市售木瓜酵素經由DEAE-Sepharose、CM-Sepharose、Sephacryl S-100管柱層析後，可獲得兩種具有幾丁質酶活性的蛋白酶，分子量分別為26kDa（F1）及28kDa（F2）。以酪蛋白為基質，F1和F2最適反應溫度、pH、熱安定性及pH安定性分別為（70℃、7、40-60℃、5-11）和（80℃、8、25-60℃、4-10）；以懸浮態幾丁質為基質，F1和F2最適反應溫度、pH、熱安定性及pH安定性分別為（50℃、4、25-50℃、5-8）和（40℃、4、30-70℃、4-7）。金屬離子對酵素影響，以酪蛋白為基質，F1和F2受Cu2+、Fe2+、 Zn2+、Mn2+、EDTA和PMSF抑制，以懸浮態幾丁質為基質，F1 和F2分別受（Cu2+、Fe2+、 Zn2+、Mn2+）和（Fe2+、Ca2+）抑制。F2蛋白酶與幾丁質酶活性受SDS抑制；化學合成物對酵素影響，F1和F2蛋白酶分別受到（S10C、S85O）和（S11C、S10C）抑制，F2幾丁質酶受S10C2所抑制；基質特異性之比較：F1和F2對Casein和Azoalbumin皆具有活性以外，F2對Azocasein也具有活性；以酪蛋白為基質，F1和F2的 Km與Vmax為（5 mg/mL、0.24 U/mL）和（5.26mg/mL、0.93 U/mL）；以懸浮態幾丁質為基質，F1和F2的 Km與Vmax為（66.7mg/ mL、5U/L）和（116.7 mg/ mL、30U/L）。

From commercial papain, two proteases ( F1 and F2 ) with chitinase activity were recovered by using DEAE-Sepharose ion-exchange chromatography, CM-Sepharose ion-exchange chromatography, and gel filtration on a Sephacryl S-100 column. When these two enzymes (F1 and F2) were denatured with SDS-PAGE and a reducing agent, F1 and F2 exhibited a single band at 26 kDa and 28 kDa, respectively. When casein and colloidal chitin were used as substrates for measuring the protease and chitinase activities of F1, the optimum pH, pH stability, optimum temperature, and thermal stability were (7, 5-11, 70, 40-60℃) and (4, 5-8, 50℃, 25-50℃) respectively.  Measuring with the same substrates, the optimum pH, pH stability, optimum temperature, and thermal stability of F2 were (8, 4-10, 80℃, 25-60℃) and (4, 4-7, 40℃, 30-70℃) respectively. Using casein as the substrate, F1 and F2 were inactived by Cu2+、Fe2+、 Zn2+、Mn2+、EDTA and PMSF . Using colloidal chitin as the substrate, F1 and F2 were inactived by (Cu2+,Fe2+, Zn2+,Mn2+) and (Fe2+,Ca2+). As for effects of chemical substrates on two enzymes, proteases (F1 and F2) were inactived by（S10C, S85O）and（S11C, S10C） while chitinase (F2) was inactived by S10C2. Comparison of the substrate specificity: F1 and F2 act on casein and azoalbumin, besides, F2 also acts on azocasein. With casein as the substrate, the Km and Vmax of F1 and F2 were (5 mg/mL、0.24 U/mL) and (5.26mg/mL、0.93 U/mL) respectively. With colloidal chitin as the substrate, the Km and Vmax of F1 and F2 were (66.7mg/ mL、5U/L) and (116.7 mg/mL、30U/L) respectively.

