TKU013係以烏賊軟骨粉為唯一碳/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 ureilytica。TKU013生產蛋白酶和幾丁質酶之較適培養條件為，於含有1.5 %烏賊軟骨粉、0.1 % K2HPO4及0.05 %MgSO4．7H2O之50 mL液體培養基(pH 9)，於25℃搖瓶培養3天，可同時獲得較高蛋白酶活性(0.19U/ml)和幾丁質酶活性(0.021U/mL)。將發酵所得上清液經硫酸銨沉澱、DEAE- Sepharose、Sephacryl S-100和Phenyl-Sepharose等層析步驟，可純化出二種蛋白酶(P1、P2)和一種幾丁質酶(C1)，經SDS-PAGE測得分子量分別為50 kDa、 50 kDa及60 kDa。P1、P2、C1之最適反應溫度、最適pH、熱安定性、pH安定性分別為(40℃、pH 10、<50℃、pH 7～11)，(50℃、pH 10、<40℃、pH 8～11)，(50℃、pH 6、<50℃、pH 5～8)。幾丁質酶活性受Zn2+、Cu2+完全抑制；而蛋白酶在Mg2+的存在下，約剩原活性的1/3。在2% (v/v) Tween20、Tween40或Triton X-100的存在下，P2、C1幾乎不受影響，P1則受到抑制；第4天的發酵上清液，具有較佳的DPPH自由基清除力。

Serratia ureilytia TKU013, a protease and chitinase producing strain, was isolated from the soil in the north Taiwan. The optimized condition for protease and chitinase production were found when the culture was shaken at 25℃ for 3 days in 50 mL of medium containing 1.5% squid pen powder(SPP), 0.1% K2HPO4 and 0.05% MgSO4‧7H2O at pH 9. Two proteases(P1,P2) and a chitinase(C1) were purified from the culture supernatant by chromatography procedures of DEAE-Sepharose, Sephacryl S-100 and Phenyl-Sepharose. The molecular mass of P1, P2 and C1 determined by SDS-PAGE was approximately 50 kDa , 50 kDa and 60 kDa, respectively. 
      The optimum temperature, optimum pH, thermal stabilities and pH stabilities of P1, P2 and C1 were (40℃, pH 10 <50℃, pH7~ 11),( 50℃, pH 10, <40℃,pH 8~11)and( 50℃,pH 6, <50℃,pH 5~8), respectively. The chitinase(C1)was completely inactivated by Zn2+, Cu 2+. Both proteases retained 35% of its original activity in the presence of Mg2+. C1 was activated in the presence of 2% (v/v) Tween 20, 2% (v/v) Tween 40 or 2% (v/v) Triton X-100. The protease P2 retained 80% of its original activity in the presence of 2% (v/v) Tween 20 and 2% (v/v)Tween 40, respectively. The supernatant of the fourth day showed better DPPH radical scavenging activity.

目錄

頁次
封面內頁
簽名頁
授權書
中文摘要i
英文要ii
誌謝iv
目錄v
圖目錄ix
表目錄xi

第一章 緒言1
第二章 文獻回顧2
       2.1 Serratia ureilytia之特性2
       2.2 蛋白質酶2
           2.2.1 蛋白酶於工業上之應用2
       2.3 幾丁質3
       2.4  幾丁質酶3
           2.4.1 幾丁質及幾丁聚醣之應用4
       2.5 抗氧化活性4
第三章 材料與方法5
       3.1 菌株5
       3.2 培養基(液) 5
       3.3 化學材料5
       3.4 儀器6
       3.5 菌株之篩選與分離純化7
       3.6 蛋白酶活性之測定7
       3.7幾丁質酶活性之測定8
       3.8 酵素最適培養條件探討8
           3.8.1碳/氮源之選擇8
           3.8.2 培養溫度9
           3.8.3 培養液pH9
           3.8.4 培養體積9
           3.8.5 培養時間10
       3.9 蛋白酶及幾丁質酶之純化分離10
           3.9.1 粗酵素液之製備10
           3.9.2 離子交換樹脂層析法10
           3.9.3 膠體過濾層析法11
           3.9.4 疏水性膠層析法11
       3.10 蛋白質電泳分析11
       3.11 酵素生化特性分析12
           3.11.1 酵素最適反應溫度測定12
           3.11.2 酵素熱安定的測定12
           3.11.3 酵素最適反應pH的測定13
           3.11.4 酵素pH安定性的測定13
           3.11.5 金屬離子與抑制劑對酵素活性的影響14
           3.11.6有機溶劑對酵素之活性及安定性的影響14
           3.11.7界面活性劑對酵素活性的影響14
        3.12 蛋白酶基質特異性15
        3.13 DPPH自由基清除能力之測定15
第四章 結果與討論17
        4.1 蛋白酶及幾丁質酶生產菌之篩選17
        4.2 菌株特性17
        4.3 酵素較適生產條件探討22
            4.3.1 培養溫度22
            4.3.2 培養pH22
            4.3.3 培養體積22
            4.3.4 碳/氮源選擇23
            4.3.5 碳/氮源濃度23
            4.3.6 培養時間23
            4.3.7 總結24
        4.4 酵素之純化分離36
            4.4.1 粗酵素液之製備36
            4.4.2 離子交換樹脂層析法36
            4.4.3 膠體過濾層析法37
            4.4.4 疏水性層析法37
            4.4.5 綜合結果38
        4.5酵素之分子量判定38
        4.6 酵素之生化特性分析47
            4.6.1 酵素之最適反應溫度47
            4.6.2 酵素之熱安定性47
            4.6.3 酵素之最適反應pH47
            4.6.4 酵素之pH安定性48
            4.6.5 金屬離子及抑制劑對酵素之影響48
            4.6.6 有機溶劑對酵素活性及安定性之影響49
            4.6.7 界面活性劑對酵素活性之影響49
        4.7 酵素之基質特異性50
        4.8 DPPH自由基清除能力50
第五章 結論68
參考文獻69

