186-188.
Linder, M. and Teeri, T. T. 1997. The roles and function of cellulose-binding domains. J. Biotechnol. 57:15-28.
Jeon, Y. J., and Kim, S. K. 1998. Bioactivities of chitosan oligosaccharides and their derivatives. In Advances in Chitin Science, R. H. Chen , and H. C. Chen, (Ed.), Vol. 3, p.328-333. Rita Advertising Co, ROC.
Jesus, D. L. C., Manuel, R., Jose, M. L., Antonio, H. G., Fernando, D. Jose, A. P. T., Antonio, L. and Tahia, B. 1993. Carbon source control on β-glucanase, chitobiaes and chitinase from Trichoderma harzianum. Arch. Microbiol. 159:316-322.
Jeuniaux, C. 1966. Chitinases. Methods Enzymol. 8: 644-650.
Kafetzopoulos, D., Martinou, A., and Bouriotis, V. 1993. Biocoversion of chitin to chitosan purification and characterization of chitin deacetylase from Mucor rouxii. Proc. Natl. Acad. Sci. 90:2564-2568.
Kobayashi, M., Watanabe, T., Suzuki, S., and Suzuki, M. 1990. Effect of N-acetylchitohexaose against Candida albicans infection of tumor-bearing mice. Microbiol. Immunol. 34:413-426.
Koga, D., Hirata, T., Sueshige, N., Tanaka, S., and Ide, A. 1992. Induction patterns of chitinase in yam callus by inoculation with autoalaved Fusarium oxysporum, ethylene, chitin, and chitosan oligosaccharides. Biosci. Biotech. Biochem. 56:280-285.
Koga, D., Jilka, J., and Kramer, K. J. 1983. Insect endochitinases : glycoproteins from moulting fluid, integument and pupal haemolymph of Manduca sexta L. Insect Biochem. 13: 265-305
.Krishnaveni, S., Liang, G.H., Muthukrishnan, S. and Manickman, A. 1999. Purification and partial characterization of chitinases from sorghum seeds. Plant Science. 144:1-7.
Kuranda, M. J. and Robbins, P. W. 1991. Chitinase is required for cell depuration during growth of Saccharomyces cerevisiae. J. Biol. Chem. 266:19758-19767.
Lindsay, G. J. H. 1985. Development of gastric chitinase activity in rainbow trout (Salmo gairdneri). Aquaculture 48:258-290.
Matsumoto, T., Ono, Y., Watanabe, T., Mikami, T., Suzuki, S., Suzuki, M., Matajira, Y., and Sakai, K. 1996. Effect of long term oral administration of chitin, partially degraded chitin and N-acetylchitoligosaccharide on immunological and blood enzyme levels in aged mice. Chitin chitosan research, Proceedings of the 10th symposium, Japanese society for chitin and chitosan. 2:94-95
Manson, F. D. C., Fletcher, T. C. and Gooday, G. W. 1992. Localization of chitinolytic enzymes in blood of turbot, Scophthalmus maximus, and their possible roles in defence. J. Fish Biol. 40:919-927.
Manson, F. D. C., Gooday, G. W., and Fletcher, T. C. 1989. The development of gastric and blood chitinase activity in the turbot, Scophthalmus maximus (L.). In Chitin and Chitosan, G. Skjak-Brek, T. Anthonsen, and P. Sandford. (Ed.) p.243-253. Elsevier Science Publishers Ltd. England.
McCarthy, K. J. and Gooday, G.W. 1992. A rapid and sensitive microassay for determination of chitinolytic activity. J. Microbiol. Mothods. 14:229-237.
Mellor, K. J., Nicholas, R. O. and Adams, D. J. 1994. Purification and characterization of chitinase from Candida albicans. FEMS Microbiol. Letts. 119:111-118.
Minke, R. and Blackwell,J. 1978. The structure of a chitin. J. Mol. Biol. 120:167.
