臺灣博碩士論文加值系統

幾丁質酵素檢測方法大致可分為兩類，一是利用受質被水解後，其物理及化學的特性進行檢測；另一種則是利用改質後的受質，經幾丁質酵素水解後檢測其反應情形，即可得知酵素活性。本研究利用化學方法將chitin azure改質成為凝膠態，藉著增加其在溶液中的表面積與懸浮性，並以SGCTS為標準品來檢測，驗證本法於幾丁質酵素檢測的上準確度與靈敏度高且不易受其他物質干擾。
蛋白質體是指細胞所有表現的蛋白質，目前常以雙向電泳來進行分析，由於分泌幾丁質酵素的微生物，其所分泌的幾丁質酵素常不只ㄧ種，因此利用蛋白質體的方法來分析，可迅速獲得等電點、分子量等訊息。蛋白質體的分析法常受限於蛋白質偵測方法之靈敏度，故本研究則在SDS-PAGE分析酵素活性作為偵測的標準，使其靈敏度大幅提升。藉由本研究所建立的方法分析野生菌種Aeromonas schubertii所產生的幾丁質酵素，來確認此法之可行性。其能產生七種幾丁質酵素，等電點分別為4.23、4.29、4.39、4.71、4.97、5.73、6.23之間，其中有兩種具SDS-耐受性，分子量皆大於35kDa。在反應pH值的變化下，可知其活性於pH 6.0環境下有較佳的表現。最後自校內土壤中篩選出具有幾丁質酵素之菌體，應用此法分析亦可知其分泌幾丁質酵素之種類。本技術的建立，不但提升偵測的靈敏度，同時也可分析多種蛋白質性質的功能，且藉著改變環境的酸鹼值，亦能暸解其對所有蛋白活性表現。且在蛋白質篩選與特性分析上，大幅縮短時間與降低成本，有利下於游之進行與進一步商業化的價值。

There are two mthods for assaying chitinase activity. One is to determine the physical or chemical properties of the product from chitin hydrolyzed by chitinase. The other is to monitor the chemical from the substrate which is modified by chemical technique catalyzed by the enzyme. In the study, the chitin azure was colloidized for surface area and suspension increase. The accuracy and sensitivity of chitin azure were validated with Streptomyces griseus. The interference from environmental factors was also low in the chitinase assay with colloid chitin azure.
The molecular weights and isoelectrofocusing points of chitinases from microbes can be identifiedned by proteomics techniques. The activity and characteristics described above of chitinases can be obtained by activity proteomics established in the study. In addition, the sensitivity of the techniques was also 107 times more than that of Coommassie bright blue staining. Seven chitinases of pI 4.23, 4.29, 4.39, 4.71, 4.97, 5.73, and 6.23, were identified from Aeromonas schubertii with the activity proteomics techniques. Two of them were SDS-resistant. The activity was also detected on pH 5, 6, 7,8 or 9. The activities of the chitinases from Aeromonas schubertii were high in the pH6.
Finally, the approach was applied on the microbes screened from oils of MingChi Uninvesity of.Technology The characteristics of the chitinases were also obtained. Conclusively, the activity proteomics was feasible to analyze chitinase. In future, other enzymes analyzed with activity proteomics will be developed. And the techniques will be applied in industry.

