幾丁質是地球上含量第二多的聚合物，具有線性的多醣結構，由N-乙醯基-D-葡萄糖胺組成。幾丁質為節肢動物外骨骼的主要成分，例如：昆蟲和甲殼類動物，其水解產物，殼聚醣和幾丁質寡醣，可應用於食品市場。此外，某些具有特定長度的寡醣在工業、農業和醫學領域的運用逐漸被注意。然而，由於幾丁質的結構過於穩定，所以尚未有工業化流程將幾丁質轉變為特定幾丁質寡醣。我們研究一個在本土細菌株中發現的幾丁質分解酶，具有水解幾丁質的能力，我們將其命名為Cloudtin。Cloudtin擁有一個獨特的結構，包含兩個幾丁質酶域分別分佈在N和C端。在弱酸性的條件下，Cloudtin有較好的水解能力，其主要產物為單醣和雙醣。由於幾丁質寡糖的應用潛力，我們對Cloudtin進行工程改造以提高其產生幾丁質寡糖的效率。我們根據結構和序列比對設計了8個突變蛋白。通過1維核磁共振、高效液相層析和動力學測定法評估不同突變蛋白的活性和最終產物，我們鑑定出Cloudtin的酶活性主要來自C端幾丁質酶域，而連接區域可能有助於幾丁質辨識。最後，我們也確定了三個能生產三醣和四醣的突變蛋白，而增加幾丁質內切酶的活性的新策略也在本研究中被討論。

Chitin, the second richest polymers in earth, has a linear polysaccharide structure, which is composed by N-acetyl-D-glucosamine. It is a principle ingredient of the exoskeletons of arthropods, such as insects and crustaceans. The hydrolysis products, such as chitosan and chitin oligosaccharides, become profitable in food market. In addition, the oligosaccharides with certain lengths starts to be noticed in the industrial, agricultural and medical interests. The conversion of chitin into chitin polysaccharides cannot be robotically achieved because of the tightly packed structure. We focused on a potential chitinase candidate identified from local bacteria strain. The chitinase is named as Cloudtin, which has demonstrated the ability in hydrolyzing chitin. Cloudtin contains a unique structure in which two chitinase domains are respectively distributed in the N- and C-terminus. Cloudtin performed efficient hydrolysis under weak acidic conditions and hydrolyzed products of mono-, and disaccharide. Due to the potent applications of chitin oligosaccharides, we intend to engineer Cloudtin to enhance its efficiency of yielding chito-oligosaccharide. We designed 8 mutants based on the structure and sequence comparisons. The activities and final products of the different mutants were evaluated by 1H NMR, HPLC and kinetic assay. We identified the enzyme activity of Cloudtin mainly derived from the C-terminal chitinase domain while the linker domain might assist the chitin recognition. Together with analysis by HPLC, we identified three mutants with enhanced ability in producing tri- and tetra-saccharide. New strategies in increasing the endo-chitinase activity has been discussed.

Chapter 1 Introduction
1.1 Chitin………………………………………………………………………...1
1.2 Chitin derivatives…………………………………………………………….2
1.3 Antioxidant activity of chitin and its derivatives…………………………….3
1.4 Anti-inflammatory effect of chitin and its derivatives……………………….4
1.5 Antimicrobial effects of chitin and its derivatives…………………………...5
1.6 Immuno-stimulating and anticancer effects………………………………….5
1.7 Application in drug delivery of chitin and its derivatives……………………6
1.8 Chitinases and its classification………………………………………………6
1.9 Aim of this study…………………………………………………………......8
Chapter 2 Materials and Methods
2.1 Protein expression and purification of Cloudtin………………...………….10
2.2 Preparation of colloidal chitin……………………………………...……….11
2.3 Optimum conditions of enzymatic hydrolysis of the colloidal chitin………11
2.4 Nuclear magnetic resonance (NMR)……………………………………......12
2.5 High performance liquid chromatography (HPLC) analysis chito-
oligosaccharide….…………………………………………….....................12
2.6 Kinetic and enzyme assays.............................................................................12
Chapter 3 Results
3.1 Overall structural property of Cloudtin..........................................................14
3.2 Truncations design and purification...............................................................15
3.3 The C-domain chitinase structure in complex with oligosaccharide.............17
3.4 Mutation design based on structure and sequence analysis...........................19
3.5 Mutation expression and purification............................................................24
3.6 Colloidal chitin...............................................................................................25
3.7 Optimum condition for hydrolyzing chitin and NMR detection....................25
3.8 HPLC analysis of chito-oligosaccharide........................................................29
3.9 Kinetic, Michaelis-Menten equation, and Linearweaver-Burk plot...............33
Chapter 4 Discussion..................................................................................................42
References...................................................................................................................46

References

Abdou, E. S., Nagy, K. S. A., &Elsabee, M. Z. (2008). Extraction and characterization of chitin and chitosan from local sources. Bioresource Technology, 99(5), 1359–1367. https://doi.org/10.1016/j.biortech.2007.01.051
Chang, K. L. B., Lee, J., &Fu, W. R. (2000). HPLC Analysis of N-acetyl-chito-oligosaccharides during the Acid Hydrolysis of Chitin. Journal of Food and Drug Analysis, 8(2), 75–83.
