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系統識別號 U0002-0607200616572200
中文論文名稱 鳳梨酵素所含蛋白酶及幾丁質酶之純化與定性
英文論文名稱 Purification and Characterization of Proteases and Chitinases from Bromelain
校院名稱 淡江大學
系所名稱(中) 生命科學研究所碩士班
系所名稱(英) Graduate Institute of Life Sciences
學年度 94
學期 2
出版年 95
研究生中文姓名 陳芊蓓
研究生英文姓名 Chien-Pei Chen
學號 693290065
學位類別 碩士
語文別 中文
口試日期 2006-06-08
論文頁數 116頁
口試委員 指導教授-王三郎
委員-陳銘凱
委員-王全祿
中文關鍵字 幾丁質酶  蛋白酶  鳳梨酵素 
英文關鍵字 chitinase  protease  Bromelain 
學科別分類 學科別醫學與生命科學生物學
中文摘要 本研究主要由鳳梨酵素純化分離幾丁質酶與蛋白酶並探討其生化特性。鳳梨酵素經離子交換及膠體過濾層析,可分離出二種蛋白酶(P1、P2)、二種幾丁質酶(C1、C2)和一個蛋白酶/幾丁質酶(PC1)的酵素。
以酪蛋白為基質所測得P1、PC1、P2之最適反應pH分別為6、7、8,最適溫度分別為40℃、70℃、60 ℃。P1在50℃以下和pH 4~7,PC1與P2分別在50℃以下與pH 4~10之間具安定性。基質特異性分析測得三種酵素所含蛋白酶對酪蛋白及偶氮酪蛋白二種基質有較高的水解活性。三種蛋白質酶的活性皆受Cu2+和Zn2+所抑制,此外,PC1和P2會被Fe2+所抑制。P2活性受EDTA和PMSF強烈抑制,而PMSF亦抑制PC1部份活性。以SDS-PAGE分析出來的分子量為26 kDa、28 kDa以及26 kDa之P1、PC1以及P2之蛋白酶。P1蛋白酶的Vmax與Km分別為0.2 U/mL與1.96 mg/mL;PC1蛋白酶的Vmax與Km分別為0.13 U/mL與5.26 mg/mL;P2蛋白酶的Vmax與Km分別為0.16 U/mL與12.7 mg/mL。Lee-S11Me和Lee-S8SO二種化學合成化學品對鳳梨酵素之蛋白酶活性具抑制效果。
C1、PC1和C2三種純化出之幾丁質酶中,PC1和C2之最適pH為4,而C1則為pH 5,最適溫度皆介於50~60 ℃。安定性方面,三者皆於偏酸,溫度25~60 ℃間較穩定。PC1和C2之幾丁質酶活性會受EDTA所抑制,此外,PC1亦會受Fe2+和Mn2+所抑制。SDS-PAGE分析C1、PC1以及C2所得的分子量分別為26 kDa、28 kDa以及28 kDa。C1幾丁質酶的Vmax與Km分別為0.04 U/mL與55.56 mM;PC1幾丁質酶的Vmax與Km分別為0.05 U/mL與28.57 mM;C2幾丁質酶的Vmax與Km分別為0.04 U/mL與125 mM。化學合成化學品則以Lee-O12SO、Lee-O7C、Lee-S10C2和Lee-O701C對鳳梨酵素之幾丁質酶活性具抑制效果。
英文摘要 The purification and characterization of chitinases and proteases from the commercial bromelain preparation was studied. Two chitinases (C1 and C2), two proteases (P1 and P2), and a chitinase/protease (PC1) were purified using DEAE-Sepharose ion-exchange chromatography, CM-Sepharose ion-exchange chromatography, and gel filtration on a Sephacryl S-100 column.
The protease of P1, PC1 and P2 was found to have a pH optimum at 6, 7 and 8, and a temperature optimum at 40℃, 70℃, 60℃, respectively, as acting on casein. The protease was stable at 25 ~ 50℃ and pH 4 ~ 7(P1), pH 4~10(PC1 and P2). Among several natural substrates tested, casein and azocasein were the best substrates. The activity of three proteases was inhibited by Cu2+ and Zn2+. In addition, the activity of PC1 and P2 protease was inhibited by Fe2+. The activity of P2 protease was strongly inhibited by EDTA and PMSF, while the activity of PC1 protease was also inhibited by PMSF. The apparent molecular mass based on SDS-PAGE was estimated 26 kDa, 28 kDa and 26 kDa for P1, PC1 and P2 protease enzyme, respectively. The Vmax and Km were 0.2 U/mL and 1.96 mg/mL, respectively, for the P1 protease enzyme, and they were 0.13 U/mL and 5.26 mg/mL, respectively, for the PC1 protease enzyme. The Vmax and Km were 0.16 U/mL and 12.7 mg/mL, respectively, for the P2 protease enzyme. The protease activity on crude enzyme was inhibited by Lee-S10Me and Lee-S8SO.