目錄
明志科技大學碩士學位論文 I
指導教授推薦書 I
明志科技大學碩士學位論文 II
口試委員會審定書 II
明志科技大學學位論文授權書 III
誌謝 IV
中文摘要 V
ABSTRACT VI
目錄 VII
表目錄 XII
圖目錄 XIII
一、緒論與文獻回顧 1
1.1 幾丁質的介紹 1
1.2 幾丁質酵素分佈、分類與用途 2
1.2.1 動物幾丁質酵素 2
1.2.2 植物幾丁質酵素 3
1.2.3 微生物幾丁質酵素 3
1.3 國際生化學會之命名 3
1.4 幾丁質酵素水解模式 4
1.5 幾丁質酵素活性分析方法 4
1.5.1. 還原醣測定法 4
1.5.2. DNS 5
1.5.3. CA 5
1.5.4. CCA 5
1.5.5. CM-chtitn-RBV 6
1.5.6. 螢光光度法 6
1.5.7. SDS-PAGE上直接分析幾丁質酵素活性-calcofluor white M2R 6
1.5.8. SDS-PAGE上直接分析幾丁質酵素活性-coomassie blue 7
1.5.9. 三明治法分析幾丁質酵素活性 7
1.5.10. SDS-PAGE上直接分析幾丁質酵素活性CM-chitin-RBV 8
1.5.11. 電子掃描測定法 8
1.6 研究目的與係由 8
二、實驗規劃 10
2.1 實驗目的 10
2.2 實驗器材 11
2.2.1 儀器 11
2.2.2 藥品與材料 12
2.3 實驗步驟 14
2.3.1 磷酸鉀緩衝液的製備 14
2.3.2 Bushnell-Haas (BH) selection medium的製備 14
2.3.3 CCA 製備 14
2.3.4 乙二醇幾丁質製備 14
2.3.5 還原醣測定法 15
2.3.5.1 Color reagent solution與GlcNAc solution製備 15
2.3.5.2 標準曲線 15
2.3.5.3 各種參數對反應的影響 15
2.3.6 各種參數對CA測定法的影響 16
2.3.7 各種參數對CCA測定法的影響 17
2.3.8 熱穩定性測試 17
2.3.9 菌種培養 18
2.3.10 蛋白質濃縮 18
2.3.11 SDS- PAGE 18
2.3.12 蛋白質含量測定 19
2.3.13 蛋白質CBB染色 19
2.3.14 蛋白質硝酸銀染色 19
2.3.15 雙向電泳 20
2.3.16 蛋白質轉漬 21
三、實驗結果 22
3.1 酵素活性測定法 22
3.2 乙二醇幾丁質品質之評估 23
3.3 靈敏度 23
3.3.1 SGCTS 23
3.3.2 Lysozyme 23
3.4 A. schubertii產生幾丁質酵素之誘導 24
3.5 幾丁質誘導A. schubertii所產生幾丁質酵素活性之確認 24
3.6 幾丁質酵素的濃縮 24
3.7 蛋白質濃度測定 25
3.8 標準品之雙向電泳與活性分析 25
3.9 純化樣品之雙向電泳與活性分析 25
3.9.1 pH 3-10之雙向電泳與活性分析 25
3.9.2 pH 4-7之雙向電泳與活性分析 26
3.9.3 pH 3-5.6之雙向電泳與活性分析 26
3.10 醱酵樣品之雙向電泳與活性分析 26
3.10.1 pH 4-7之雙向電泳與活性分析 26
3.10.2 反應緩衝液pH值與幾丁質酵素活性 27
3.11 蛋白質轉漬與N端定序 27
3.12 篩選具分泌幾丁質酵素之微生物 28
3.12.1 菌種篩選與培養 28
3.12.2 活性確認與YM10處理 28
3.12.3 雙向電泳程序與酵素活性測定法 28
四、討論 30
五、結論 35
六、參考文獻 97
表目錄
表一、磷酸鉀緩衝液的製備87
表二、不同濃度的SDS-聚丙烯醯胺膠片製作劑量表 88
表三、硝酸銀染色試劑配方與簡易操作表 89
表四、第ㄧ維等電點電泳儀參數設定表 90
表五、SDS-聚丙烯醯胺膠體電泳(SDS-PAGE)試劑配方91
表六、蛋白質含量測定92
表七 熱穩定性測試 93
表八 不同pH值下幾丁質酵素活性表現情形94
圖目錄
圖一、結構相似性 36
圖二、自然界幾丁質常見的三種構形37
圖三、植物幾丁質防禦真菌入侵意示圖38
圖四、幾丁質酵素種類39
圖五、醣類水解酵素的反應機構 :40
圖六、電子式測定法 41
圖七、酵素活性測定方式與作用時間之關係42
