Bacillus subtilis TKU007係株以蝦殼粉為主要碳源，篩選自台灣北部土壤之蛋白酶生產菌，發酵蝦殼粉所得離心上清液具有幾丁聚醣酶及蛋白酶活性。幾丁聚醣酶較適生產條件為1.5% 蝦殼粉、0.1%
K2HPO4、0.05% MgSO4．7H2O之液態培養基(pH7)於30℃振盪培養1天。發酵所得離心上清液經硫酸銨沉澱、DEAE-Sepharose及Sephacryl-S100管柱層析後，可純化出幾丁聚醣酶。經SDS-PAGE及膠體層析測定分子量分別為25 kDa及33 kDa。TKU007幾丁聚醣酶之N末端胺基酸序列含：PQNI。其最適反應pH、最適反應溫度、pH安定性、熱安定性、Km 及Vmax分別為pH 7, 37℃, pH 4-9,25-37 ℃, 0.438 mg/mL, 0.144 U/mL；其活性會受Cu2+所抑制，而2%(v/v) Tween 40或Triton X-100則不具抑制效果。
B. subtilis TKU007生產耐界面活性劑、耐有機溶劑及對鹼性安定性高之絲胺酸蛋白酶之較適培養條件為1.5% 蝦殼粉、0.1% K2HPO4
、0.05% MgSO4．7H2O之液態培養基(pH7)於30 ℃培養2天。發酵所得離心上清液離心，經硫酸銨沉澱、DEAE-Sepharose、Phenyl Sepharose及Sephacryl S-100管柱層析後，純化出一分子量經SDS-PAGE及膠體層析測定為28 kDa及30 kDa之蛋白酶，且培養第一天時發現TKU007蛋白酶會造成幾丁聚醣酶活性降低。TKU007蛋白酶之N末端胺基酸序列為:AQSVPYGISQIKAPALGSQG。其最適反應pH、最適反應溫度、pH安定性、熱安定性、Km 及Vmax分別為pH 7-11, 50 ℃, pH 5-11, 50 ℃, 0.13 mg/mL, 0.86 U/mL；在25%有機溶劑存在下，於25℃及4℃放置10天，蛋白酶仍維持原活性80%以上。此外，蛋白酶在2% Tween 20、2% Tween 40及2 mM SDS存在下分別還維持原活性之100%、100%及69%。

The chitosanase and protease-producing bacterium, Bacillus subtilis TKU007, was isolated from soil in the north Taiwan. The supernatant of the culture medium contains the protease and the chitosanase activity. The optimized culture condition for production of the chitosanase was composed of 1.5% SSP,0.1% K2HPO4,0.05% MgSO4．7H2O at pH 7 and incubation in 250mL Erlenmeyer flask containing 200mL kept shaking at 30℃for one day. The chitosanase was purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose, and Sephacryl-S100. The molecular mass of TKU007 chitosanase determined by SDS-PAGE and gel filtration was approximately 25 kDa and 33 kDa, respectively. The N-terminal amino acid sequence of the protease contains PQNI. The optimum pH, optimum temperature, pH stability, thermal stability, Km, and Vmax of TKU007 chitosanase were pH7, 37℃, pH4-9, 25-37℃, 0.438 mg/mL, and 0.144 U/mL, respectively. TKU007 chitosanase was inactivated by Cu2+,but not by 2%Tween 40, and 2%Triton X-100. 
An extracellular serine protease with novel properties of surfactant-
stable, solvent-stable, and alkaliphilic was purified from B. subtilis TKU007. The optimized culture condition for production of the protease was composed of 1.5% SSP,0.1% K2HPO4, 0.05% MgSO4．7H2O at pH 7 and incubation in 250mL Erlenmeyer flask containing 100mL kept shaking at 30℃ for two days. The protease was purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose, Phenyl Sepharose, and Sephacryl-S100. The molecular mass of TKU007 protease determined by SDS-PAGE and gel filtration was approximately 28 kDa and 30 kDa, respectively. The TKU007 protease showed suppressing effect on the chitosanase, which was apparent at the first day. The N-terminal amino acid sequence of the protease was: AQSVPYGISQIKAPALGSQG. The optimum pH, optimum temperature, pH stability, thermal stability, Km, and Vmax of TKU007 protease were pH 7-11, 50℃, pH 5-11, 50℃, 0.13 mg/mL, and 0.86 U/mL, respectively. More than 80% of the original activity was retained even after preincubation for 10 days at 25℃or 4℃ in the presence of 25% tested organic solvents. Additionally, the TKU007 protease retained 100%, 100%, and 69% of its original activity in the presence of 2% Tween 20, 2% Tween 40, or 2 mM SDS, respectively.