目 錄

授權書
口試委員審議通過委員簽名表
謝誌 --------------------------------  I
中文摘要 ---------------------------  Ⅱ
英文摘要 ---------------------------  Ⅲ
目 錄 -------------------------------- Ⅴ
圖目錄 ----------------------------- Ⅵ
表目錄 ------------------------------- Ⅶ

緒論 -------------------------------------  1
材料與方法 ----------------------------- 13
結果與討論 --------------------------------- 20
結論與展望 --------------------------------- 29
圖表 -------------------------------------- 30
參考文獻 ---------------------------------- 61
 
圖 目 錄
圖 一	  植物幾丁質酶的分類 ------------------------------------------  6
圖 二	  pH值對粗酵素液蛋白酶與幾丁質酶活性之影響 ---------  31
圖 三 	  粗酵素液經過DEAE Sepharose CL-6B分離之結果  -----  32
圖 四 	  DEAE Sepharose後之酵素液經過CM-Sepharose分
         離之結果 -----------------------------------------------------------  33
圖 五 	  F1以Sephacryl S-100分離之結果 --------------------------  34
圖 六 	  F2以Sephacryl S-100分離之結果 --------------------------  35
圖 七 	  以SDS-PAGE測定木瓜酵素純化過程中具蛋白酶與
   幾丁質酶酵素液之分子量 --------------------------------------  36
圖 八	  分子量標準品和F1蛋白質於Sephacryl S-100之層
         析圖譜 --------------------------------------------------------------  37
圖 九	  分子量標準品和F2蛋白質於Sephacryl S-100之層
         析圖譜 --------------------------------------------------------------  38
圖 十	  溫度對F1與F2蛋白酶活性之影響 -------------------------  39
圖十一	  蛋白酶（F1與F2）之熱安定性 ---------------------------- 40 
圖十二	  pH值對F1與F2蛋白酶活性之影響 -------------------------  41
圖十三	  蛋白酶（F1與F2）之pH安定性 ------------------------------  42
圖十四	  化學物質對粗酵素、F1與F2蛋白酶活性之影響 ---------  43
圖十五	  蛋白酶（F1）Lineweaver-Burk雙倒數作圖之Km
         與Vmax -------------------------------------------------------------  44
圖十六	  蛋白酶（F2）Lineweaver-Burk雙倒數作圖之Km
         與Vmax -------------------------------------------------------------  45
圖十七	  溫度對F1與F2幾丁質酶活性之影響 ---------------------  46
圖十八	  幾丁質酶（F1與F2）之熱安定性 ------------------------  47
圖十九	  pH值對F1與F2幾丁質酶活性之影響 ----------------------  48
圖二十	  幾丁質酶（F1與F2）之pH安定性 -------------------------  49
圖二十一 化學物質對粗酵素、F1與F2幾丁質酶活性之影響 --------  50
圖二十二 幾丁質酶（F1）Lineweaver-Burk雙倒數作圖之
Km與Vmax  ------------------------------------------------------  51
圖二十三 幾丁質酶（F2）Lineweaver-Burk雙倒數作圖之
Km與Vmax  ------------------------------------------------------  52
 
表 目 錄

表 一	 蛋白酶在食品工業上的應用-------------------------------------  4
表 二	 蛋白酶之應用 --------------------------------------------------  5
表 三	 幾丁質和幾丁聚醣之應用 ---------------------------------------  8
表 四	 N-乙醯幾丁寡糖之應用 ------------------------------------------  9
表 五	 木瓜酵素之純化總表 --------------------------------------  53
表 六	 不同木瓜蛋白酶分子量之比較----------------------------------  54
表 七	 金屬離子對F1與F2蛋白酶與幾丁質酶活性之影響---------  55
表 八	 蛋白酶抑制劑對F1與F2蛋白酶活性之影響------------------  56
表 九	 界面活性劑對F1和F2蛋白酶與幾丁質酶活性之影響----  57
表 十	 粗酵素液、F1、F2蛋白酶對不同基質之水解能力比較-------  58
表十一	 F1與F2蛋白酶特性之比較---------------------------------------  59
表十二	 F1與F2幾丁質酶特性之比較 ----------------------------------  60

1. Mckee RA, S. Smith: Purification of proteinases from Carica papaya. Phytochemistry 1986, 25:2283-2287.
2. Amon R: Papain. Methods Enzymol 1970, 19:226-244.
3. Kunimitsu DK, Yasunobu KT: Chymopapain. IV. The chromatographic fractionation of partially purified chymopapain and the characterization of crystalline chymopapain B. Biochim Biophys Acta 1967, 139:405-417.
4. Ebata M, Yasunobu KT: Chymopapain. I. Isolation, crystallization, and preliminary characterization. J Biol Chem 1962, 237:1086-1094.