圖八、酵素活性測定方式與酵素活性單位之關係43
圖九、酵素活性測定方式與反應環境pH值之關係44
圖十、乙二醇幾丁質成品45
圖十一、乙二醇幾丁質品質測定(一)46
圖十二、乙二醇幾丁質品質測定(二)47
圖十三、SGCTS之銀染靈敏度48
圖十四、以SGCTS測定酵素活性測定法之靈敏度49
圖十五、以Lysozyme之銀染靈敏度50
圖十六、以Lysozyme測定酵素活性測定法之靈敏度 51
圖十七、經挑選Aeromonas schubertii單株菌落 52
圖十八、經幾丁質誘發A. schubertii幾丁質酵素之活性測定53
圖十九、經幾丁質誘發A. schubertii幾丁質酵素之活性測定54
圖二十、經幾丁質誘發A. schubertii幾丁質酵素之蛋白質濃縮分析55
圖二十一、經幾丁質誘發A. schubertii幾丁質酵素之濃縮56
圖二十二、純化之ASCTS 之SDS-PAGE分析57
圖二十三、純化之ASCTS 活性測定法分析58
圖二十四、純化之ASCTS 以活性測定法分析水解形態59
圖二十五、BSA與Lysozyme之雙向電泳(pH 3-10 NL)分析(一)60
圖二十六、BSA與Lysozyme之雙向電泳(pH 3-10 NL)分析(二)61
圖二十七、BSA與Lysozyme酵素活性測定(pH 3-10 NL) (一)62
圖二十八、BSA與Lysozyme酵素活性測定(pH 3-10 NL) (二)63
圖二十九、幾丁質誘發並純化後之A. schubertii胞外蛋白質之雙向電泳(pH 3-10)分析64
圖三十、經幾丁質誘發並純化後A. schubertii.幾丁質酵素活性分析(pH 3-10) 65
圖三十一、幾丁質誘發並純化後A. schubertii胞外蛋白質之雙向電泳(pH 4-7)分析66
圖三十二、經幾丁質誘發並純化後A. schubertii幾丁質酵素活性(pH 4-7)67
圖三十三、幾丁質誘發並純化後A. schubertii胞外蛋白質之雙向電泳(pH 3-5.6)分析68
圖三十四、經幾丁質誘發並純化後A. schubertii幾丁質酵素活性(pH 4-7)69
圖三十五、幾丁質誘發A. schubertii所產生之胞外蛋白質之雙向電泳(pH 4-7)分析70
圖三十六、經幾丁質誘發A. schubertii所產生幾丁質酵素活性(pH 4-7)71
圖三十七、經幾丁質誘發A. schubertii所產生幾丁質酵素活性於pH 6.0反應緩衝液(pH 4-7)72
圖三十八、經幾丁質誘發A. schubertii所產生幾丁質酵素活性於pH 7.0反應緩衝液(pH 4-7)73
圖三十九、經幾丁質誘發A. schubertii所產生幾丁質酵素活性於pH 8.0反應緩衝液(pH 4-7)74
圖四十、經幾丁質誘發A. schubertii所產生幾丁質酵素活性於pH 9.0反應緩衝液(pH 4-7)75
圖四十一、轉漬醱酵液中之蛋白於PVDF膜上76
圖四十二、轉漬已純化之蛋白於PVDF膜上77
圖四十三、由土壤中篩選具分泌幾丁質酵素之微生物78
圖四十四、經幾丁質誘導之上清液與YM10處理後胞外蛋白分析 79
圖四十五、經幾丁質誘導之上清液與YM10處理後酵素活性分析 80
圖四十六、未知微生物1經幾丁質誘發所產生的醱酵液蛋白質之雙向電泳(pH 3-10NL)分析81
圖四十七、未知微生物1經幾丁質誘發所產生幾丁質酵素之酵素活性測定法(pH 3-10NL)分析82
圖四十八、未知微生物2經幾丁質誘發所產生的醱酵液蛋白質之雙向電泳(pH 3-10NL)分析83
圖四十九、未知微生物2經幾丁質誘發所產生幾丁質酵素之酵素活性測定法(pH 3-10NL)分析84
圖五十、未知微生物3經幾丁質誘發所產生的醱酵液蛋白質之雙向電泳(pH 3-10NL)分析85
圖五十一、未知微生物3經幾丁質誘發所產生幾丁質酵素之酵素活性測定法(pH 3-10NL)分析86

1.Wikipedia, The free encyclopedia, http://en.wikipedia.org/wiki/Henri_Braconnot
2.陳錦坤、許清輝、李錦榆、林忠亮、方炳勳、黃冬梨、吳奇生。 2001.在電泳片上直接分析Chitinase活性的新方法。 明志技術學院90年度技術與教學研討會。21-23.
