TKU017 係以蝦殼粉為唯一碳/氮源，篩選自台灣北部土壤之一株蛋白酶及幾丁質酶生產菌，經鑑定為 Serratia sp.。TKU017 生產蛋白酶及幾丁質酶之較適培養條件為含有 2% 蝦殼粉、0.1% K2HPO4 及 0.05% MgSO4．7H2O 之 100 mL 液態培養基 (pH 5) 在 25℃ 搖瓶培養 3 天。所得發酵上清液，經硫酸銨沉澱、DEAE-Sepharose、Phenyl-Sepharose及Sephacryl S-100等層析步驟，純化出一種蛋白酶及一種幾丁質酶，經 SDS-PAGE 測其分子量分別為 53 kDa 及 65 kDa。於最適反應 pH、最適反應溫度、pH安定、熱安定性方面，蛋白酶的為 pH 9、40℃、pH 5-11 及 ＜40℃；幾丁質酶的為 pH 5、50℃、pH 5-7 及 ＜50℃。蛋白酶活性受 EDTA 所抑制，為金屬型蛋白酶，幾丁質酶活性則受 Cu2+、Mn2+、SDS 所抑制。

The protease and chitinase producing strain, Serratia sp. TKU017, cultured by using shrimp shell powder as the only carbon/nitrogen source, was isolated from the soil in the northern Taiwan. The optimized condition for the production of the protease and chitinase was found when the culture was shaken at 150 rpm at 25℃for 3 days in 100mL of medium contain 2% shrimp shell powder (SSP), 0.1 % K2HPO4 and 0.05 % MgSO4．7H2O (pH5). One protease and one chitinase were purified by chromatography procedures of DEAE-Sepharose, Phenyl-Sepharose, and Sephacryl S-100. The molecular mass of the protease and the chitinase determined by SDS-PAGE was approximately 53 kDa and 65 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of protease were pH 9, 40℃, pH 5-11, and ＜40℃, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of chitinase were pH 5, 50℃, pH 5-7, and及＜50℃, respectively. The protease activity was inactivated by EDTA；The chitinase activity was inactivated by Cu2+, Mn2+ and SDS.