The chitinase of PC1 and C2 was found to have a pH optimum at 4, but the chitinase of C1 was at 5; all of them were found to have a temperature optimum at 50 ~ 60℃ as acting on suspended chitin. The chitinase was stable at 25 ~ 60℃ and preferential towards acidity. The activity of PC1 and C2 chitinase was inhibited by EDTA; while the activity of PC1 chitinase was inhibited by Fe2+ and Mn2+. The apparent molecular mass based on SDS-PAGE was estimated 26 kDa, 28 kDa and 28 kDa for C1, PC1 and C2 chitinase enzyme, respectively. The Vmax and Km were 0.04 U/mL and 55.56 mM, respectively, for the C1 chitinase enzyme, and they were 0.05 U/mL and 28.57 mM for the PC1 chitinase enzyme. The Vmax and Km were 0.04 U/mL and 125 mM, respectively, for the C2 chitinase enzyme. The chitinase activity on crude enzyme was inhibited by Lee-O12SO、Lee-O7C、Lee-S10C2 and Lee-O701C.
論文目次 封面內頁
授權書
論文口試委員審議通過委員簽名表
誌謝……………………………………………………………………I
中文摘要………………………………………………………………II
英文摘要………………………………………………………………III
目錄…………………………………………………………………… V
圖目錄………………………………………………………………VIII
表目錄…………………………………………………………………XI

第一章 緒論…………………………………………………………1
第二章 文獻整理……………………………………………………3
2.1 鳳梨酵素……………………………………………………3
2.1.1 鳳梨酵素的來源………………………………………3
2.1.2 鳳梨酵素的成份………………………………………3
2.1.3 鳳梨酵素之生理活性與保健功能……………………4
2.2 幾丁聚合物化學結構與物化性質…………………………5
2.2.1 幾丁聚合物之起源……………………………………5
2.2.2 幾丁質…………………………………………………7
2.2.3 幾丁聚醣………………………………………………8
2.2.4 N-乙醯幾丁寡醣及幾丁寡醣…………………………12
2.3 幾丁質酶……………………………………………………12
2.3.1 幾丁質酶的分類………………………………………12
2.3.2 幾丁質酶之物化性質與天然分佈……………………15
2.3.3 幾丁質酶的一般性質…………………………………16
2.3.4 植物幾丁質酶之功能與特性…………………………17
2.3.5 N-乙醯幾丁寡醣的特性與應用………………………18
2.4 蛋白質之酵素水解…………………………………………20
2.4.1 蛋白酶…………………………………………………20
2.4.2 蛋白酶的分佈…………………………………………21
2.4.3 蛋白酶的一般性質……………………………………22
2.4.4 蛋白酶的分類…………………………………………23
2.4.5 蛋白酶的應用…………………………………………25
第三章 材料與方法………………………………………………30
3.1 實驗材料……………………………………………………30
3.1.1 酵素來源………………………………………………30
3.1.2 化學藥品及耗材………………………………………30
3.1.3 膠體材料………………………………………………31
3.1.4 儀器設備……………………………………………………31
3.2 實驗方法……………………………………………………31
3.2.1 鳳梨酵素之部份性質探討……………………………31
3.2.2 蛋白酶之純化分離及定性……………………………35
3.2.3 幾丁質酶之純化分離及定性…………………………41
第四章 結果與討論………………………………………………43
4.1 鳳梨酵素之部份性質探討…………………………………43
4.1.1 基質特異性……………………………………………43
4.1.2 各種化學合成化合物對酵素活性之影響……………43
4.2 蛋白酶之純化分離及定性…………………………………48
4.2.1 酵素純化………………………………………………48
4.2.2 酵素定性………………………………………………49
4.3 幾丁質酶之純化分離及定性………………………………76
4.3.1 酵素純化………………………………………………76
4.3.2 酵素定性……………………………………………………80
第五章 結論………………………………………………………99
參考資料……………………………………………………………101

圖 目 錄
圖2.1 鳳梨果實至鳳梨莖的剖面…………………………………4
圖2.2 以蝦蟹殼製備幾丁質的流程簡圖…………………………9
圖2.3 α-chitin之結構圖…………………………………………10
圖2.4 β-chitin結構圖……………………………………………11
圖2.5 幾丁質分解酵素之作用機制………………………………14
圖4.1 不同的化學合成化學品(1mg/mL)對鳳梨酵素酵
素所含蛋白酶與幾丁質酶活性之影響……………………45
圖4.2 化學合成化學品(S8SO和S11Me)濃度對鳳梨酵
素蛋白酶活性之影響………………………………………46
圖4.3 化學合成化學品(O12SO、S10C2、O7C和O701C)
