Bacillus amyloliquefaciens V656係株分離自台灣中部土壤之蛋白酶生產菌。本研究以B. amyloliquefaciens V656發酵全脂奶粉生產蛋白酶。B. amyloliquefaciens V656生產蛋白酶之較適培養條件為0.5% milk powder，0.05% MgSO4．7H2O，0.1% K2HPO4 ,於pH 7，100mL，30℃,所得蛋白酶活性為55.15 U/mL。B. amyloliquefaciens V656發酵全脂奶粉所得上清液經硫酸銨沉澱、DEAE-Sepharos陰離子交換層析法及Sephacryl-S100膠體過濾層析，可純化出一種蛋白酶。此蛋白酶之最適反應pH、最適反應溫度、pH安定性及熱安定性分別為7、50℃、6-9及50℃。所得蛋白酶經SDS-PAGE電泳分析測得分子量為29 KDa，EDTA及馬麥特鐵對其具抑制效果。

A protease producing microorganism, Bacillus amyloliquefaciens V656, capable of utilizing whole milk powder as nutrition source was screened out from the soils in central Taiwan. In this research, whole milk powder was fermented by B. amyloliquefaciens V656 to produce protease. An optimal protease activity55.15 U/mL was obtained with a medium containing 0.5% milk powder，0.05% MgSO4．7H2O，0.1% K2HPO4 at pH 7,100 mL, 30℃.A protease was purified from the culture supernatant with ammonium sulfate precipitation followed by DEAE-Sepharose column chromatogrephy and Sephacryl-S100 gel chromatography. The purified protease was stable within pH 6-9. The optimal temperature and pH for the protease were 50℃ and 7 respectively. The molecular weight of the protease was 29 KDa assayed by SDS-PAGE. EDTA and marmite irons showed inhibitory effect on this protease.

論文口試委源審議通過委員簽名表、授權書
中文論文提要 I
英文論文提要 II

第一章　研究目的 1
第二章　文獻回顧 2
2.1 發酵作用 2
2.1.1發酵作用的起源 5
2.1.2結論 14
2.1.3主要研究者 14
2.1.4參考資料 15
2.2 酵素之應用 21
2.3 蛋白酶 27 2.3.1 發現與簡介 27
2.3.2 命名與其分類依據 28
2.4 一般性質 30
2.4.1熱變性 30
2.4.2表面變性 30
2.4.3壓力變性 30
2.4.4 pH變性 31
2.4.5有機溶劑變性 31
2.5附錄 蛋白質研究歷史 32
第三章 研究材料與方法 36
3.1材料及藥品 36
3.2研究設備與器材 37
3.3實驗菌株 38
3.4實驗方法 38
3.4.1 蛋白酶活性之測定 38
3.4.2蛋白質定量分析 39
3.5蛋白酶最適生長條件探討 39
3.5.1碳源濃度對酵素的影響 39
3.5.2 基礎培養基起始之pH對酵素的影響 39
3.5.3溶氧量對酵素的影響 39
3.5.4 培養時間對酵素的影響 40
3.5.5 微量金屬對酵素的影響 40
3.5.6 培養溫度 40
3.6 蛋白酶之分離純化 40
3.6.1 大量培養 40
3.6.2 上清液的製備 40
3.6.3硫酸銨沉澱 41
3.6.4陰離子交換樹脂（Anion exchange chromatography）41
3.6.5膠體過濾層析（Gel filtration chromatography）41
3.7 蛋白酶之生化性質探討 41
3.7.1酵素之最適反應pH測定 41
3.7.2 酵素之pH安定性測定 42
3.7.3 酵素之最適反應溫度測定 42
3.7.4 酵素之熱安定性測定 42
3.7.5 蛋白質電泳分子量的測定 43
3.7.6 等電點測定 45
3.7.7 金屬離子及蛋白酶抑制劑對酵素活性的影響 46
3.7.8 有機溶劑對酵素活性之影響 46
3.7.9 CaCl2存在下之熱安定性 47
3.7.10 測何種金屬離子為酵素之輔酶 47
第四章 研究結果與討論 49
4.1 Bacillus amyloliquefaciens V656生產蛋白酶最適培養條件之探討 49
4.1.1 氮源 49
4.1.2基礎起始培養基之pH值 49
4.1.3培養液充填體積 50
4.1.4培養時間對酵素活性的影響 50
4.1.5金屬離子及EDTA對酵素活性的影響 50
4.1.6培養溫度對酵素活性的影響 51
4.1.7綜合結果 51
4.2 Bacillus amyloliquefaciens V656之酵素純化 56
4.2.1 Diethylaminoethyl（DEAE）Sepharose CL-6B 陰離子層析法 57
4.2.2 Sephacryl S-100 gel filtration 57
4.2.3酵素之純化概要表 57
4.3 Bacillus amyloliquefaciens V656酵素生化性質的分析 61
4.3.1 酵素之最適反應pH值測定 61
4.3.2 酵素之pH安定性測定 61
4.3.3 酵素之最適反應溫度測定 61
4.3.4 酵素之溫度穩定性測定 61
4.3.5 酵素之等電點測定 61
4.3.6 酵素之Km及Vmax值測定 62
4.3.7 酵素之分子量測定 68
4.3.8金屬離子及抑制試劑對酵素活性的影響 69
4.3.9 有機溶劑對酵素活性之影響 71
4.3.10 CaCl2存在下之熱安定性 72
4.3.11 測何種金屬離子為酵素之輔酶 73
第五章 結論 78
第六章 參考文獻 79