圖 目 錄   頁次
圖4.1  S. ureilytia TKU013之顯微照片……………………………..18
圖4.2  S. ureilytia TKU013之16S rDNA部分序列.…......………...19
圖4.3 溫度對TKU013生產蛋白酶之影響………………………...25
圖4.4 溫度對TKU013生產幾丁質酶之影響…..………………….26
圖4.5 pH對TKU013生產蛋白酶之影響…………………………..27
圖4.6 pH對TKU013生產幾丁質酶之影響………………………...28
圖4.7 培養體積對TKU013生產蛋白酶之影響……………….…..29
圖4.8  培養體積對TKU013生產幾丁質酶之影響….………...…..30
圖4.9 不同碳/氮源對TKU013生產蛋白酶之影響………...……...31
圖4.10 不同碳/氮源對TKU013生產幾丁質酶之影響…..…..…...32
圖4.11 SPP濃度對TKU013生產蛋白酶之影響…………………..33
圖4.12 SPP濃度對TKU013生產幾丁質酶之影響………………..34
圖4.13 S. ureilytia TKU013生產蛋白酶及幾丁質酶活性之生長曲線............................................................................................. 35
圖4.14 S. ureilytia TKU013所生產酵素之純化分離流程圖……….39
圖  4.15 TKU013蛋白酶及幾丁質酶之 DEAE-Sepharose CL-6B層析圖譜………………..................................................................40
圖4.16 蛋白酶(P1)及幾丁質酶(C1)之Sephacryl S-100酵素層析圖譜……………………………………………………….…….41
圖4.17 蛋白酶(P1)及幾丁質酶(C1)之Phenyl-Sepharose之酵素層析圖譜………………………………………………………..…42
圖4.18 蛋白酶(P2)之Sephacryl S-100層析圖譜..……………..…..43
圖4.19 P1、P2、C1於SDS-PAGE之分子量分析.………………..46
圖4.20 酵素之最適反應溫度………………………….……………52
圖4.21 酵素之熱安定性…………………………………………….53
圖4.22 酵素之最適反應pH………………………………………...54
圖4.23 酵素之pH安定性…………………………………………..55
圖4.24 不同之有機溶劑（最終濃度20%，v/v）對蛋白酶活性之影        
       響……………………………………………………………58
圖4.25 不同之有機溶劑（最終濃度20%，v/v）對幾丁質酶活性之  
       影響…………………………………………………………59
圖4.26 不同之有機溶劑（最終濃度20%，v/v）對蛋白酶安定性之
       影響…………………………………………………………60
圖4.27 不同之有機溶劑（最終濃度20%，v/v）對幾丁質酶安定性
       之影響………………………………………………………61
圖4.28 0-4天發酵上清液之DPPH自由基清除能力………………63
圖4.29 不同濃度第4天發酵上清液之DPPH自由基清除能力….64