濃度對鳳梨酵素幾丁質酶活性之影響……………………47
圖 4.4 鳳梨酵素所含蛋白酶和幾丁質酶之DEAE-Sepharose  
CL-6B 管柱之層析圖………………………………………50
圖4.5 鳳梨酵素所含蛋白酶和幾丁質酶之CM-Sepharose
CL-6B 管柱層析圖……………………………………………51
圖 4.6 蛋白酶P1於Sephacryl S-100之膠體層析圖………………52
圖4.7 PC1區分所含蛋白酶和幾丁質酶之Sephacryl
S-100之膠體層析圖…………………………………………53
圖4.8 蛋白酶P2之Sephacryl S-100之膠體層析圖………………54
圖4.9 以SDS-PAGE測定鳳梨酵素純化過程中具蛋
白酶與幾丁質酶活性波峰酵素液之分子量…………………56
圖4.10 溫度對P1、PC1與P2蛋白酶活性之影響……………………60
圖4.11 溫度對P1、PC1與P2蛋白酶安定性之影響…………………61
圖4.12 pH對P1、PC1與P2蛋白酶活性之影響………………………62
圖4.13 pH對於P1、PC1與P2蛋白酶安定性之影響…………………63
圖4.14 鳳梨酵素所含蛋白酶對不同基質水解能力之
比較…………………………………………………………67
圖4.15 不同基質濃度對鳳梨酵素、P1、PC1與P2之蛋白酶活
性影響………………………………………………………70
圖4.16 蛋白酶(crude enzyme)Lineweaver-Burk 雙倒數
作圖之Km與Vmax……………………………………………71
圖4.17 蛋白酶(P1)Lineweaver-Burk 雙倒數作圖之
Km與Vmax……………………………………………………72
圖4.18 蛋白酶(PC1)Lineweaver-Burk 雙倒數作圖之
Km與Vmax ……………………………………………………73
圖4.19 蛋白酶(P2)Lineweaver-Burk 雙倒數作圖之
Km與Vmax……………………………………………………74
圖4.20 幾丁質酶C1於Sephacryl S-100之膠體層析圖……………77
圖4.21 幾丁質酶C2於Sephacryl S-100之膠體層析圖……………78
圖4.22 溫度對C1、PC1及C2幾丁質酶活性之影響…………………86
圖4.23 溫度對C1、PC1及C2幾丁質酶安定性之影響………………87
圖4.24 pH對C1、PC1及C2幾丁質酶活性之影響……………………88
圖4.25 pH 對C1、PC1及C2幾丁質酶安定性之影響………………89
圖4.26 鳳梨酵素所含幾丁質酶對不同基質水解能力之比較……92
圖4.27 不同基質濃度對鳳梨酵素、C1、PC1及C2之幾丁質
酶活性影響……………………………………………………93
圖4.28 幾丁質酶(crude enzyme)Lineweaver-Burk
雙倒數作圖之Km與Vmax……………………………………94
圖4.29 幾丁質酶(C1)Lineweaver-Burk 雙倒數作圖
之Km與Vmax…………………………………………………95
圖4.30 幾丁質酶(PC1)Lineweaver-Burk 雙倒數作圖之
Km與Vmax……………………………………………………96
圖4.31 幾丁質酶(C2)Lineweaver-Burk 雙倒數作圖之
Km與Vmax……………………………………………………97

表 目 錄
表2.1 自然界存在之蛋白酶……………………………………27
表2.2 蛋白酶之分類……………………………………………29
表3.1 標準蛋白質………………………………………………40
表4.1 鳳梨酵素之基質特異性…………………………………44
表4.2 鳳梨酵素之蛋白酶純化總表……………………………55
表4.3 各種金屬離子及鹽類對鳳梨酵素、P1、PC1及P2
蛋白酶活性之影響……………………………………64
表4.4 界面活性劑對鳳梨酵素、P1、PC1及P2蛋白酶活
性之影響…………………………………………………65
表4.5 化學合成化學品對酵素P1、PC1及P2之蛋白酶活性
影響………………………………………………75
表4.6 鳳梨酵素之幾丁質酶純化總表…………………………79
表4.7 各種金屬離子及鹽類對鳳梨酵素、C1、PC1及C2
幾丁質酶活性之影響…………………………………90
表4.8 界面活性劑對鳳梨酵素、C1、PC1及C2幾丁質酶活
性之影響………………………………………………91
表4.9 化學合成化學品對C1、PC1及C2之幾丁質酶活
性影響…………………………………………………98
表5.1 蛋白酶之生化特性…………………………………………99
表5.2 幾丁質酶之生化特……………………………………99

參考文獻 1. 王立禾,阮立昂譯,1977。蛋白質構造與功能。第133~148頁。復漢出版社。
2. 王鳳英編譯,1993。界面活性劑的原理與應用。第3~139頁。高立圖書出版社,再版。台北。
3. 王三郎,1996。水產資源利用學。高立圖書出版社。
4. 王三郎,2000。生物技術。高立圖書出版社。
5. 高德一,2005。Bacillus sp. TKU004所生產耐有機溶劑蛋白酶之純化及定性。大葉大學生物產業科技學系碩士論文。
6. 張薏苓,2000。鳳梨酵素粗製品水解幾丁聚醣之研究。靜宜大學食品營養學系研究所碩士論文。
7. 張濱、鄭曉珮,2003。鳳梨酵素:大自然的營養聖品。元氣齋出版社,台北市。
8. 張文智,2003。利用Bacillus cereus 發酵蝦蟹殼廢棄物所生產抗真菌酵素之分離純化及其應用。國立中興大學食品科學研究所博士論文。
9. 張瓊瑋,2004。Aeromonas sp. DYU-T007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系碩士論文。
10. 曾惠婷,2005。幾丁聚醣之鳳梨酵素水解產物於抗腫瘤及抗菌之效果探討。淡江大學生命科學系碩士論文。
11. 莊榮輝,1985。水稻蔗糖合成酶之研究。國立台灣大學農業化學研究所博士論文。
12. 莊榮輝,2000。酵素純化方法,酵素化學與分析-酵素化學實驗。國立台灣大學濃業化學系生物化學實驗室,台北。
13. 陳澄河,2004。化腐朽為神奇-淺談幾丁類物之特性與應用。生物資源 生物技術。6(3/4):24-34。
14. 賴威安,2000。Bacillus sp. P-6中蛋白酶的生產與性質分析。國立中與大學食品科學系碩士論文。