圖目錄

圖 4.1.1氮源和培養天數對Bacillus amyloliquefaciensV656蛋白酶生產能之影響 52
圖 4.1.2 起始pH值對於Bacillus amyloliquefaciensV656蛋白酶生產能之影響 53
圖 4.1.3 溶氧量對Bacillus amyloliquefaciensV656蛋白酶生產能之影響 54
圖 4.1.6 培養溫度對Bacillus amyloliquefaciensV656蛋白酶生產能之影響 55
圖4.2.1 DEAE Sepharose CL-6B之蛋白酶陰離子層析法 58
圖4.2.2 Sephacryl S-100之蛋白酶過濾層析法 59
圖4.3.1蛋白酶之最適反應(pH 7) 63
圖4.3.2蛋白酶之PH安定性( pH 7) 64
圖4.3.3蛋白酶之最適反應溫度 (30℃) 65
圖4.3.4蛋白酶之熱安定性 (30℃) 66
圖4.3.5蛋白酶之等電點層析圖譜 67
圖4.3.7蛋白酶之SDS-PAGE 68
圖4.3.8金屬離子及抑制試劑對酵素活性的影響 74
圖4.3.9 有機溶劑對酵素活性之影響 75
圖4.3.10 CaCl2存在下之熱安定性 76
圖 4.3.11 測何種金屬離子為酵素之輔酶 77

表目錄

表2.5附錄 蛋白質研究歷史 32
表3.1電泳片之配置組成 44
表4.2.1 Bacillus amyloliquefaciensV656蛋白酶之純化表 60
表4.1.5 各種濃度之金屬離子及EDTA對蛋白酶活性的影響 70

1.Kumar S,Sharma R,Saharan R(2005)Extracellular acid protease from Rhizopus oryzae:purification and characterization. Biochemistry 40:1701-1705.

2.Rossland E,Langsrud T,Einar P,Sorhaug T(2005)Production of antimicrobial metabolites by strains of Lactobacillus or Lactococcus co-cultured with Bacillus cereus in milk.Journal of Food Microbiology 98:193-200.

3.Rossland E,Langsrud T ,Sorhaug T(2005)Influence of controlled lactic fermentation on growth and sporulation of Bacillus cereus in milk.Journal of Food Microbiology.

4.JOO H.S.,Koo Y.M.,Choi J.W.,Chang C.S(2005)Stabilization method of an alkaline protease from inactivation by heat,SDS and hydrogen peroxide, Journal of Food Microbiology.36:766-772.

5.Rueckert A,Ronimus S,Morgan H(2005) Development of a rapid detection and enumeration method for thermophilic bacilli milk powders. Journal of Microbiological Methods 60:155-167 .

6.Vyver W,Dawson D,Casey N,Tricarico J(2005)Effect of glycosylation on the stability of fungal xylanase exposed to protease or rumen fluid in vitro.Journal of Food Microbiology 116:259-269.