Molano, J., Duran, A. and Cabib, E. 1977. A rapid and sensitive assay for chitinase using tritiated chitin. Anal. Biochem. 83:648-656.
Moncrief, J. S., R. Obiso, L. A. Barroso, J. J. Kling, R. L. Wright, R. l. Van Tassell, D. M. Lyerly, and T.D. Wilkins. 1994. The enterotoxin of Bacteroides fragilisis is a matrix metalloprotease. Infect. Immune. 63:175-181.
Morgan, W. T. J. and Elson, L. A. 1934. A colormetric method for the determination of N-acetylglucosamine and N-acetylchondrosamine. Biochem. J. 28:988-995.
Morgavi, D. P., Sakarada, M., Tomita, Y. and Onodera, R. 1996. Electrophoretic forms of chitinolytic and lysozyme activities in Ruminal Protozoa. Curr. Microbiol. 32:15-118.
Morimoto, K., K. Tetsuya, K. Sakka and K. Ohmiya. 1997. Cloning, sequencing, and expression of the gene encoding Clostridium paraputrificum chitinase chiB and analysis of the function of novel Cadherin-like domains and a chitin-binding domain. J. Bacteriol. 179:7306-7314.
Murao, S., Kawada, T., Itoh, H., Oyama, H. and Shin, T. 1992. Purification and characterization of a novel type of chitinase from Vibrio alginolyticus TK-22. Biosci. Biotech. Biochem. 56:368-369.
Muzzarelli, R. A. A. 1996. Chitosan-based dietary foods. Carbohydr. Polym..29:309-316.
Nishimura, Y., Watanabe, Y., Hong, J. M.,Takeda, H., Wada, M., and Yukawa, M. 1997. Intestinal absorption of 14C-chitosan in rats. Chitin Chitosan Research. 3:55-61.
O’Brien, M. and Colwell, R. R. 1987. A rapid test for chitinase activity that uses 4-Methylumbelliferyl-N-acety-D-glucosaminide. Appl. Environ. Microbiol.
Ohtakara, A., Izume, M. and Mitsutomi, M. 1988. Action of microbial chitinases on chitosan with different degrees of deacetylation. Agric. Biol. Chem. 52:3181-3182.
Okamoto, Y., Nose, M., Miyatyke, K., Sekine, J., Oura, R., Shigemasa, Y., Minami, S. Physical changes of chitin and chitosan in canine gastrointestinal tract. Carbohydrate polymers. 44:211-215.
Okutani, K., and Kimata, M. 1964. Studies on chitinolytic enzyme present in aquatic animals-Ⅲ. Distribution of chitinase in digestive organs of a few kinds of aquatic animals. Bulletin of the Japanese Society of Scientific Fisheries. 30:574-576.
Olsen, M.A., Blix, A.S., Utsi, T.H.A., Sormo, W., and Mathiesen, S.D. 2000. Chitinolytic bacteria in the minke whale forestomach.Can. J. Microbiol. 46:85-94.
Osswald, W. F., McDonald, R. E., Nieda, R. P. and Mayer, R.T. 1992. Quantitative fluorometric analysis of plant and microbial chitosanase. Anal. Biochem. 204:40-46
Otakara, A.1961. Studies on the chitinolytic enzyme of Black-koji mold. Part Ⅱ. Purification of chitinase Agric Biol. Chem. 25:54-60.
Pel, R., Wijngaard, A. J. V. D., Epping, E., and Gottschal, J. C. 1990. Comparison of the chitinolytic properties of Clostridium sp. strain 9.1 and a chitin-degrading bacterium from the intestinal tract of the plaice, Pleuronectes platessa (L.). J. Gen.Microbiol. 136:695-704.
Petschow, B. W. and Talbott, R.D. 1991. Response of bifidobacterium species to growth promoters in human and cow milk. Pediatr. Res. 29:208-213.