目錄
	頁次
授權書
簽名頁
誌謝	I
中文摘要	II
英文摘要	III
目錄	IV
圖目錄	IX
表目錄	XI

第一章 緒言	1
第二章 文獻回顧	2
     2.1幾丁質與幾丁聚醣之構造及應用	2
        2.1.1幾丁質與幾丁聚醣之構造	2
2.1.2幾丁質與幾丁聚醣之應用	3
     2.2 幾丁質酶與幾丁聚醣酶之分布及功能	3
        2.2.1 幾丁質酶	3
        2.2.2 幾丁聚醣酶	3
     2.3蛋白酶	3
        2.3.1蛋白酶分類	4
        2.3.2蛋白酶之應用	4
     2. 4 抗氧化活性	5
第三章 材料與方法	7
     3.1 實驗菌株	7
     3.2 實驗材料	7
     3.3 實驗儀器	8
     3.4 生產菌株之篩選	8
     3.5幾丁質之製備	9
     3.6 幾丁質酶之活性測定	9
     3.7蛋白酶之活性測定	10
     3.8 酵素較適生產條件探討	10
        3.8.1 碳/氮源種類	10
        3.8.2 碳/氮源濃度	10
        3.8.3培養溫度	11
        3.8.4培養基酸鹼值	11
        3.8.5 培養體積	11
        3.8.6 培養時間	11
     3.9 酵素之分離純化	11
        3.9.1 粗酵素液之製備	11
        3.9.2 陰離子交換層析	12
        3.9.3 疏水性層析	12
        3.9.4 膠體過濾層析	13
     3.10 蛋白質電泳分析	13
     3.11蛋白酶活性電泳	13
     3.12 蛋白質定量分析	14
     3.13 酵素之特性分析	14
        3.13.1 酵素最適反應溫度	14
        3.13.2 酵素之熱安定性	15
        3.13.3 酵素最適反應pH	15
        3.13.4 酵素之pH安定性	15
        3.13.5 金屬離子與抑制劑對酵素活性之影響	16
        3.13.6 界面活性劑對酵素活性之影響	16
        3.13.7蛋白酶之基質特異性	16
        3.13. 8幾丁質酶之基質特異性	17
     3.14 DPPH自由基清除能力之測定	17
     3.15薄層色層層析	18
第四章 結果與討論	19
     4.1蛋白酶及幾丁質酶生產菌之篩選	19
     4.2菌株特性	19
     4.3酵素較適生產條件探討	23
     4.4蛋白酶及幾丁質酶之分離純化	35
        4.4.1 粗酵素液製備	35
        4.4.2 陰離子交換層析	35
        4.4.3 疏水性層析	35
        4.4.4 膠體過濾層析	36
        4.4.5 綜合結果	36
     4.5蛋白酶及幾丁質酶之分子量測定	37
        4.5.1蛋白酶-SDS-PAGE (CBR染色法)	37
        4.5.2蛋白酶活性染色	37
        4.5.3幾丁質酶- SDS-PAGE (銀染法) 	37
        4.5.4綜合結果	37
     4.6蛋白酶及幾丁質酶之特性分析	51
        4.6.1幾丁質酶之最適反應溫度及熱安定性	51
        4.6.2蛋白酶之最適反應溫度及熱安定性	51
        4.6.3幾丁質酶之最適反應 pH 及 pH 安定性	51
        4.6.4蛋白酶之最適反應pH及pH安定性	51
        4.6.5金屬離子對幾丁質酶之影響	51
        4.6.6金屬離子對蛋白酶之影響	51
        4.6.7界面活性劑對幾丁質酶之影響	52
        4.6.8界面活性劑對蛋白酶之影響	52
     4.7蛋白酶之基質特異性	52
     4.8幾丁質酶之基質特異性	53
     4.9抗氧化力分析	53
第五章 結論	68
參考文獻	69










圖目錄
	頁次
圖2.1 幾丁質、幾丁聚醣及纖維素之構造	6
圖4.1 Serratia sp. TKU017 之顯微鏡照片	20
圖4.2 不同碳/氮源對 TKU017 幾丁質酶生產之影響	25
圖4.3 不同碳/氮源對 TKU017 蛋白酶生產之影響	26
圖4.4 SSP 濃度對 TKU017 幾丁質酶生產之影響	27
圖4.5 SSP 濃度對 TKU017 蛋白酶生產之影響	28
圖4.6溫度對 TKU017 蛋白酶生產之影響	29
圖4.7 pH 對 TKU017 幾丁質酶生產之影響	30
圖4.8 pH 對 TKU017 蛋白酶生產之影響	31
圖4.9培養體積對 TKU017 幾丁質酶生產之影響	32
圖4.10培養體積對 TKU017 蛋白酶生產之影響	33
圖4.11 Serratia sp. TKU017於 SSP 培養基生產蛋白酶及幾丁質酶之生長曲線圖	34
圖4.12 Serratia sp. TKU017 所生產酵素之純化分離流程圖	39
圖4.13 TKU017 之蛋白酶及幾丁質酶 DEAE-Sepharose CL-6B 層析圖譜	40
圖4.14 TKU017 之幾丁質酶 Phenyl-Sepharose 層析圖譜	41
圖4.15 TKU017 之幾丁質酶 Sephacryl S-100 層析圖譜	42
圖4.16 TKU017 之蛋白酶 Phenyl-Sepharose 層析圖譜	43
圖4.17 TKU017 之蛋白酶 Sephacryl S-100 層析圖譜	44
圖4.18 TKU017 蛋白酶 SDS-PAGE 之分子量分析及活性染色	47
圖4.19 TKU017 幾丁質酶 SDS-PAGE 之分子量分析	48
圖4.20 pH對TKU017幾丁質酶酵素活性之影響	55
圖4.21溫度對TKU017幾丁質酶酵素活性之影響	56
圖4.22 pH對TKU017蛋白酶酵素活性之影響	57
圖4.23溫度對TKU017蛋白酶酵素活性之影響	58
圖4.24薄層色層分析TKU017發酵上清液之抗氧化物質	66
圖4.25發酵不同培養基所得DPPH清除率的比較	67

