15. Adler-Nissen, J. 1986. Enzymatic hydrolysis of food proteins. pp. 19-20. Elselier Applied Science Publ. London and New York.
16. American Academy of Pediatrics Committee on Nutrition. 1989. Hypoallergenic infant formulas. Pediatrics. 83: 1068-1069.
17. Arakane, Y., and D. Koga. 1999. Purification and characterization of a novel chitinase isozyme from yam tuber. Biosci. Biotechnol. Biochem. 63: 1895-1901.
18. Araki, Y., and E. Ito. 1974. A pathway of chitosan formation in Mucor rouxii: enzymatic deacetylation of chitin, Biochem. Biophys. Res.Commun. 56: 669-675.
19. Bagnasco, P., L. D. L. Fuente, G. Gualtieri, F. Noya, and A. Arias. 1998. Fluorescent Pseudomonas spp. As biocontrol agent against forage legume root pathogenic fungi. Soil Viol. Biochem. 30: 1317-1322.
20. Beuth, J. and J. M. Braun. 2005. Modulation of murine tumor growth and colonization by bromelainw, an extract of the pineapple plant (Ananas comosum L.). In Vivo. Mar-Apr, 19(2): 483-485.
21. Beynon, R. J. and J. S. Bond. 1989. Proteolytic enzymes: a piratical approach. pp. 57-65. IRL Press.
22. Bollag, D. M., M. D. Rozycki, and S. J. Edelstein. 1996. Protein methods. Wiley-Liss Inc, USA, New York.
23. Boller, T., A. Gehri, F. Mauch, and U. Vogeli. 1983. Chitinase in bean leaves: induction by ethylene, purification, properties and possible function. Planta. 157: 22-31.
24. Boyce, C. O. L. 1986. NOVO'S handbook of practical biotechnology. A Publication of NOVO Industry A/S Enzymes Division Bagsvaerd, Denmark.
25. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-54.
26. Carroad, D. A., and R. A. Tom. 1978. Bioconversion of shellfish chitin waste: process conception and selection of microorganism. J. Food Sci. 43: 1158-1161.
27. Cohen-Kupiec, R., and J. Chet. 1998. The molecular biology of chitin digest. Curr. Opin. Biotech. 9: 270-277.
28. Collinge, D. B., K. M. Kragh, J. D. Mikkelsen, K. K. Nielsen, U. Rasmussen, and K. Vad. 1993. Plant chitinase. Plant J., 3: 31-40.
29. Cosio, I. G., R. A. Fisher, and D. A. Carroad. 1982. Bioconversion of shellfish chitin waste: waste pretreatment, enzyme production, process design, and economic analysis. J. Food Sci. 47: 901-906.
30. Deane, E. E., J.M. Whipps, J. M. Lynch, and J. F. Peberdy. 1998. The purification and characterization of a Trichoderma harzianum exochitinase. Biochem. Biophys. Acta. 1383: 101-110.
31. Deshpande, M. V. 1986. Enzymatic degradation of chitin and its biological application. J. Sci. Ind. Res. 45: 273-275.
32. Drenth, J., J.N. Jansonius, R. Koekoek, H. M. Swen, and B.G. Wolthers. 1968. Structure of papain. Nature. 218: 929-932.
33. Eijsink, V. G. H., O.R. Veltman, W. Aukema, G. Vriend, and G. Venema. 1995. Structural determinants of the stability of thermolysin-like proteinases. Structural Biol. 2: 374-379.