5. Kimmel JR, Smith EL: Crystalline papain. I. Preparation, specificity, and activation. J Biol Chem 1954, 207:515-531.
6. Howard JB, Glazer AN: Studies of the physicochemical and enzymatic properties of papaya lysozyme. J Biol Chem 1967, 242:5715-5723.
7. Azarkan M, Amrani A, Nijs M, Vandermeers A, Zerhouni S, Smolders N, Looze Y: Carica papaya latex is a rich source of a class II chitinase. Phytochemistry 1997, 46:1319-1325.
8. Shimazaki A, Makino Y, Omichi K, Odani S, Hase S: A new sugar chain of the proteinase inhibitor from latex of Carica papaya. J Biochem (Tokyo) 1999, 125:560-565.
9. Odani S, Yokokawa Y, Takeda H, Abe S, Odani S: The primary structure and characterization of carbohydrate chains of the extracellular glycoprotein proteinase inhibitor from latex of Carica papaya. Eur J Biochem 1996, 241:77-82.
10. Zerhouni S, Amrani A, Nijs M, Smolders N, Azarkan M, Vincentelli J, Looze Y: Purification and characterization of papaya glutamine cyclotransferase, a plant enzyme highly resistant to chemical, acid and thermal denaturation. Biochim Biophys Acta 1998, 1387:275-290.
11. Gololobov M, Song I, Wang W, Bateman RC, Jr.: Steady-state kinetics of glutamine cyclotransferase. Arch Biochem Biophys 1994, 309:300-307.
12. G. Wilson: Some properties of an endo 1,3 β glucanase from papain. BIOCHEM.SOC.TRANS. 1974, 2:1115-1116.
13. Song I, Taylor M, Baker K, Bateman RC, Jr.: Inhibition of cysteine proteinases by Carica papaya cystatin produced in Escherichia coli. Gene 1995, 162:221-224.
14. R. GIORDANI AM, R. VERGER: Tributyroylglycerol hydrolase activity in Carica papaya and other latices. Phytochemistry 1991, 30:1069-1072.
15. Silva LG, Garcia O, Lopes MT, Salas CE: Changes in protein profile during coagulation of latex from Carica papaya. Braz J Med Biol Res 1997, 30:615-619.
16. Hung TH, Chang YM, Sung HY, Chang CT: Purification and characterization of hydrolase with chitinase and chitosanase activity from commercial stem bromelain. J Agric Food Chem 2002, 50:4666-4673.
17. Wang S, Wu J, Rao P, Ng TB, Ye X: A chitinase with antifungal activity from the mung bean. Protein Expr Purif 2005, 40:230-236.
18. Arakane Y, Hoshika H, Kawashima N, Fujiya-Tsujimoto C, Sasaki Y, Koga D: Comparison of chitinase isozymes from yam tuber--enzymatic factor controlling the lytic activity of chitinases. Biosci Biotechnol Biochem 2000, 64:723-730.
19. Martin MN: The Latex of Hevea brasiliensis Contains High Levels of Both Chitinases and Chitinases/Lysozymes. Plant Physiol 1991, 95:469-476.
20. Ponath Y, Vollberg H, Hahlbrock K, Kombrink E: Two differentially regulated class II chitinases from parsley. Biol Chem 2000, 381:667-678.
21. Sasaki C, Itoh Y, Takehara H, Kuhara S, Fukamizo T: Family 19 chitinase from rice (Oryza sativa L.): substrate-binding subsites demonstrated by kinetic and molecular modeling studies. Plant Mol Biol 2003, 52:43-52.
22. Jung HW, Hwang BK: Isolation, partial sequencing, and expression of pathogenesis-related cDNA genes from pepper leaves infected by Xanthomonas campestris pv. vesicatoria. Mol Plant Microbe Interact 2000, 13:136-142.
23. Stintzi A, Heitz T, Prasad V, Wiedemann-Merdinoglu S, Kauffmann S, Geoffroy P, Legrand M, Fritig B: Plant 'pathogenesis-related' proteins and their role in defense against pathogens. Biochimie 1993, 75:687-706.
24. Hartley BS: Proteolytic enzymes. Annu Rev Biochem 1960, 29:45-72.
25. Mitchel RE, Chaiken IM, Smith EL: The complete amino acid sequence of papain. Additions and corrections. J Biol Chem 1970, 245:3485-3492.