7.Azeredo L,Freire D,Soares R,Leite S,Ceolh R (2004)Production
and partial Characterization of thermophilic proteasefrom
Streptomyces sp.isolated from Brazilian cerrado soil.Enzymeand
Microbial Technology,34：354-358.

8.Ashok S,Osaku C,Rocha S,Carlos K (2003) Characterization and Stability of protease from Penicillium sp. produced bysolid-state fermentation. Enzyme and Microbial Technology 32：246-251.

9.Nishiya Y,Hibi T,Oda J(2002)A purification method of the diagnostic enzyme Bacillus uricase using magnetic beads and non-specific protease.Journal of Food Microbiology,25:426-429.

10.Wang S.L.,Shin I.L.,Liang T.W.,Wang C.H(2002) Purification and Characterization of Two Antifungal Chitinases Extracellularly Produced by Bacillus amyloliquefaciens V656 in a Shrimp and Crab Shell Powder Medium.J. Agric.Food Chem ,50：2241-2248.

11.Wang S.Y.,Moyne A.L.,Wu S.J.,Locy D.,Singh N.K (2001)Purification and characterization of a Bacillus cereusexochitinase. Enzyme and Microbial Technology 28：492-498.

12.KAWACHI I,FUJIEDA T,UJITA M,SHII Y,YAMAGISHI K,SATO H,FUNAG
T,HARA H (2001) Purification and Propertiea of Extracellular
Chitinasea from the Parasitic FungusIsaria japonica. JOURNAL OF
BIOSCIENCE AND BIOENGINEERING Vol.92,No.6,544-549.

13.Aikat K,Bhattacharyya B(2001)Protease production in solid
state fermentation with Liquid medium recycling in a stacked
platereactor and in a packed bed reactor by a local strain of
Rhizopus oryzae Process Biochemistry 36：1059-1068.

14.Wang S.L.,Yang J.K.,Shin I.L.,Tzeng Y.M(2000) Production and purification of protease from a Bacillus subtilis that can
deproteinize crustacean wastes Enzyme and Microbial Technology 26 ：406–413.

15.Sookkheo B,Sinchaikul S,Phutrakul S ,and Chen S.T(2000) Purification and Characterization of the Highyl Thermostable Proteases form Bacillus stearothermophilus TLS33.

16.Atanabe W,Yamada M,Nakagawa S,Hirose T,Noguchi A,Yasuda M and Ishikawa H.(1999) Purification and characterization of organic solvent-stable protease from organic solvent-tolerant Pseudomonas aeruginosa PTS-01.Journal of Bioscience and Bioengineering 87：61-68.

17.Babiker M,Banat A,Kameyama Y,Yoshioka A,Koga D. (1999) Purification and characterization of A 54 kDa chitinase from Bombyx mori. Insect Biochemistry and Molecular Biology 29：537-547.


18.Yamaoka H,Hayashi H,Karita S,Kimura T,Sakka K,Ohmiya K
(1999)Purification and Some Properties of a Chitinase from
Xanthomonas sp.Strain AK.Journal of Bioscience and
Bioengineering 88:328-330.

19.Kang S,Park S, Lee G (1999) Purification and characterization of a Novel Chitinase from the entomopathogenicfungus Metarhizium anisopliae. Journal of Invertebrate Pathology 73：276-281.

20.Yang F.C.,Lin I.H (1998) Production of acid protease using thin stillage from a rice-spirit distillery by Aspergillus niger. Enzyme and Microbial Technology 23：397-402.

21.Babe L.,Schmide B (1998) Purification and biochemical nalysis of WprA,a 52-kDa serine protease rsecreted by B.subtilis as an activecomplexwithits23-kDapropeptide.Biochimiophys.Actas.1386：211-219.

22.Liao C,Callus E (1998)Biochemicaland genetic characterization of an Extracellularprotease from Pseudomonas fluorescens Microb,64：914-921.

23.Wang S.L.,Chio S.H (1998) Deproteinization of shrimp and crab shell with the protease of Pseudomonas aeruginosa K-187.Enzyme and Microbial Technolopy 22:629-633.

24.REMISHIH N.,CDONALD K(1997) Purification and Characterization of Chitinase from Transformed Callus Suspension Cultures of Trichosanthes kirilowii Maxim Journal of Fermentation and Bioengineering.84:128-34.