封面內頁
簽名頁
授權書	
中文摘要	I
英文摘要	III
誌謝	V
目錄	VI
圖目錄	XII
表目錄	XIV

第一章 緒論	1
第二章 文獻回顧	2
       2.1枯草桿菌之應用………………………………………….2
       2.2幾丁質、幾丁聚醣及其寡醣之製備與應用..…….……2 
          2.2.1幾丁質………………………………………………2
          2.2.2幾丁聚醣……………………………………………3
          2.2.3 N-乙醯幾丁寡醣與幾丁寡醣…………...………6
          2.2.4 N-乙醯幾丁寡醣與幾丁寡醣之生理活性……….8
       2.3幾丁質及幾丁聚醣水解酵素……………………….…..8
       2.3.1幾丁質酶……………………………………….….….8
       2.3.2幾丁聚醣酶…………………………………………….9
       2.3.3其他可水解幾丁聚醣之酵素………………………….9
       2.4蛋白酶…………………………………………………..12
          2.4.1蛋白酶之簡介…………………………………….12
          2.4.2蛋白酶之分類…………………………………….12
          2.4.3蛋白酶在工業上之應用………………………….13
       2.5酵素在有機溶劑中之行為……………………………..14
          2.5.1蛋白酶在有機溶劑中之安定性與合成反應…….14

第三章 材料與方法………..………………..…………………….17
       3.1實驗菌株………………………………………………..17
       3.2實驗材料………………………………………………..17
       3.3實驗儀器………………………………………………..18
       3.4生產菌株之篩選………………………………………..19
       3.5蛋白酶之活性測定……………………………………..19
       3.6幾丁聚醣酶之活性測定………………………….…….20
       3.7蛋白酶之較適培養條件探討……………………….….20
          3.7.1碳源之選擇……………………………………….20
          3.7.2培養基之酸鹼值……………...………….…….20
          3.7.3培養溫度………………………………………….21
          3.7.4培養基之通氣量………………………….………21
          3.7.5較適培養時間…………………………….………21
       3.8酵素之純化分離………..……………..………………21
          3.8.1粗酵素液之製備…………………….……………21
          3.8.2離子交換樹脂層析法………………….…………22
          3.8.3疏水性層析法……………….……………………22
          3.8.4膠體過濾層析法………………………….………22
       3.9分子量標定……………………………...…………….23
       3.10蛋白質電泳分析……………………….………………24
       3.11蛋白質定量分析…………………………………….…24
       3.12酵素生化特性分析………………………….…………24
           3.12.1酵素最適溫度………………………………….24
           3.12.2酵素熱安定…………………………………….24
           3.12.3酵素最適pH……….………….……………….25
           3.12.4酵素pH安定性………………………………….25
           3.12.5金屬離子與抑制劑對酵素活性之影響……….25
           3.12.6界面活性劑對酵素活性之影響……………….26
           3.12.7酵素動力學性質………………………..…….26
           3.12.8蛋白酶之基質特異性………….………………26
       3.13有機溶劑對酵素活性之影響…….……………………27
       3.14粗酵素液之蛋白酶及幾丁聚醣酶活性消長……….…27
       3.15蛋白質轉印…………………………………………….28
       3.16利用Bacillus subtilis TKU007之發酵液進行小白菜生長測試……………………………………......................28
           3.16.1小白菜之預培養……………..……………….28
           3.16.2促進小白菜生長試驗…………..…………….28
第四章 結果與討論………………………………………………...30
       4.1蛋白酶生產菌之篩選…………………..………………30
       4.2菌株特性………………………….…………………….30
       4.3碳源之選擇……………………………………………..30
       4.4蛋白酶較適生長條件探討……………………………..35
       4.5粗酵素液之蛋白酶及幾丁聚醣酶活性關係…..………36
       4.6蛋白酶之純化分離……………………..………………37
          4.6.1粗酵素液製備……………………..…………….37
          4.6.2離子交換樹脂層析法…..……………………….38
          4.6.3疏水性層析法………………………….…………38
          4.6.4膠體過濾層析法………………..……………….38
          4.6.5綜合結果…………………………..…………… 39
       4.7蛋白酶之分子量判定………………………..…………39
          4.7.1膠體過濾層析法………………………………….39
          4.7.2 SDS-PAGE………………………………………..40
          4.7.3綜合結果………………………………….………40
       4.8純化後蛋白酶之生化特性分析…………….………….40
          4.8.1蛋白酶反應最適溫度及熱安定性……………….40
          4.8.2蛋白酶之最適反應pH及pH安定性………….....41
          4.8.3各種化學藥品對蛋白酶之影響…….……………41
          4.8.4各種界面活性劑對蛋白酶之影響……………….41
          4.8.5蛋白酶之基質特異性………………………….…42
          4.8.6蛋白酶之酵素動力學…………………………….42
       4.9有機溶劑對蛋白酶活性及安定性影響…………….….42
       4.10蛋白酶N端序列分析………………………………....43
       4.11幾丁聚醣酶之純化分離……………………………….76
           4.11.1粗酵素液製備………………………………….76
           4.11.2離子交換樹脂層析法……….…………………76
           4.11.3膠體過濾層析法……………….………………76
           4.11.4綜合結果…………………………………….…77
       4.12幾丁聚醣酶之分子量判定……………..……..…….77
           4.12.1膠體過濾層析法…………………………..….77
           4.12.2 SDS-PAGE………………………………..……77
           4.12.3綜合結果………………………………..…….78
       4.13純化後幾丁聚醣酶之生化特性分析……….……..…78
           4.13.1純化後幾丁聚醣酶之最適溫度及熱安定性...78
           4.13.2幾丁聚醣酶之最適反應pH及pH安定性….....78
           4.13.3各種化學藥品對幾丁聚醣酶之影響…..…...79
           4.13.4各種界面活性劑對幾丁聚醣酶之影響.………79
           4.13.5幾丁聚醣酶之酵素動力學………………..….79
       4.14有機溶劑對幾丁聚醣酶活性及安定性影響…….……79
       4.15幾丁聚醣酶N端序列分析……………………………..80
       4.16利用B. subtilis TKU007抗腫瘤測試……………….81
       4.17利用B. subtilis TKU007之發酵液進行小白菜生長測試
           ………………………………………………………….81