3.林楷洺。2005. Cloning and Expression of SDS-resistantendochitinase from Aeromonas schubertii. 長庚大學生化與生醫工程研究所碩士論文。
4.Bade, M. L. and Stinson, A. 1979. Molting fluid chitinase a homotropic allosteric enzyme. Biochemical and Biophysical Research Communications. 87: 349-353.
5.Brameld, K. A., Shrader, W. D., Imperiali, B.and Goddard, W. A. 3rd.1998 . Substrate assistance in the mechanism of family 18 chitinases: theoretical studies of potential intermediates and inhibitors. Journal of Molecular Biology. 280: 913-923.
6.Bourgault, R. and Bewly D., 2002. Gel diffusion assay for endo-β-mannase and methylesterase can underestimate enzyme activity due to proteolytic degradation a remedy. Analytical Biochemistry.300: 87-93.
7.Cabello, F., Jorrin, J.V.and Tena, M. 1994. Chitinase and beta-1,3-glucanase activities in chickpea (Cicer arietinum)—induction of different isoenzymes in response to wounding and ethephon. Physiol. Plant. 92: 654-660.
8.Faisal, M., Schafhauser D. Y., Garreis, K. A., Elsayed, E. and La Peyre,J. F. 1999. Isolation and characterization of Perkinsus marinus protase using bacitratcin-sepharose affinity chromatography. Comparative Biochemistry and Physiology Part B. 123: 417-426.
9.Flach, J., Pilet, P. E. and Jolles, P. 1992. What’s new in chitinase research. Experientia. 48: 701-716.
10.Gerhardt, L. B. D., Sachetto-Martins, G., Contarini, M. G., Sandroni, M., Ferreira, R. D., de Lima, V. M., Cordeiro, M. C., de Oliveira, D. E.and Margis-Pinheiro, M. 1997. Arabidopsis thaliana class IV chitinase is early induced during the interaction with Xanthomonas campestris. FEBS Lett. 419: 69–75.
11.Gómez, M. R., Rojas, L. I. A, Rojas, N.G. A. and Cruz, R. C., 2004. Colloidal chitin stained with Remazol Brilliant Blue R, a useful substrate to select chitinolytic microorganisms and to evaluate chitinases. Journal of Microbiological Methods. 56: 213– 219.
12.Guo, S. H., Chen, J. K. and Lee, W. C. 2004. Purification and characterization of extracellular chitinase from Aeromonas schubertii. Enzyme and Microbial Technology. 35: 550-556.
13.Hadwiger, L. A., Fristensky, B. and Riggle, R.C. 1984. “ Chitosan, A natural regulator in plant-fungal pathogen interactions, increases crop yied. Chitin, Chitosan and Related Enzymes. Academic press, New York. 291-302.
14.Hackman, R. H. and Goldberg, M. 1964. New substrates for use with chitinases. Analytical Biochemistry. 8: 397-401.
15.Henrissat, B. 1991. A classificaton of glycosyl hydrolases based on amino acid sequence similarities. The Journal of Biochemistry. 280: 309-316.