34. El-Gharbawi, M. and J. R. Whitaker. 1963. Fractionation and partial characterization of the proteolytic enzymes of Stem bromelain. Biochemistry. 2(3): 476-481.
35. Fox, P. F., P. A. Morrisey, and D. M. Mulvihill. 1982. Chemical and enzymatic modification of food protein. In “Developments in Food Proteins, I” pp. 1-60. ed. Hudson, B. J., Elsevier Appl. Sci., NJ, U.S.A.
36. Fujinaga, M., R. J. Read, A. Sielecki, W. Ardelt, M. Jr. Laskowski, and M. N. G. James. 1982. Refined crystal structure of the molecular comples of Streptomyces griseus protease B, A serine protease, with the third domain of the ovomucoid inhibitor from turkey. Proc. Natl. Acad. Sci. USA 79: 4868-4872.
37. Fujiwara, N., K. Yamamoto, and A. Masui. 1991. Utilization of a thermostable alkaline protease from an alkalophilic thermophile for the recovery of silver from used X-ray film. J. Ferment Bioeng. 72: 306-308.
38. Fujiwara, N., and K. Yamamoto. 1987. Decomposition of gelatin layers on X-ray films by the alkaline protease from Bacillus sp. Hakkokogaku. 65: 531-534.
39. Gajju, H., T. C. Bhalla, and H. O. Agarwal. 1996. Utilization of thermostable alkaline protease from Bacillus coagulans PB-77 for silver recovery from used X-ray films. INL Proceedings of 37th Annual Conference of Association of Microbiologists of India, December 4-6, Chennai, India, (Abstract no. IM-4), pp. 79.
40. Gildberg, A. 1993. Enzymatic processing of marine raw materials, Process Biochem. 28: 1-15.
41. Gildberg, A., and J. Raa. 1977. Properties of propionic acid/formic acid preserved silage of cod viscera. J. Sci. Food Agric. 28: 647-653.
42. Godfrey, T. 1986. Comparison of key characteristics of industrial enzyme by type and source. In: Industrial Enzymology. pp. 466-557. (ed. T. Godfrey and J. Reichelt). Macmillan Publishers Ltd, New York.
43. Gooday, G. W. 1990. Physiology of microbial degradation of chitin and chitosan. Biodegradation 1: 177-190.
44. Gould, J. B. 1975. Enzyme data, In: Handbook of enzyme niotechnology (Wise-man, A., ed.). pp. 128-162. Ellis Horwood Ltd., Chichester.
45. Graham, L. S., and K. Vad. 1994. Plant chitinase. Can. J. Bot. 72: 1057-1083.
46. Gupta, M. N. 1992. Enzyme function in organic solvents. Eur. J. Biochem. 203: 25-32.
47. Hale, L. P. 2004. Proteolytic activity and immunogenicity of oral bromelain within the gastrointestinal tract of mice. Int Immunopharmacol 4: 255-264.
48. Hale, L. P., P. K. Greer, C. T. Trinh, and C. L. James. 2005. Proteinase activity and stability of natural bromelain preparations. Int Immunopharmacol. 5: 783-793.
49. Hackman, R. H. 1954. Austr. J. Biol. Sci. 7: 168-178.
50. Hartley, B. S. 1960. Proteolytic enzymes. Ann. Rev. Biochem. 29: 45.
51. Heinicke, R. M., and W. A. Gortner. 1957. Stem bromelain – a new protease preparation from pineapple plants. Econ. Bot. 11: 225-234.
52. Hirano, S., T. Yamamoto, M. Hayashi, T. Nishida, and H. Inui. 1990. Chitinase activity in seeds coated with chitosan derivates. Agric. Biol. Chem. 54: 2719-2720.
53. Hou, W. C., Y. C. Chen, and Y. H. Lin. 1998. Chitinase activity of sweet potato (Ipomoea batatas [L.] Lam var. Tainong 57). Bot. Bull. Acad. Sin. 39: 93-97.
54. Hung, T. H., Y. M. Chang, H. Y. Sung, and C. T. Chang. 2002. Purification and characterization of hydrolase with chitinase and chitosanase activity from commercial stem bromelain. J Agric Food Chem. 50:4666-73.
55. Huber, R., and W. Bode. 1978. Structure basis of the activation and action of trypsion. Acc. Chem. Res. 11: 114-122.
56. Imoto, T., and K. Yagishita. 1971. A simple activity measurement by lysozyme. Agric. Biol. Chem. 35: 1154-6.
57. James, J., and B. K. Simpson. 1996. Application of Enzymes in Food Processing, Crit. Rev. in Food Sci. Nutr. 36(5): 437-463.
58. Jeuniaux, C. 1966. Methods in enzymology. 8: 644-654. Academic Press, New York.
59. Jeuniaux, C. 1964. Free chitin and masked chitin in invertebrate skeletal structures. Arch. Int. Physiol. Biochem. 72: 329-330.