Cheba, B. A., Zaghloul, T. I., El-Massry, M. H., &El-Mahdy, A. R. (2017). Kinetics Properties of Marine Chitinase from Novel Red Sea Strain of Bacillus. Procedia Engineering, 181, 146–152. https://doi.org/10.1016/j.proeng.2017.02.383
Chung, Y. C., Su, Y. P., Chen, C. C., Jia, G., Wang, H. L., Wu, J. C. G., &Lin, J. G. (2004). Relationship between antibacterial activity of chitosan and surface characteristics of cell wall. Acta Pharmacologica Sinica, 25(7), 932–936.
DaSilva, C. A., Chalouni, C., Williams, A., Hartl, D., Lee, C. G., &Elias, J. A. (2009). Chitin Is a Size-Dependent Regulator of Macrophage TNF and IL-10 Production. The Journal of Immunology, 182(6), 3573–3582. https://doi.org/10.4049/jimmunol.0802113
Devlieghere, F., Vermeulen, A., &Debevere, J. (2004). Chitosan: Antimicrobial activity, interactions with food components and applicability as a coating on fruit and vegetables. Food Microbiology, 21(6), 703–714. https://doi.org/10.1016/j.fm.2004.02.008
Helander, I. M., Nurmiaho-Lassila, E. L., Ahvenainen, R., Rhoades, J., &Roller, S. (2001). Chitosan disrupts the barrier properties of the outer membrane of Gram-negative bacteria. International Journal of Food Microbiology, 71(2–3), 235–244. https://doi.org/10.1016/S0168-1605(01)00609-2
Henrissat, B., &Bairoch, A. (1993). New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochemical Journal, 293(3), 781–788. https://doi.org/10.1042/bj2930781
Huang, R., Mendis, E., Rajapakse, N., &Kim, S. K. (2006). Strong electronic charge as an important factor for anticancer activity of chitooligosaccharides (COS). Life Sciences, 78(20), 2399–2408. https://doi.org/10.1016/j.lfs.2005.09.039
Hurtado-Guerrero, R., &vanAalten, D. M. F. (2007). Structure of Saccharomyces cerevisiae Chitinase 1 and Screening-Based Discovery of Potent Inhibitors. Chemistry and Biology, 14(5), 589–599. https://doi.org/10.1016/j.chembiol.2007.03.015
Janes, K. A., Fresneau, M. P., Marazuela, A., Fabra, A., &Alonso, J. (2001). Janes-2001-Chitosan nanoparticl.pdf. Journal of Controlled Release, 73, 255–267. Retrieved from www.elsevier.com
Je, J. Y., &Kim, S. K. (2006). Chitosan derivatives killed bacteria by disrupting the outer and inner membrane. Journal of Agricultural and Food Chemistry, 54(18), 6629–6633. https://doi.org/10.1021/jf061310p
Jeon, Y. J., &Kim, S. K. (2000). Continuous production of chitooligosaccharides using a dual reactor system. Process Biochemistry, 35(6), 623–632. https://doi.org/10.1016/S0032-9592(99)00118-1
Kezuka, Y., Ohishi, M., Itoh, Y., Watanabe, J., Mitsutomi, M., Watanabe, T., &Nonaka, T. (2006). Structural Studies of a Two-domain Chitinase from Streptomyces griseus HUT6037. Journal of Molecular Biology, 358(2), 472–484. https://doi.org/10.1016/j.jmb.2006.02.013
Kim, M. S., Hyun, J. Y., Mi, K. Y., Kim, N. S., Bum, S. S., &Kim, H. M. (2004). Inhibitory effect of water-soluble chitosan on TNF-α and IL-8 secretion from HMC-1 cells. Immunopharmacology and Immunotoxicology, 26(3), 401–409. https://doi.org/10.1081/IPH-200026887
Kobayashi, M., &Suzuki, S. (1990). Effect of N-Acetylchitohexaose against Candida Infection of Tumor-Bearing Mice albicans and Masuko SUZUKI *, of Microbiology , and 2Second of Hygienic Chemistry , Tohoku College of Pharmacy , Sendai , Miyagi ( Accepted for publication ., 34(5), 413–426.