16.Hsu, S. C. and Lockwood, J. L. 1975. Powered chitin agar as a selective medium for enumeration of Actinomycetes in water and soil. Applied and Environmental Microbiology. 29: 422-426.
17.Imoto, T. and Yagishita, K. 1971. A simple activity measurement of lysozyme. Agricultural and biological chemistry. 35: 1154-1156.
18.Inglis, P. W. and Peberdy, J. F. 1997. Production and purification of a chitinase from Ewingella americana, a recently described pathogen of the mushroom, Agaricus bisporus. FEMS Microbiology Letters. 157: 189-194.
19.Jeuniaux, C. 1966. Methods in enzymology. Academic Press, New York. 8: 644-654.
20.Joshi, M. B., Rogers M. E., Shakarian, A. M., Yamage, M., Al-Harthi, S. A., Bates, P. A. and Dwyer, D. M. 2005. Molecular characterization, expression, and in vivo analysis of LmexCht1: the chitinase of the human pathogen, Leishmania mexicana. Journal of Biological Chemistry. 280: 3847-3861.
21.Karp, M. W., and Karp, L. C., 2004. Chitin Checking — Novel Insights into Asthma. New England Journal of Medicine. 351: 1455-1457.
22.Ken, A. B. and William, A. G. 1998. The role of enzyme distortion in the single displacement mechanism of family 19 chitinases. 95: 4276-4281.
23.Khan, A., Williams, K., Molloy, M. P. and Nevalainen, H. 2003. Purification and characterization of a serine protease and chitinases from Paecilomyces lilacinus and detection of chitinase activity on 2D gels. Protein Expression and Purification. 32: 210-220.
24.Knorr, D. 1984. Use of chitinous polymers in food-A challenge for food research and development. Food Technology. 38: 85-97.
25.Kuranda, M. J., and Robbin, P. W. 1991. Chitinase is required for cell separation during growth of Saccharomyces cerevisiae. Journal of Biological Chemistry. 266: 19758-19767.
26.Laura, R.C., Marı´a del, C. M.C., Sergio, H., Sergio, R., Keiko, S. 2006.Enzymatic hydrolysis of chitin in the production of oligosaccharides using Lecanicillium fungicola chitinases. Process Biochemistry. 41: 1106–1110.
27.Lee, Y. K. and Hwang, B. K. 1996. Differential induction and accumulation of β-1-3glucanase and chitinase isoforms in the intercellular space and leaf tissues of pepper by Xanthomonas campestris pv. vesicatoria infection. Journal of Phytopathology. 144: 79–87.
28.Lin, J., Xiao, X., Zeng, X. and Wang, F. 2006. Expression, characterization and mutagenesis of the gene encoding -N-acetylglucosaminidase from Aeromonas caviae CB101. Enzyme and Microbial Technology. 38: 765–771.
29.Liu, B. L., Kao, P. M., Tzeng, Y. M., Feng, K. C. 2003. Production of chitinase from Verticillium lecanii F091 using submerged fermentation. Enzyme and Microbial Technology. 33: 410-415.
30.Louise, E. D. and Peter, J. B. 2004. Acidic mammalian chitinase – a potential target for asthma therapy. Pharmacological Sciences . 25: 509-511.
31.Lo, H. F., Lin, L. L. Chen, H. L., Hsu W. H. and Cheng, C. T. 2001. Enzymic properties of a SDS-resistant Bacillus sp. TS-23 α-amylase produced by recombinant Escherichia coli. Process Biochemistry. 36: 743-750.
32.Luis, V. and Ray, H. 2004. Development of a method for the detection and quantification of total chitinase activity by digital analysis. Journal of Microbiological Methods. 59: 7–14.
33.Maeda, H.and Ishida, N. 1967. Specificity of binding of hexopyranosyl polysaccharides with fluorescent brightener. The Journal of Biochemistry. 62: 276–278.