60. Jones, B. L., D. Fontanini, M. Jarvinen, and A. Pekkarinen. 1997. Simplified endoproteinase assays using gelatin or azogelatin. Anal. Biochem. 263: 214-220.
61. Kafetzopoulos, D., A. Martinou, and V. Bouriotis. 1993. Bioconversion of chitin to chitosan: purification and characterization of chitin deacetylase from Mucor rouxii, Proc. Natl. Acad. Sci. 90: 2564-2568.
62. Karadzic, I., A. Masui, and N. Fujiwara. 2004. Purification and characterization of a protease from Pseudomonas aeruginosa grown in cutting oil. J. Biosci. Bioeng. 3: 145-152.
63. Knorr, D. 1984. Use of chitinous polymer in food. Food Technol. 1: 85-88.
64. Koga, D., T. Tsukamoto, N. Sueshige, T. Usumi, and A. Ido. 1989. Kinetics of chitinase from yam, Dioscorea opposite thumb. Agric. Biol. Chem. 53(12): 3121-3126.
65. Koga, D., K. Mizuki, A. Ide, M. Kono, T. Matsui, and C. Shimizu. 1990. Kinetics of a chitinase from a prawn, Penzeus japonicus. Agric Biol. Chem. 54: 2505-2512.
66. Koga, D., T. Hirata, N. Sueshige, S. Tanaka, and A. Ide. 1992. Induction patterns of chitinases in yam callus by inoculation with autoclaved Fusarium oxysporum, ethylene, and chitin and chitosan oligosaccharides, Biosci, Biotech. Biochem. 56: 280-285.
67. Koga, D. 1996. Comparative biochemistry of insect and plant chitinaseIn: RAA Muzzarelli (ed): Chitin enzymology, 2: Atec, Grottammare, 85-94.
68. Kono, M., T. Matsui, and C. Shimizu. 1987. Purification and some properties of chitinase from the stomach of red sea bream Pagrus major. Nippon Suisan Gakkaishi. 53: 131-136.
69. Kono, M., T. Matsui, C. Shimizu, and D. Koga. 1990. Purification and some properties of chitinase from the stomach of Japanese ell, Anguilla japonica. Agric. Biol. Chem. 54: 973-978.
70. Kristinsson, H. G., and B. A. Rasco. 2000a. Fish protein hydrolysates: production, biochemical, and functional properties. Crit. Rev. Food Sci. Nutr. 40(1): 43-81.
71. Laemmli, U. K. 1970. Cleavage of structural during assembly of the head of bacteriophage T4. Nature. 227: 680-685.
72. Lahl, W. J., and S. D. Braun. 1994. Enzymatic production of protein hydrolysates food use. Food Technol. 48(10): 68-71.
73. Laskowski, M. Jr. and I. Kato. 1980. Protein inhibitors of proteinases. Annu. Rev. Biochem. 49: 593-626.
74. Light,A.. and J. Greenberg. 1965. The sequence of 26 amino acid residues at the amino terminus of papain. J. Biol. Chem. 204: 258-265.
75. Matsumiya, M., and A. Mochizuki. 1995. Purification and some properties of chitinase from the stomach of common mackerel Scomber japonicus. Bull Coll Agr Vet Med, Nihon Univ. 52: 131-136.
76. Maurer, H. R. 2001. Bromelain: biochemistry, pharmacology and medical use. Cell Mol. Life Sci. Aug; 58(9): 1234-1245.
77. Metaig, C., E. Grabowska, K. Eckert, K. Rehse, and H. R. Maurer. 1999. Bromelain proteases reduce human platelet aggregation in vitro, adhesion to bovine endothelial cells and thrombus formation in rat vessels in vivo. In Vivo. Jan-Feb; 13(1): 7-12.
78. Moneral, J., and E. T. Reese. 1969. The chitinase of Serratia marcescens. Can. J. Microbiol. 15: 689-696.
79. Morita, Y., Q. Hasan, T. Sakaguchi, Y. Murakami, K. Yokoyama, and E. Tamiya. 1998. Proterties of a cold-active protease from psychrotrophic Flavobacterium balustinum P104. Appl. Microbiol. Biotechnol. 50: 669-675.
80. Mulder, F. A. A., D. Schipper, R. Bott, and R. Boelens. 1999. Altered flexibility in the substrate-binding site of related native and engineered highalkaling Bacillus subtilisins. J. Mol. Biol. 292: 111-123.
81. Murachi, T., and H. Neurath. 1960. Fractionation and specificity studies on stem bromelain. Journal of Biological Chemistry. 235(1): 99-107.
82. Murao, S., T. Kawada, H. Itoh, H. Oyama, and T. Shin. 1992. Purification and characterization of a novel type of chitinase from Vibrio alginolyticus TK-22. Biosci. Biotech. Biochem. 56: 368-369.
83. Murao, S., T. Kawada, H. Itoh, H. Dyama, and T. Shin. 1992. Purification and characterization of a novel type of chitinase from Vibrio alginolyticus TK-22. Biosci. Biothec. Biochem. 56: 368-369.