表 目 錄  頁次
表4.1 API鑑定系統分析結果………..……………………...………20
表4.2 TKU013之較適生產條件.........................................................24
表4.3 S. ureilytia TKU013蛋白酶P1及P2之純化概要…………….44
表4.4 S. ureilytia TKU013幾丁質酶C1之純化概要.……………...45
表4.5 各種物質對酵素活性之影響…...............................................56
表4.6 不同界面活性劑對酵素活性之影響…...……………………57
表4.7 各種基質對蛋白酶活性之影響…………………………..….62
表4.8 各種微生物生產蛋白酶之特性比較………………………...68
表4.9各種微生物生產幾丁質酶之特性比較………………………69

1.賴威安。Bacillus sp. P-6中蛋白酶的生產與性質分析。2000。國立中興大學食品科學研究所碩士論文。台中。
2.陳泰州嗜水性產氣單胞桿菌Aeromonas hydrophila臨床株CKH-29溶血素之純化與分析。2001：第114-115頁。國立台灣大學農業化學研究所碩士論文，台北。
3.Anwar A, Saleemuddin M (1998) Alkaline protease：a review. Biore Technol. 64：175-183.
4.Allan CR , Hadiger LA (1979) The fungicidal effect of chitosan on fungi of varying cell wall composition. Experi mycol. 3：285-287.
5.Andrew TG, Michael GH, Adrienne H (2005) Production of chitinolytic enzymes by Serratia marcescens QMB1466 using various chitinous substrates. J Chem Technol Biotechnol. 80：28-34.
6.Baumann U, Bauer M, Létoffé S, Delepelaire P, Wandersman C (1995) Crystal Structure of a Complex Between Serratia marcescens Metallo-protease and an Inhibitor from Erwinia chrysanthemi. J. Mol. Biol. 248：653-661.
7.Burd W, Yourkevich O, Voskoboev AJ, K.-H. van Pée (1995) Purification and properties of a non-heam chloroperoxidase from Serratia marcescens. FEMS Microbiol. Lett. 129：255-260.
8.B Bhadra, P Roy, Chakraborty R(2005) Serratia ureilytica sp. nov., a novel urea-utilizing species. Int J Syst Evol Microbiol.55： 2155-2158.
9.Cody RM(1989) Disdtribution of chitinase and chitobiase in Bacillus. Curr. Microbiol. 19：201-205.
10.Demidyuk IV, Kalashnikov AE, Gromova TY, Gasanov EV, Safina DR, Zabolotskaya MV, Rudenskaya GN, Kostrov SV (2006) Cloning, sequencing, expression, and characterization of protealysin, a novel neutral proteinase from Serratia proteamaculans representing a new group of thermolysin-like proteases with short N-terminal region of precursor. Protein Expr Purif. 47：551-561.
11.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.
12.Gal SW, Choi JY, Kim CY, Cheong YH, Choi YJ, Bahk JD, Lee SY, Cho MJ (1997) Isolation and characterization of 54-kDa and 22-kDa chitinase genes of Serratia marcescens KCTC2172. FEMS Microbiol. Lett. 151：197-204.
13.Gal SW, Choi JY, Kim CY, Cheong YH, Choi YJ, Bahk JD, Lee SY, Cho MJ (1998) Cloning of the 52-kDa chitinase gene from Serratia marcescens KCTC2172 and its proteolytic cleavage into an active 35-kDa enzyme. FEMS Microbiol. Lett. 160：151-158.
14.Gupta A, Roy I, Khare SK, Gupta MN (2005) Purification and characterization of a solvent stable protease from Pseudomonas aeruginosa PseA. J. Chromatography A. 1069：155-161.
15.Gupta M.N (1992) Enzyme function in organic solvents. Eur. J. Biochem. 203：25-32.
16.Imoto T, Yagishita K (1971) A simple activity measurement by lysozyme. Agric. Biol. Chem.35：1154-1156.
17.Ito N, Hiroze M, Fukushima G, Tauda H, Shira T, Tatematsu M (1986) Studies on antioxidant; their carcinogenic and modifying effects on chemical carcinogensis. F. Chem. Toxicol. 24：1071-1081.
18.Juang RH, Chang YD, Sung HY, Su JC (1984) Oven-drying method for polyacrylamide gel slab packed in cellophane sandwich. Anal. Biochem. 141：348-350.
19.Jonse BL, Fontanini D, Jarvinen M, Pekkarinen A (1997) Simplified endoproteinase assays using gelatin or azogelatin. Anal. Biochem. 263：214-220.
20.Kim HS, Timmis KN, Golyshin PN (2007) Characterization of a chitinolytic enzyme from Serratia sp. KCK isolated from kimchi juice. Appl Microbiol Biotechnol. DOI：1007.
21.Karadzic I, Masui A, Fujiwara N (2004) Purification and Characterization of a protease from Pseudomonas aeruginosa grown in cutting oil. J. Biosci. Bioeng. 98：145-152.