34.McCreath, K. J. and Gooday, G. W. 1992. A rapid and sensitive microassay for determination of chitinolytic activity. Journal of Microbiological Methods.14: 229-237.
35.Minke, R. and Blackwell, I. 1978. The structure of α-chitin. Journal of Molecular Biology. 120: 167-181.
36.Ohtakara, A. 1963. Studies on the chitinolytic enzymes of black-kogi mold. Part V. Participation of two different enymes in the decompodition of glycochtitn to constituent amionsugar. Agricultural and Biological Chemistry. 27: 450-460.
37.Ohtakara, A., Mitsutomi M.and Uchida Y. 1979. Purification and some properties of chitinase from Vibrio sp. Journal Fermentation Technology.57: 196-177.
38.Rahimi, S., Perry, R. N.and Wright, D. J. 1998. Detection of chitinases in potato plants following infection with the potato cyst nematodes, Globodera rostochiensis and G-pallida. Nematologica. 44: 181-193.
39.Renkema, G. H., Boot, R. G., Au, F. L., Donker-Koopman W. E., Strijland A., Anton O. M., Martin H. and Johannes M. A. 1998. Chitotriosidase, a chitinase, and the 39-kDa human cartilage glycoprotein, a chitin-binding lectin, are homologues of family 18 glycosyl hydrolases secreted by human macrophages. Eur. Journal of Biochemistry. 251: 504-509.
40.Sekiguchi, J., Matsumiya, M. and Mochizuki, A., 1995. Distribuction of chitinolytic enzymes in seaweeds. Fisherier Science. 61: 876-881.
41.Tronsmo, a., and Harman, G. E. 1993. Detection and quantification of N-Acetyl-β-D-glucosaminidas Chitobiosidase, and endochitinase in solution and on gel. Analytical Biochemistry. 208: 74-79.
42.Trudel, T. and Asselin, A. 1989. Detection of chitinase activity after polyacrylamide gel electrophoresis. Analytical Biochemistry. 178: 362-366.
43.Van Loon, L.C. and Van Kammen, A. 1970. Polyacrylamide disk electrophoresis of the soluble leaf proteins from Nicotiana tabacum var. ‘‘Samsum’’ and ‘‘Samsum NN’’. Virology. 40: 199–211.
44.Van der W., A.J., Qian, X. M. and Botha, A. M. 1998. Differential induction of apoplastic peroxidase and chitinase activities in susceptible and resistant wheat cultivars by Russian wheat aphid infestation. Plant Cell Reports. 18: 132–137.
45.Wang, S. Y., Moyne, A. L., George, T., Wu, S. J., Robert, D. L. and Narendra K. S. 2001. Purification and characterization of a Bacillus cereus exochitinase. Enzyme and Microbial Technology. 28: 492–498.
46.Yamada, H. and Imoto, T. 1981. Aconvenient synthesis of glycochitin, a substrate of lysozyme. Carbohydrate Research. 92: 160-162.
47.Zhu, B. C. R. and Laine, R. A. 1997. Depolymerization of chitin with chitinases. In Chitin handbook. (ed. Muzzarelli R. A. A. and Peter M. G.)
147-151.
48.Zhang, Y.Y. and Punja, Z. K. 1994. Induction and characterization of chitinase isoforms in cucumber (Cucumis sativus L.): effect of elicitors, wounding and pathogen inoculation. Plant Sci. 99: 141– 150.
49.Zhang, H., Huang, X., Fukamizo, T., Muthukrishnan, S.and Kramer, J. K., 2002. Site-directed mutagenesis and functional analysis of an active site tryptophan of insect chitinase. Insect Biochemistry and Molecular Biology. 32: 1477–1488.
50.Zhu, Z., Zheng, T., Homer, R. J., Kim, Y. K., Chen, N. Y., Cohn, L., Hamid, Q., and Elias1 J. A. 2004. Acidic Mammalian Chitinase in Asthmatic Th2 Inflammation and IL-13 Pathway Activation. science. 304: 1678-1682.