84. Muzzarelli, R. A. A., G. and R. Roccheti. 1978. Isolation of lysozyme on chitosan. Biotech. Bioeng. 20:87-94.
85. Muzzarelli, R. A. A. 1985. Determination of the degree of acetylation of chitosan by first derivative ultraviolet spectrometry. Carbohydr. Polymer. 5:461-469.
86. Mynott, T. L., A. Ladhams, P. Scarmato, and C. R. Engwerda. 1999. Bromelain, from pineapple stem, proteolytically blocks activation of extracellular regulated kinase-2 in T cells. J. Immunol. 163: 2568-2575.
87. Nakamura, T., Y. Syukunobe, T. Sakarai, and T. Idota. 1993. Enzymatic production of hypoallergenic peptides from casein. Milchwissenschaft. 48: 11-14.
88. Nam, M. Y., Y. H. Shon, S. K. Kim, C. H. Kim, T. R. Jeong, and K. S. Nam. 2000. Effect of chitosan oligosaccharides on polyamine metabolism for chemopreventive activity. J. Chit. Chitos. 5: 15-18.
89. Nam, M. Y., Y. H. Shon, S. K. Kim, C. H. Kim, and K. S. Nam. 1999. Inhibitory effect of chitosan oligosaccharides on the growth of tumor cells. J. Chit. Chitos. 4: 184-188.
90. Neuhaus, J. M. 1999. Plant chitinase (PR-3, PR-4, PR-8, PR-11). In “Pathogenesis-Related proteins in Plants”, eds. Datta, S. K. and Muthukrishnan, S., CRC Press, Bokca Raton. pp. 77-105.
91. Neurath, H. 1984. Evolution of proteolytic enzymes by Hans. Science. 24: 350.
92. Osswald, W. F., J. P. Shapiro, H. Doostdar, R. E. McDonald, R. P. Niedz, C. J. Nairn, J. Hearm, and R. T. Mayer. 1994. Identification and characterization of acidic hydrolases with chitinase and chitosanase activities from sweet orange callus tissue. Plant Cell Physiol. 35: 811-820.
93. Overdijk, B. and G. J. V. Steijk. 1994.Human serum contains a chitinase: identification of an enzyme, formerly described as 4-methylumbelliferyl-tetra-N-acetylchitotetraoside hydrolase (MU-TACT hydrolase). Glycobiol. 4: 797-803.
94. Ota, S., T. H. Fu, and R. Hirohata. 1961. Studies on bromelain II. Its activation and fractionation. Journal of Biochemistry. 49(6): 532-537.
95. Reed, G. 1975. Enzymes in food processing. 2nd ed pp. 263. academic Press.
96. Roberts, G. A. F. 1992. Chitin Chemistry. MacMillan Press, London.
97. Roby, D., A. Gadell, and A. Toppan. 1987. Chitin oligosaccharides as elicitors of chitinase activity in melon plants. Biochem. Biophys. Res. Commun. 143: 885-887.
98. Rose, B., C. Herder, H. Loffler, G. Meierhoff, N. C. Sehloot, M. Walz, and S. Martin. 2006. Dose-dependent induction of IL-6 by Plant-derived proteases in vitro. Clin. Exp. Immunol. Jan; 143(1): 85-92.
99. Rowan A. D., D. J. Buttle, and A. J. Barrett . 1988. Ananain: a novel cysteine proteinase found in pineapple stem. Arch Biochem Biophys. 267: 262-70.
100. Shigemasa, Y., K. Saito, H. Sashiwa, and H. Saimoto. 1994. Enzymatic degradation of chitins and partially deacetylated chitins. Int. J. Biol. Microbiol. 16: 43-49.
101. Shimahara K., Y. Takiguchi, K. Ohkouchi, and O. Okada. 1984. Chitin, Chitosan, and related enzymes, ed. P. J. Zikakis. 239-255, Academic press, Oriando.
102. Singh L. R., Th. P. Devi, and S. K. Devi. 2004. Purification and characterization of a pineapple crown leaf thiol protease. Preparative Biochem. Biotech. 34(1): 25-43.
103. Singh, P. P., Y. C. Shin, C. S. Park, and Y. R. Chung. 1999. Biological control of Fusarium wilt of cucumber by chitinolytic bactera. Phytopathol. 89: 92-99.
104. Sormorin, O., and N. Nishi. 1979. Studies on chitin preparation of benzyl and benzoyl chitins. Polym. J. 2:391-498.
105. Suzuki, K., T. Mikami, Y. Okawa, A. Tokoro, S. Suzuki, and M. Suzuki. 1986. Antitumor effect of hexa-N-acetyl-chitohexaose. Carbohydr. Res. 151: 403-410.
106. Tagawa, K., and K. Okazaiki. 1991. Isolation and some culture conditions of Streptomyces species which produce enzyme lysing Aspergillus niger cell wall. J. Ferment. Bioeng. 71: 230-236.