22.Kumar CG, Takagi H (1999) Microbial alkaline protease：from a bioindustrial viewpoint. Biotechnol. Adv. 17：561-594.
23.Kreig NR (1986) Facultatively Anaerobic Gram-Negative Rods, Systematic Bacteriology. 1：477-484.
24.Kothary MH, McCardell BA, Frazar CD, Deer D, Tall BD (2007) Characterization of the zinc-containing metalloprotease (zpx) and development of a species-specific detection method for Enterobacter sakazakii. Appl Environ Microbiol.
25.Kumarasamy Y, Nahar L, Cox1PJ, Jaspars M, Sarker SD (2003) Bioactivity of secoiridoid glycosides from Centaurium erythraea. Phytomedicine. 10: 344-347.
26.Lahl WJ, Braun SD (1994) Enzymatic production of protein      hydrolysates for food use. Food technol48：68-71.
27.Marty KB, Williams CL, Guynn LJ, Benedik MJ, Blanke SR(2002) Characterization of a Cytotoxic Factor in Culture Filtrates of Serratia marcescens. Inf. Immu. 70：1121-1128.
28.Mohamed SA, Fahmy AS, Mohamed TM, Hamdy SM (2005) Proteases in egg, miracidium and adult of Fasciola gigantica. Characterization of serine and cysteine proteases from adult. Comp. Biochem. Physiol. B. Biochem. Mol. Biol.142：192-200.
29.Mortimer PS, Heinz S, Hans GT, Albert B, Hans GS (1981) The Genus Serratia. The Prokaryotes.1192-1193.
30.Mikhailova AG, Likhareva VV, Khairullin RF, Lubenets NL, Rumsh LD, Demidyuk IV, Kostrov SV (2006) Psychrophilic trypsin-type protease from Serratia proteamaculans. Biochem. (Mosc). 71：563-570.
31.Romero FJ, García LA, Salas JA, Díaz M, Quirós LM (2001) Production, purification and partial characterization of two extracellular proteases from Serratia marcescens grown in whey. Process Biochem. 36：507-515.
32.Salamone PR, Wodzinski RJ (1997) Production, purification and characterization of a 50-kDa extracellular metalloprotease from Serratia marcescens. Appl Microbiol Biotechnol. 48：317-324.
33.Suzuki K, Sugawara N, Suzuki M, Uchiyama T, Katouno F, Nikaidou N, Watanabe T (2002) Chitinases A, B, and C1 of Serratia marcescens 2170 produced by recombinant Escherichia coli: enzymatic properties and synergism on chitin degradation. Biosci. Biotechnol. Biochem. 66：1075-1083.
34.Sasaki YF, 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. Mutation Research/Genetic Toxicol. Environ. Mutage. 519：103-119.
35.Shimada, K. et.al. (1992) J. Agric. Food Chem. 40：945-948.
36.Todd EW (1949) Quantitative studies on the total plasmin and trypsin inhibitor of human blood serum. J. Exp. Med. 39：295-308.
37.Tiwari RP, Singh J, Grewal JS, Grover V, Gupta N (2000) Purification and characterisation of haemolysin from Serratia marcescens B-91. Indian J Med Res. 112：186-190.
38.Wang SL, Kao TY, Wang CL, Yen YH, Chern MK, Chen YH (2006) A solvent stable metalloprotease produced by Bacillus sp. TKU004 and its application in the deproteinization of squid pen for β-chitin preparation. Enzyme Microbial Technol. 39：724-731.
39.Wang SL, Yeh PY (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.
40.Watanabe K, Sakai J, Hayano K (2003) Bacterial extracellular protease activities in field soils under different fertilizer managements. Can. J. Microbiol. 49：305-312.
41.Woytowich AE, Selvaraj G, Khachatourians GG (2000) Analysis of the chiB gene of Serratia liquefaciens. J. Biotechnol. 80：277–283.
42.Yoo JJ, Lee YS, Song CY, Kim BS (2004) Purification and characterization of a 43-kilodalton extracellular serine proteinase from Cryptococcus neoformans. J. Clin. Microbiol. 42：722-726.
43.Zhang Y, Bak DD, Heid H, Geider K (1999) Molecular Characterization of a Protease Secreted by Erwinia amylovora. J. Mol. Biol. 289：1239-1251.
44.Zakaria MB, Muda WMW, Abdullah MP (1995) Chitin and chitosan the versatile environmentally friendly modern materials. Penerbit University Kebangsaan Malaysia. 123：275-282.