Place, A. R. 1996. The biochemical basis and ecological significance of chitin digestion. In Chitin Enzymology, R. A. A. Muzzarelli (Ed.), vol.2 p.39-54. Atec Edizioni-Grottammare, Italy.
Ravi Kumar, M. N. V. 2000. A review of chitin and chitosan application. Reative and functional polymers. 46:1-27.
Reissing, J. L., Strominger, J. L. and Leloir, L. F. 1955. A modification colormetric method for the estimation of N-acetylaminosugars. J. Biol. Chem. 217:959-966.
Richard, J. O. JR., A. O. Azghani, and T. D. Wilkins. 1997. The Bacteroides fragilis fragilysin disrupts the paracellular barrier of epithelial cells. Infect. Immune. 65:1431-1439.
Roberts, A. K., Chierici, R., Sawtzki, G., Hill, M. J., Volpato, S., and Vigi, V. 1992. Supplementation of an adapted formula with bovin lactoferrin: 1. Effect on the infant faecal flora. Acta. Paediatr. 81:119-124.
Roberts, R. L. and Cabib, E. 1982. Serratia marcescens chitinaes:one-step purification and use for the determination of chitin. Anal. Biochem. 127:402-412.
Sabapathy, U. and Teo, L. H. 1993. A quantitative study of some digestive enzymes in the rabbitfish, Siganus canaliculatus and the sea bass, Lates calcarifer. J. Fish Biol. 42:595-602.
Sakai, K., Katsumi, R., Isobe, A., and Nanji, F. 1991. Purification and hydrolytic action of a chitosanase from Nocardia orientalis. Biochem. Biophys. Acta. 1079:65-72.
Sakai, K., Narihara, M., Kasama, Y., Wakayama, M. and Moriguchi, M. 1994. Purification and characterization of thermostable β-N-acetylhexosaminidase of Bacillus stearothermophilus CH-4 isolated from chitin-containing compost. Appl. Environ. Microbiol. 60:2911-2915.
Salmine, S., Isolauri, E. and Onnela, T. 1995. Gut flora in normal and disordered atates. Chemotherapy. 41:5-15.
Scheltinga, A.C.T., Hennig, M., Dijkstra, B. W. 1996. The 1.8 A resolution structure of hevanine, a plant chitinase /lysozyme, and analysis of glycosyl hydrolase family 18. J. Mol. Biol. 262:243-257.
Sera, H. and Okutani, K. 1968. Studies on chitinolytic enzyme present in aquatic animals-Ⅶ. The chitinolytic enzyme present in a sea bream, Acanthopagrus schlegedi. Bulletin of the Japanese Society of Scientific Fisheries. 34:920-924.
Shahidi, F., Arachchi, J. K. V., and Jeon, Y. J. 1999. Food applications of chitin and chitosans. Food Sci. Technol. 10:37-51.
Shimosaka, M., Nogawa, M., Ohno, Y. and Okazaki, M. 1993. Chitosanase from the plant pathigenic fungus, Fusarium solani sp. Phaseoli- purification and some properties. Biosci. Biotech. Biochem. 57:231-235.
Simpson, B. K., Gagne, N. Ashie, I. N. A., and Noroozi, E. 1997. Utilization of chitosan for raw shrimp. Food Biotechnology 11(1):25.
Stankiewicz, B. A., Briggs, D. E. G., Evershed, R. P., Flannery, M. B., and Wuttke, M. 1997. Preservation of chitin in 25-million-year-old fossils. Science 276:1541-1543.
St leger, R.J., Staples, R. C. and Roberts, D.W. 1993. Entomopathogenic isolates of Metarbizium anisopliae, Beauveria bassiana and Aspergillus flavus produce multiple extracellular chitinase isoenzymes. J. Invertebr .Pathol. 61:81-84.
Streichsbier, F. 1983. Utilization of chitn as sole carbon and nitrogen source by Chromobacterium violaceum. FEMS Microbiol. Letts. 19:129-132.