107. Taira, T., N. Toma, and M. Ishihara. 2005. Purification, characterization, and antifungal activity of chitinases from pineapple (Ananas comosus) leaf. Biosci. Biotechnol. Biochem. 69(1): 189-196.
108. Takahashi, N., Y. Yasuda, K. Goto, T. Miyake, and T. Murachi. 1973. Multiple molecular form of stem bromelain. Isolation and characterization of two closely related components, SB1 and SB2. Journal of Biochemistry. 74: 355-373.
109. Takayanagi, T., K. Ajisaka, Y. Takiguchi, and K. Shimahara. 1991. Isolation and characterization of thermostable chitinases from Bacillus licheniformis X-7u. Biochem. Biophys. Acta. 1078: 404-410.
110. Takegawa, K., B. Mikami, S. Iwahara, Y. Marita, K. Yamamoto, and T. Tochikura. 1991. Complete amino acid sequence of endo-β-N-acetylglucosaminidase from Flavobacterium sp. Eur. J. Biochem. 202: 175-180.
111. Timmis, K., G. Hobbs, and R. C. W. Berkeley. 1974. Chitinolytic Clostridia isolated from marine mud. Can. J. Microbiol. 20: 1284-1285.
112. Tokoro, A., N. Tatewaki, K. Suzuki, T. Mikami, S. Suzuki, and M. Suzuki. 1998. Growth-ingibitory effect of hexa-N-acetylchitohexaose and chitohexaose against meth-A solid tumor. Chem. Pharm. Bull. 36: 784-790.
113. Tsujibo, H., K. Minoura, K. Miyamoto, H. Endo, M. Moriwaki, and Y. Inamori. 1993. Purification and properties of a thermostable chitinase from Streptomyces thermoviolaceus OPC-520. Gene. 134: 113-117.
114. Tsutomu, T. A. Kasumi, T. Yasuyaki, and S. Venzo. 1991. Isolation and characterization of thermostable chitinase from Bacillus licheniformis. Biochem. Biophy. Acta. 1078: 404-410.
115. Todd, E. W. 1949. Quantitative studies on the total plasmin and trypsin inhibitor of human blood serum. J. Exp. Med. 39: 295-308.
116. Taylor, M. M., D. G. Bailey, and S. H. Feairheller. 1987. A review of the use of enzymes in the tannery. J. Am. Leather Chem. Assoc. 82: 153-165.
117. Usui, T., Y. Hayashi, F. Nanjo, K. Sakai, and Y. Ishido. 1987. Transglycoylation reaction of a chitinase purified from Nocardio orientalis. Biochem. Biophys. Acta. 9923: 302-309.
118. Vad, K., J. D. Mikkelsen, and D. B. Collinge. 1991. Induction, purification and characterization of chitinase isolated from pea leaves inoculated with Ascochyta pisi. Planta. 184: 24-29.
119. Wang, S. L. and W. T. Chang. 1997. Purification and characterization of two bifunctional chitinase/lysozymes extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium. Appl. Environ. Microbiol. 63: 380-386.
120. Wang, S. Y., A. Moyne, G. Thottappilly, S. Wu, R. D. Locy, and N. K. Singh. 2001. Purification and characterization of a Bacillus cereus exochitinase. Enzyme Bicrob. Technol. 28: 492-498.
121. Ward, O. P. 1985. Proteolytic enzymes. In: Blanch, H. W., Drew, S., Wang, D. I.,editors. Comprehensive Biotechnology, The Principles, Applications and Regulations of Biotechnology in Industry, Volume 3. Oxford and New York: Pergamon Press. Pp. 709-818.
122. Wortman, A. T., C. C. Somerville, and R. R. Colwell. 1986. Chitinase determinants of Vibrio vulnificus: gene cloning and applications of a chitinase probe. Appl. Environ. Microbiol. 52: 142-145.
123. Wynne, E. C. and J. M. Pemberton. 1986. Cloning of a gene cluster from Cellvibrio mixtures which codes for cellulose, chitinase, amylase and pectinase. Appl. Environ. Microbiol. 52: 1362-1367.
124. Yabuki, M., K. Mizushina, T. Amatatsu, A. Ando, T. Fujii, M. Shimada, and M. Yamashita. 1986. Purification and characterization of chitinase and chitobiase produced by Aeromonas hydropila subsp.anaerogenes A52. J. Gen. Appl. Microbiol. 32: 25-38.
125. Yang, Y., S. Kuramitsu, and K. Hamaguchi. 1981. Hydrolsis of 4-methyl-umbelliferyl-N-acetyl-chito-oligosaccharides catalyzed by human lysozyme. J. Biochem. 89: 1357-1364.
126. Ye X. and T. B. Ng. 2005. A chitinase with antifungal activity from the mung bean. Protein Express. Purif. 40: 230-236.

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