表目錄
	頁次
表4.1 16S rDNA 部份核酸序列分析	21
表4.2 API生化生理試驗分析結果	22
表4.3 Serratia sp. TKU017幾丁質酶純化總表	45
表4.4 Serratia sp. TKU017蛋白酶純化總表	46
表4.5 TKU017蛋白酶胜肽質譜鑑定結果	49
表4.6化學品對TKU017酵素之影響	59
表4.7界面活性劑對TKU017酵素之影響	60
表4.8微生物來源之幾丁質酶/幾丁聚醣酶特性比較	61
表4.9 Serratia spp.之蛋白酶特性比較	62
表4.10 Serratia sp.TKU017所生產幾丁質酶及蛋白酶之特性	63
表4.11 TKU017蛋白酶之基質特異性	64
表4.12 TKU017幾丁質酶之基質特異性	65

1.	Alfonso, C., Martinez, M.J., Reyes, F., 1992. Purification and properties of two endo-chtiosanases from Mucor rouxii implicated in its wall degradation. FEBS Lett. 95, 187-194.

2.	Allan, C.R., Hadiger, L.A., 1979. The fungicidal effect of chitosan on fungi of varying cell wall composition. Exp Mycol. 3, 285-287.

3.	Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389-3402.

4.	Anwar, A., Saleemuddin, M., 1998. Alkaline protease: a review. Biores Technol. 64:175-183.

5.	Barrett, A.J., Rawlings, N.D., Woessner, J.F., 2003. The Handbook of Proteolytic Enzymes, 2nd ed. Academic Press.

6.	Chen, X., Xia, W., Yu, X., 2005. Purification and characterization of two types of chitosanase from Aspergillus sp. CJ22-326. Food Res Int. 38, 315-322.

7.	Deshpande, M.V., 1986. Enzymatic degradation of chitin and its biological application. J Sci Ind Res. 45, 273-277.

8.	Fenton, D.M., Eveleigh, D.E., 1981. Purification and mode of action of a chitosanase from Penicillium islandicum. J Gen Microbiol. 126, 151-165.
9.	Frankowski, J., Lorito, M., Scala, F., Schmid, R., Berg, G., Bahl, H., 2001. Purification and properties of two chitinolytic enzymes of Serratia plymuthica HRO-C48. Arch Microbiol. 176, 421-426.

10.	He, H., Chen, X., Sun C., Zhang, Y., Gao, P., 2006. Preparation and functional evaluation of oligopeptide-enriched hydrolysate from shrimp (Acetes chinensis) treated with crude protease from Bacillus sp. SM98011. Biores Technol. 97, 385-390.

11.	Heussen, C., Dowdle, E.B., 1980. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal Biochem. 102, 196-202.

12.	Imoto, T., Yagishita, K., 1971. A simple activity measurement by lysozyme. Agric Biol Chem.35, 1154-1156.

13.	Jeuniaux, C., 1966. Chitinases. Methods Enzymol. 8, 644-650.

14.	Jo, Y.Y., Jo, K.J., Jin, Y.L., Kim, K.Y., Shim, J.H., Kim, Y.W., Park, R.D., 2003. Characterization and kinetics of 45kDa chitosanase from Bacillus sp. P16. Biosci Biotechnol Biochem. 67, 1875-1882.

15.	Jones, B.L., Fontanini, D., Jarvinen, M., Pekkarinen, A., 1997. Simplified endoproteinase assays using gelatin or azogelatin. Anal Biochem. 263, 214-220.

16.	Kalisz, M.H., 1988. Microbial proteinases. Adv Biochem Eng Biotechnol. 36, 17-55

17.	Kaodkura, K., Rokutani, A., Yamamoto, M., Ikegami, T., Sugita, H., Itoi, S., Hakamata, W., Oku, T., Nishio, T., 2007. Purification and characterization of Vibrio parahaemolyticus extracellular chitinase and chitin oligosaccharide deacetylase involved in the production of heterodisaccharide from chitin. Appl Microbiol Biotechnol. 75, 357-365.