Studer, M., Flück, K. and Zimmermanm,W. 1992. Production of chitinase by Aphanocladium album grown on crystalline and colloidal chitin. FEMS Microbiol. Letts. 99:213-216.
Sugita, H., Kumazawa, J., and Deguchi, Y. 1996. Production of chitinase and β- N- acetylglucosaminidase by intestinal bacteria of pinnipedian animals. Lett. Appl. Microbiol. 23:275-278.
Suzuki, K., Tokoro, A., Okawa, Y., Suzuki, S., and Suzuki, M. 1985. Enhancing effects of N-acetyl-chito-oligosaccharides on the active oxygen-generating and microbicidal activities of peritoneal exudate cells in mice. Chem. Pharm. Bull. 33:886-888.
Svitil, A. L. and Kirchman, D. L..1998. A chitin-binding domain in a marine bacterial chitinase and other microbial chitinase: implications for the ecology and evolution of 1,4-β-glycanases. Microbio. 144:1299-1308.
Takahashi, M., Tsukiyama, T. and Suzuki, T. 1993. Purifications and some properties of chitinase produced by Vibrio sp. J. Ferment. Bioeng. 75:457-459.
Tanaka, Y., Tanioka, S., Tanaka, M., Tanigawa, T., Kitamura, Y., Minami, S., Okamoto, Y., Miyashita, M., and Nanno, M. 1997. Effects of chitin and chitosan particles on BALB/c mice by oral and parenteral administration. Biomaterials 18:591-595.
Tannock, G. W. 1995. Microecology of the gastrointestinal tract in relation to lactic acid bacteria. Int. Diary Journal. 5:1059-1070.
Terada, A., Hara, H., Sato, D., Higashi, T., Nakayama, S., Sakamoto, K., Ishioka, E., Maezaki, Y., Tsugita, T., Takekawa, T., and Mitsuoka, T. 1995. Effect of dietary chitosan on faecal microbiota and faecal metabolites of human. Microb. Ecol. Health Dis. 8:15-21.
Terwisscha van Scheltinga, A. C., S. Armand, K. H. Kalk,A. Isogai, B. Henrissat and B. W. Dijkstra. 1995. Stereochemistry of chintin hydrolysis by a plant chitinase/ lysozyme/ and X-ray structure of a complex with allosamidin: Evidence for substract assisted catalysis. Biochem. 34: 15619-15623.
Tjoelker, L. W., Gosting, L., Frey, S., Hunter, C. L., Trong, H. L., Steiner, B., Brammer, H. and Gray P. W. 2000. Structural and functional definition of the human chitinase chitin-binding domain. J. Biol. Chem. 275(1):514-520
Tokoro, A., Kobayashi, M., Tatewaki, N., Suzuki, K., Okawa, Y., Mikami, T., Suzuki, S., and Suzuki, M. 1989. Protective effect of N-acetyl chitohexaose on Listeria monocytogenes infection in mice. Microbiol. Immunol. 33:357-367.
.Tronsmo, A. and Harman, G. E. 1993. Detection and quantification of N-acetyl-β-D-glucosaminidase, chitobiosidase, and endochitinase in solutions and on gels. Anal. Biochem. 208:74-79.
Tsujibo, H., Yoshida, Y., Imada, C., Okami, Y., Miyamoto, K. and Inamori, Y. 1991. Isolation and characterization of a chitin degrading marine bacterium belonging to the genus Alteromonas. Nippon Suisan Gakkaishi. 57:2127-2131.
Tsujibo, H., Orikoshi, H., Imada, C., Okami, Y. Miyamoto, K., and Inamori, Y. 1993. Site-directed mutagenesis of chitinase from Aeromonas sp. Strain O-7. Biosci. Biotech. Biochem. 57:1396-1397.
Tsujibo, H., Yoshida, Y., and Miyamoto, K. 1992. Purification, properties, and partial amino acid sequence of chitinaes from a marine Alteromonas sp.strain O-7. Can. J. Microbiol. 38:891-897.