26. Kamphuis IG, Drenth J, Baker EN: Thiol proteases. Comparative studies based on the high-resolution structures of papain and actinidin, and on amino acid sequence information for cathepsins B and H, and stem bromelain. J Mol Biol 1985, 182:317-329.
27. 王正仁 , 陳啟祥,  陳孟伶: 水解酵素在工業上的利用. 生物產業 Bioindustry. Edited by; 1999:1-11. vol 10.
28. Henrissat B, Bairoch A: New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 1993, 293 (Pt 3):781-788.
29. Henrissat B: A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 1991, 280 (Pt 2):309-316.
30. Wiweger M, Farbos I, Ingouff M, Lagercrantz U, Von Arnold S: Expression of Chia4-Pa chitinase genes during somatic and zygotic embryo development in Norway spruce (Picea abies): similarities and differences between gymnosperm and angiosperm class IV chitinases. J Exp Bot 2003, 54:2691-2699.
31. Ohno T, Armand S, Hata T, Nikaidou N, Henrissat B, Mitsutomi M, Watanabe T: A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT 6037. J Bacteriol 1996, 178:5065-5070.
32. Beintema JJ, Terwisscha van Scheltinga AC: Plant lysozymes. Exs 1996, 75:75-86.
33. Beintema JJ: Structural features of plant chitinases and chitin-binding proteins. FEBS Lett 1994, 350:159-163.
34. Shinshi H, Neuhas JM, Ryals J, Meins F, Jr.: Structure of a tobacco endochitinase gene: evidence that different chitinase genes can arise by transposition of sequences encoding a cysteine-rich domain. Plant Mol Biol 1990, 14:357-368.
35. Araki T, Torikata T: Structural classification of plant chitinases: two subclasses in class I and class II chitinases. Biosci Biotechnol Biochem 1995, 59:336-338.
36. Hamel F, Boivin R, Tremblay C, Bellemare G: Structural and evolutionary relationships among chitinases of flowering plants. J Mol Evol 1997, 44:614-624.
37. Iseli B, Boller T, Neuhaus JM: The N-terminal cysteine-rich domain of tobacco class I chitinase is essential for chitin binding but not for catalytic or antifungal activity. Plant Physiol 1993, 103:221-226.
38. Subroto T, Sufiati S, Beintema JJ: Papaya (Carica papaya) lysozyme is a member of the family 19 (basic, class II) chitinases. J Mol Evol 1999, 49:819-821.
39. Gomez L AI, Casado R, Aragoncillo C.: Seed chitinases. Seed Science Research 2002, 12:217-230.
40. Collinge DB, Kragh KM, Mikkelsen JD, Nielsen KK, Rasmussen U, Vad K: Plant chitinases. Plant J 1993, 3:31-40.
41. Nielsen KK, Mikkelsen JD, Kragh KM, Bojsen K: An acidic class III chitinase in sugar beet: induction by Cercospora beticola, characterization, and expression in transgenic tobacco plants. Mol Plant Microbe Interact 1993, 6:495-506.
42. Jekel PA, Hartmann BH, Beintema JJ: The primary structure of hevamine, an enzyme with lysozyme/chitinase activity from Hevea brasiliensis latex. Eur J Biochem 1991, 200:123-130.
43. Melchers LS, Apotheker-de Groot M, van der Knaap JA, Ponstein AS, Sela-Buurlage MB, Bol JF, Cornelissen BJ, van den Elzen PJ, Linthorst HJ: A new class of tobacco chitinases homologous to bacterial exo-chitinases displays antifungal activity. Plant J 1994, 5:469-480.
44. 江晃榮: 生體高分子(幾丁質,膠原蛋白)在食品工業上的應用. 食品資訊 1998, 150:19-25.
45. 張瓊瑋: Aeromonas sp. DYU-Too7與本土菌株JR1之幾丁質分解酶純化與特性分析: 大葉大學生物產業科技學所碩士論文: 2004.
46. Knorr D: Use of chitinous polymer in food. Food Technol, 1984, 1:85-89.
47. 郭建良: 低分子量幾丁聚醣對雙插桿菌及其他細菌之影響: 國立台灣大學食品科技研究所碩士論文: 1995.