18.	Kim, H.S., Timmis, K.N., Golyshin, P.N., 2007. Characterization of a chitinolytic enzyme from Serratia sp. KCK isolated from kimchi juice. Appl Microbiol Biotechnol. 75, 1275-1283.

19.	Knorr, D., 1984. Use of chitinous polymer in food. Food Technol. 1, 85-89.

20.	Kumar, C.G., Takagi, H., 1999. Microbial alkaline protease: from a bioindustrial viewpoint. Biotechnol Adv. 17, 561-594.

21.	Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227, 680-685.

22.	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, 527-534.

23.	Lin, H.Y., Chou, C.C., 2004. Antioxidative activities of water-soluble disaccharide chitosan derivatives. Food Res Int. 37, 883-889.

24.	Marty, K.B., Williams, C.L., Guynn, L.J., Benedik, M.J., Blanke, S.R., 2002. Characterization of a cytotoxic factor in culture filtrates of Serratia marcescens. Infect Immum. 70, 1121-1128.
25.	Mohamed, S.A., Fahmy, A.S., Mohamed, T.M., Hamdy, S.M., 2005. Proteases in egg, miracidium and adult of Fasciola gigantic characterization of serine and cysteine proteases from adult. Comp Biochem Physiol B Biochem Mol Biol. 142, 192-200.

26.	Muzzarelli, R.A., Barontini, G., Rocchetti, R., 1976. Immobilized enzymes on chitosan columns: alpha-chymotrypsin and acid phosphatase. Biotechnol Bioeng. 18, 1445-1454.

27.	Muzzarelli, R.A., Barontini, G., Rocchetti, R., 1978. Isolation of lysozyme on chitosan. Biotechnol Bioeng. 20, 87-92.

28.	Nawani, N.N., Kapadnis, B.P., 2001. One-step purification of chitinase from Serratia marcescens NK1, a soil isolate. J Appl Microbiol. 90, 803-808.

29.	Ohtakara, A., Izume, M., 1987. Preparation of D-glucosamine oligosaccharide by enzymatic hydrolysis of chitosan. J Agric Food Chem. 51, 1189-1191.

30.	Ohtakara, A., Matsunaga, H., Mitsutomi, M., 1990. Action pattern of Streptomyces grisous chitinase on partially N-acetylated chitosan. Agric Biol Chem. 54, 3191-3199.

31.	Phadatare, S.U., Rao, M., Deshpande, V., 1993. High activity alkaline protease from Conidiobolus coronatus (NCL86.8.20): enzyme production and compatibility with commercial detergents. Enzyme Microb Technol. 15, 72-76.

32.	Pinero Estrada, J.E., Bermejo Besco`s, P., Villar del Fresno, A.M., 2001. Antioxidant activity of different fractions of Spriulina platensis protean extract. Farmaco [Sci]. 56, 497-500.

33.	Pugnaloni, A., Solmi, R., Muzzarelli, R., Emanuelli, M., Moroni, F., Gazzanelli, G., Baldassarre, V., Biagini, G., 1988. Chitosan. Biochemical structural characteristics and bio-morphology. Boll Soc Ital Biol Sper. 64, 101-108.

34.	Rao, M.B., Tanksale, A.M., Ghatge, M.S., Deshpande, V.V., 1998. Molecular and biotechnological aspects of microbial proteases. Microbiol  Mole Biol Rev. 62, 597-635.

35.	Rawlings, N.D., Barrett, A.J., 1993. Evolutionary families of peptidases. Biochem J. 290, 205-218.

36.	Roberts, R.L., Cabib, E., 1982. Serratia marcescens chitinase: one step purification and use for the determination of chitin. Anal Biochem. 127, 402-412.

37.	Romero, F.J., García, L.A., Salas, J.A., Díaz, M., Quirós, L.M., 2001. Production, purification and partial characterization of two extracellular proteases from Serratia marcescens grown in whey. Process Biochem. 36, 507-515.