Lavall, R. L., Assis, O. B. G., &Campana-Filho, S. P. (2007). β-Chitin from the pens of Loligo sp.: Extraction and characterization. Bioresource Technology, 98(13), 2465–2472. https://doi.org/10.1016/j.biortech.2006.09.002
Maeda, Y., &Kimura, Y. (2004). Antitumor Effects of Various Low-Molecular-Weight Chitosans Are Due to Increased Natural Killer Activity of Intestinal Intraepithelial Lymphocytes in Sarcoma 180–Bearing Mice. The Journal of Nutrition, 134(4), 945–950. https://doi.org/10.1093/jn/134.4.945
Mangas-Sánchez, J., &Adlercreutz, P. (2015). Enzymatic preparation of oligosaccharides by transglycosylation: A comparative study of glucosidases. Journal of Molecular Catalysis B: Enzymatic, 122, 51–55. https://doi.org/10.1016/j.molcatb.2015.08.014
Moon, J. S., Kim, H. K., Koo, H. C., Joo, Y. S., Nam, H. M., Park, Y. H., &Kang, M.Il. (2007). The antibacterial and immunostimulative effect of chitosan-oligosaccharides against infection by Staphylococcus aureus isolated from bovine mastitis. Applied Microbiology and Biotechnology, 75(5), 989–998. https://doi.org/10.1007/s00253-007-0898-8
Ngo, D. N., Lee, S. H., Kim, M. M., &Kim, S. K. (2009). Production of chitin oligosaccharides with different molecular weights and their antioxidant effect in RAW 264.7 cells. Journal of Functional Foods, 1(2), 188–198. https://doi.org/10.1016/j.jff.2009.01.008
Rahman, I., Biswas, S. K., &Kode, A. (2006). Oxidant and antioxidant balance in the airways and airway diseases. European Journal of Pharmacology, 533(1–3), 222–239. https://doi.org/10.1016/j.ejphar.2005.12.087
Sahai, A. S., &Manocha, M. S. (1993). Chitinases of fungi and plants: their involvement in morphogenesis and host-parasite interaction. FEMS Microbiology Reviews, 11(4), 317–338. https://doi.org/10.1111/j.1574-6976.1993.tb00004.x
Suzuki, K. O., Mikami, T., Okawa, Y., Tokoro, A., &Suzuki, M. (1986). Note Antitumor effect of hexa-N-acetylchitohexaose and chitohexaose*, 151, 403–408.
Umemoto, N., Kanda, Y., Ohnuma, T., Osawa, T., Numata, T., Sakuda, S., …Fukamizo, T. (2015). Crystal structures and inhibitor binding properties of plant class v chitinases: The cycad enzyme exhibits unique structural and functional features. Plant Journal, 82(1), 54–66. https://doi.org/10.1111/tpj.12785
Wang, S. L., &Chang, W. T. (1997). Purification and characterization of two bifunctional chitinases/lysozymes extracellularly produced by Pseudomonas aeruginosa K- 187 in a shrimp and crab shell powder medium. Applied and Environmental Microbiology, 63(2), 380–386.
Zhu, X., Cai, J., Yang, J., &Su, Q. (2005). Determination of glucosamine in impure chitin samples by high-performance liquid chromatography. Carbohydrate Research, 340(10), 1732–1738. https://doi.org/10.1016/j.carres.2005.01.045
Zhu, X. Y., Zhao, Y., Zhang, H. D., Wang, W. X., Cong, H. H., &Yin, H. (2019). Characterization of the specific mode of action of a chitin deacetylase and separation of the partially acetylated chitosan oligosaccharides. Marine Drugs, 17(2), 6–10. https://doi.org/10.3390/md17020074