Tsukada, K., Matsumoto, T., Aizawa, K., Tokoro, A., Naruse, R., Suzuki, S., and Suzuki, M. 1990. Antimetastatic and growth-inhibitory effects of N-acetylchitohexaose in mice bearing Lewis lung carcinoma. Jpn. J. Cancer Res. 81:259-265.
Uchida, Y., Izume, M., and Ohtakara, A. 1988. Preparation of chitosan oligomers with purified chitosanase and its application. In Chitin and Chitosan, G. Skjak-Brek, T. Anthonsen, and P. Sandford. (Ed.), p.373-382. Elsevier Science Publishers Ltd, England.
Ueno, H., Miyashita, K., Sawada, Y. and Oba, Y. 1990. Purification and some properties of extracellular chitinase from Streptomyces sp. S-84. J. Gen. Appl. Nicrobiol. 36:377-392.
Uhrich, K. E., Cannizzaro, S. M., Langer, R.S., Shakesheff, K. M. 1999. Polymeric systems for controlled drug release. Chem. Rev. 99:3181.
Ulhoa, C.J. and Peberdy, J.F. 1991. Regulation of chitinase synthesis in Trichoderma harzianum. J. Gen. Microbiol. 137:2163-2169.
Vrba, J., Simek, K., Nedoma, J. and Hartman, P. 1993. 4-Methylumbelliferyl-β-N-acetylglucosaminide hydrolysis by a hight-affinity enzyme, a putative marker of protozoan bacterivory. Appl. Environ. Microbiol. 59:3091-3101.
Warren, R. A. J. 1996. Microbial hydrolysis of polysacchaides. Annu Rev Microbiol., 50:183-212.
Watanabe, T., Oyanagi, W., Suzuki, K. and Tanaka, H. 1990. Chitinase system of Bacillus circulans WL-12 and importance of chitinase A1 in chitin degradation. J. Bacteriol. 172: 4017-4022.
Watanabe, T., M. Uchida, K. Kobori and H. Tanaka. 1994. Site-directed mutagenesis of the Asp-197 and Asp-202 residues in chitinase Al of Bacillus circulans WL-12. Biosci. Biotech. Biochem. 58:2283-2285.
Watanabe, T., K. Suzuki, W. Oyanagi, K. Ohnish and H. Tanaka. 1990. Gene cloning of chintinase Al from Bacillus circulans WL-12 revealed its evolutionary relationship to Serratia chitinase and to the type Ⅲ homology units of fibronectin. J. Biol. Chem. 265:15659-15665.
Wortman, A.T., Somerville, C. C. and Colwell, R.R. 1986. Chitinase determinants of Vibrio vulnificus : gene cloning and applications of a chitinaes probe. Appl. Environ. Microbiol. 52:142-145.
Wu, M. L., Chuang, Y. C., Chen, J. P., Chen, C. S., and Chang, M. C. 2001. Identification and characterization of the three chitin-binding domains within the multidomain chitinase Chi92 from Aeromonas hydrophila JP101. Appl. Environ. Microbiol. 67(11): 5100-5106.
Yabuki, M., Mizushina, K., Amatatsu, T., Ando, A., Fuji, T., Shimada, M. and Yamashita, M. 1986. Purification and characterization of chitinase and chitobiase produced by Aeromonas hydrophila subsp. Anaergenes A52. J. Gen. Appl. Microbiol. 32:35-38.
Yamada, A., Shibbuya, N., Kodama, O., and Akatsuka, T. 1993. Induction of phytoalexin formation in suspension-cultured rice cells by N-acetylchitooligosaccharides. Biosci. Biotech. Biochem. 57:405-409.
Yanai, K., Takaya, N., Kojima, N., Horiuchi, H., Ohta, A. and Takagi. M. 1992. Purification of two chitinase from Rhizopus oligosporus isolation and sequencing of the encoding genes. J. Bacteriol. 174:7398-7406.