48. Tokoro A, Kobayashi M, Tatewaki N, Suzuki K, Okawa Y, Mikami T, Suzuki S, Suzuki M: Protective effect of N-acetyl chitohexaose on Listeria monocytogenes infection in mice. Microbiol Immunol 1989, 33:357-367.
49. Tokoro A, Tatewaki N, Suzuki K, Mikami T, Suzuki S, Suzuki M: Growth-inhibitory effect of hexa-N-acetylchitohexaose and chitohexaose against Meth-A solid tumor. Chem Pharm Bull (Tokyo) 1988, 36:784-790.
50. Smith ELaJRK: The Enzymes. Edited by Boyer PD, H. Lardy and K. Myrback, eds.; 1960:133. 
51. 陳自珍: 食品酵素學. 台南: 復文書局; 1981.
52. K. Sangeetha TEA: Chemical modification of papain for use in alkaline medium. Journal of Molecular Catalysis B: Enzymatic 2006, 38:171–177.
53. Schewimmer S: Source book of enzymology. In partVII. Enzyme action and the textural quality of foods. Edited by: The Avi. Pub. Co.; 1981:481-496. 
54. Schimada A, E. Yazawa and S. Arai: Preparation of peoteinacous surfactants by enzymatic modification and evaluation of their functional properties in a concentrated emulsion system. Agric. Biol. Chem. 1982, 46:173-182.
55. Yamachita M, S. Arai and M. Fujimaki: A novelone-step process for enzymatic incorporation of amino acidinto proteins ：Application to soy protein and flour for enchancing their methionine level. Agric. Biol. Chem. 1979, 43:1065-1068.
56. Finley  JW, W.L.Stanley,G.G. Watlers: Removal of chill haze from beer with papain immobilized on chitin. Bioteach. and Bioeng. 
 1977, 19:1895-1897.
57. Shilpa S. Khaparde RSS: Chemically modified papain for applications in detergent formulations. Biores. Technol. 2001, 78:1-4.
58. G.Martignone  RM, M.Roaldi,Y.M.Galante: Leather world; 1997,11.
59. 張文重: 蛋白質分解酵素. 台北: 環球書社印行; 1976.
60. 甘偉松: 番木瓜: 國立中國醫藥研究所出版; 1970.
61. 王德男: 台灣木瓜栽培之回顧與展望. 台灣果樹之生產及研究發展研討會專刊. Edited by; 1991:357-371. 
62. Sardinas JL: Calf rennt substitute. Process Biochem 1976, 11:10-17.
63. Scheimmer S: Industrial production and utilization of enzymes from flowering plants. Econ.Bot. 1954, 8:99-133.
64. San-Lang Wang T-CY, Ing-Lung Shih: Production of antifungal compounds by Pseudomonas aeruginosa K-187 using shrimp and crab shell powder as a carbon source. Enzyme and Microbial Technology  1999, 25:142-148.
65. Imoto IaY, K.: A sample activity measurement by lysozyme. Agric. Biol. Chem. 1971, 35:1154-1156.
66. Skelton GS: The relation between milk-clotting and proteolytic activity in papain studies. methods Enzymols. 1970, 40:170-172.
67. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72:248-254.
68. 莊榮輝: 酵素化學實驗. 台北: 國立台灣大學農業化學系生物化學實驗室; 2000.
69. koga DT, T. Sueshige, N. Usumi, T.  Ide, A.: Kinetics of chitinase from yam, Dioscorea opposita. Agric. Biol. Chem. 1989, 53:3121-3126.
70. Glazer AN, E. L. Smith: Papain and other plants sulfhydryl proteolytic enzymes. The Enzyme 1971, 3:501-547.
71. Sluyterman LA: The effect of methanol, urea and other solutes on the action of papain. Biochim Biophys Acta 1967, 139:418-429.
72. Brubacher LJ, Bender ML: The preparation and properties of trans-cinnamoyl-papain. J Am Chem Soc 1966, 88:5871-5880.
73. Glazer AN, Barel AO, Howard JB, Brown DM: Isolation and characterization of fig lysozyme. J Biol Chem 1969, 244:3583-3589.