25.Brautigam M,Hertel C,Hammes W(1997) Evidence for natural transformation of Bacillus subtilis in foodstuffs.155:93-98.

26.Anwar A,Saleemuddin M(1997) Alkaline pH actiog digestive
enzymeof the polyphagous insect pest Spilosoma obliqua：
stability and potential as detergent additives Biotechnol.Appl.
Biochem.25：43-46.

27.Chen H.C.,Hsu M.F.,Jiang S.T (1997)Purification and characterization of an exo-N,N-diacetylchitobiohydrolase-like enzyme from Cellulomonas flavigena NTOU1.Enzyme and Microbial Technology 20:191-197.

28.Savi A,Tuong A,Maftouf M,Pereillo J.Capdevielle Y,Cuillemost J.Maffread J. (1996) Purification and characterization of a novel protease from culture filtrates of a Streptomyces sp. FEMS Microbiology Letters 141：213-220.

29.Ikasari L,Mitchell D(1996) Leaching andcharacterization
of Rhizopus oligosporus acid protease
fromsolid-statefermentation. Enzyme Microb.Technol,19：
171-175.

30.OHISHI K,YAMAGISHI M,OHTA T,SUZUKI M,IZUMIDA H,SANOH N , TAN MIW A(1996) Purification and Properties of Two Chitinases from Vibro alginolyticus H-8. JOURNAL OF FERMENTATION AND BIOENGINEERING.82:588-600.

31.Ikasari L., Mitchell A (1996) Leaching and characterization of Rhizopus oligosporus acid protease from solid-state fermentation. Enzyme and Microbial Technology 19：171-175.

32.Sakurada M,MorgaviP,Komatani M,Tomita A,Onodera R (1996)
Production andcharacterization of cytosolic chitinase from
Piromycescommunis OTS1. FEMS Microbiology Letters 137：75-78.

33.Reichard U,Monod M,Ruchel R (1995)Molecular cloning and
sequencing of the gene encoding an Extracellular aspartic
proteinase from Aspergillus fumigatus. FEMS Microbiology
Letters 130：69-74.

34.Planchot Y, Colonna P (1995) Purification and Characterization of extracellular alpha-amylase from Aspergillusfumigatus. Carbohydrate Research 272：97-109.

35.Elango C, Polacheck E(1982) Endo-chitinase,a mannan-associated eneyme from Saccharoment cerevisiae.Journal Biol.Chem 257:1392-1397.

36.D.M Fenton ,D.E Eveleigh (1981) Purfication and mode ofaction of a chitionase from Penicillium islandicum.Journal Microbiol.126: 151-165.

37.PANNIFER A.D,WONG Y.A,SCHWARZENBACHER R,RENATUS R,PETOSA A, BIENKOWSKA J,LACY G,COLLIER R.J,PARK S, LEPPLA S,HANNA P & R. C(2001) LIDDINGTON.Crystal structure of the anthrax lethal factor, Nature 414: 229-233.

38.BRADLEY K.A,MOGRIDGE J,MOUREZ M,COLLIER R & YOUNG J(2001) Identification of the cellular receptor for anthrax toxin, Nature 414: 225-229 .

39. MOUREZ M,KANE R,MOGRIDGE Y,METALLO B,DESCHATELETS P, SELLMAN U, WHITESIDES L & COLLIER U(2001) Designing a polyvalent inhibitor of anthrax toxin,Nature Biotechnology 19:958–961 .

40.劉錦芳 (2003) LCF007細菌所生產蛋白質分解酵素純化之研究，台
灣彰化。

41.宋賢一 (2002) 生物化學實驗原理與方法，藝軒圖書出版社。

42.陳石根、周潤琦 (2002) 酶學，九州圖書有限公司。

43.王三郎 (2000) 應用微生物學，高立圖書出版社。

44.王三郎 (2000) 生物技術，高立圖書出版社。

45.莊榮輝 (2000) 酵素化學實驗 ，國立台灣大學農業化學系。

46.梁慈雯 (2000) Bacillus subtilis V656所生產微生物抑制物質之研究,私立大葉大學食品工程研究所碩士論文,台灣彰化。

47.蔡文城 (2000) 微生物學，藝軒圖書出版社。