38.	Saito, M., Tabeta, R., Oogawa, K., 1987. High-resolution solid-state 13C NMR study of chitosan and its salts with acid: conformation characterization of polymorph and helical structures as viewed from the conformation-dependent 13C chemical shifts. Macromol Chem. 20-24.
39.	Salamone, P.R., Wodzinski, R.J., 1997. Production, purification and characterization of a 50 kDa extracellular metalloprotease from Serratia marcescens. Appl Microbiol Biotechnol. 48, 317-324.

40.	Sannan, T., Kurrta, K., Lwakura, Y., 1976. Studies on chitin effect of deacetylation on solubility. Macromol Chem. 177, 3589-3592.

41.	Sasaki, Y.F., Kawaguchi, S., Kamaya, A., Ohshita, M., Kabasawa, K., Iwama, K., 2002. The comet assay with 8 mouse organs: results with 39 currently used food additives. Mutat Res. 519, 103-119.

42.	Sedmack, J.J., Grossberg, S.E., 1977. A rapid, sensitive, and versatile assay for protein using coomassie brilliant blue G250. Anal Biochem. 79, 544-552.

43.	Sini, T.K., Santhosh, S., Mathew, P.T., 2007. Study on the production of chitin and chitosan from shrimp shell by using Bacillus subtilis fermentation. Carbohydr Res. 342, 2423-2429.

44.	Sutrisno, A., Ueda, M., Abe, Y., Nakazawa, M., Miyatake, K., 2004. A chitinase with high activity toward partially N-acetylated chitosan from a new, moderately thermophilic, chitn-degrading bacterium, Ralstonia sp. A-471. Appl Microbiol Biotechnol. 63, 398-406.

45.	Todd, E.W., 1949. Quantitative studies on the total plasmin and trypsin inhibitor of human blood serum. J Exp Med. 39, 295–308.

46.	Toei, K., Kohara, T., 1976. A conductometric method for colloid titrations. Anal Chim Acta.83, 59-65.
47.	Wang, S.L., Chang, W.T., 1997. Purification and characterization of two bi-functional chitinase/lysozymes extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium. Appl Environ Microbiol. 63, 380–386.

48.	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, 1171-1179.

49.	Wang, S.L., Kao, T.Y., Wang, C.L., Yen, Y.H., Chern, M.K., Chen, Y.H., 2006. A solvent stable metalloprotease produced by Bacillus sp. TKU004 and its application in the deproteinization of squid pen for β-chitin preparation. Enzyme Microb Technol. 39, 724-731.

50.	Wang, S.L., Lin, C.L., Liang, T.W., Liu, K.C., Kuo, Y.H., 2008b. Conversion of squid pen by Serratia ureilytica for the production of enzymes and antioxidants. Biores Technol. in press.

51.	Wang, S.L., Lin, H.T., Liang, T.W., Chen, Y.J., Yen, Y.H., Guo, S.P., 2008c. Reclamation of chitinous materials by bromelain for the preparation of antitumor and antifungal materials. Biores Technol. 99, 4386-4393.

52.	Wang, S.L., Peng, J.H., Liang, T.W., Liu, K.C., 2008d. Purification and characterization of a chitosanase from Serratia marcescens TKU011. Carbohydr Res. 343, 1316-1323.


53.	Wang, S.L., Yeh, P.Y., 2006. Production of a surfactant- and solvent- stable alkaliphilic protease by bioconversion of shrimp shell wastes fermented by Bacillus subtilis TKU007. Process Biochem. 41, 1545-1552.

54.	Watanabe, K., Sakai, J., Hayano, K., 2003. Bacterial extracellular protease activities in field soils under different fertilizer managements. Can J Microbiol. 49, 305-312.

55.	Xing, R., Yu, H., Liu, S., Zhang, W., Zhang, Q., Li, Z., Li, P., 2005. Antioxidant activity of differently regioselective chitosan sulfates in vitro. Bioorg Med Chem. 13, 1387–1392.

56.	Yamaguchi, T., Takamura, H., Matoba, T., Terao, J., 1998. HPLC method for evalution of the free radical-scavenging activity of foods by using 1,1-diphenyl-2-picrylhydrazyl. Biosci Biotechnol Biochem. 62, 1201-1204.