第五章 結論與未來展望…………………………………………..101
參考文獻…………………………………………………………….102
附錄………………………………………………………………….116

圖 目 錄
                      頁次
圖2.1幾丁質、幾丁聚醣之製備	4
圖2.2幾丁質、幾丁聚醣、纖維素之構造	5
圖4.1 Bacillus subtilis TKU007之顯微照片……………………31
圖4.2 16S rDNA部份鹼基序列分析……………………………....32
圖4.3 MIDI微生物脂肪酸組成分析鑑定系統分析結果........	.33
圖4.4 VITEK鑑定套組分析結果	.............................34
圖4.5蝦殼粉末添加對TKU007產生蛋白酶活性之影響	..........44
圖4.6不同碳源對於TKU007產生蛋白酶活性之影響...........	.45
圖4.7溫度對TKU007產生蛋白酶活性之影響.................	.46
圖4.8 pH對TKU007產生蛋白酶活性之影響	...................47
圖4.9通氣量對TKU007產生蛋白酶活性及幾丁聚醣酶之影響.....48
圖4.10不同通氣量培養一天之SDS-PAGE....................	49
圖4.11 Bacillus subtilis TKU007培養於蝦殼培養基所產蛋白酶
       及幾丁聚醣酶變化情形……………………………………50
圖4.12蛋白酶與幾丁聚醣酶於37℃下反應後之SDS-PAGE......	51
圖4.13 Bacillus subtilis TKU007所生產蛋白酶之純化分離流程
       圖	..............................................54
圖4.14 DEAE-Sepharose CL-6B 之蛋白酶層析圖譜..........	55
圖4.15 Phenyl Sepharose 6 Fast Flow之蛋白酶層析圖譜...	56
圖4.16 Sephacryl S-100之蛋白酶層析圖譜................	57
圖4.17分子量標準品和蛋白酶於Sephacryl S-100之層析圖譜..59
圖4.18蛋白酶於SDS-PAGE之分子量分析…………………….....60
圖4.19蛋白酶之最適反應溫度…………………………………...61
圖4.20蛋白酶之熱安定性………………………………………...62
圖4.21蛋白酶之最適反應pH……………………………………...63
圖4.22蛋白酶之pH安定性…………………………………….....64
圖4.23蛋白酶於50℃之Lineweaver-Burk Plot………………….65
圖4.24有機溶劑對蛋白酶活性之影響…………………………...70
圖4.25蛋白酶對有機溶劑之安定性……………………………...71
圖4.26 Bacillus subtilis TKU007所生產幾丁聚醣酶純化分離流
       程圖………………………………………………………..82
圖4.27 DEAE-Sepharose CL-6B 之幾丁聚醣酶層析圖譜……...83
圖4.28 Sephacryl S-100 之幾丁聚醣酶層析圖譜………………84
圖4.29分子量標準品和幾丁聚醣酶於Sephacryl S-100之層析圖
       譜…………………………………………………………..86
圖4.30幾丁聚醣酶於SDS-PAGE之分子量分析……………….....87
圖4.31幾丁聚醣酶之最適反應溫度……………………………...88
圖4.32幾丁聚醣酶之熱安定性…………………………………...89
圖4.33幾丁聚醣酶之最適反應pH………………………………...90
圖4.34幾丁聚醣酶之pH安定性……………………………….....91
圖4.35幾丁聚醣酶於37℃之Lineweaver-Burk Plot…………...97
圖4.36有機溶劑對幾丁聚醣酶活性之影響………….…………..98
圖4.37幾丁聚醣酶對有機溶劑之安定性………….……………..99


表 目 錄
頁次
表2.1幾丁質、幾丁聚醣的應用…………………………………...7
表2.2幾丁質酶之來源與特性……………………………………..10
表2.3各種酵素對幾丁聚醣酶之降解反應…………..……………11
表2.4一般常用有機溶劑之logP值…………………..….……….16
表3.1蛋白質轉印所需溶液配方………………………………..…29
表4.1比較蛋白酶與幾丁聚醣酶活性關係………………………..52
表4.2模擬各階段之蛋白量、蛋白酶、幾丁聚醣酶……………..53
表4.3 Bacillus subtilis TKU007蛋白酶之純化總表………….58
表4.4各種化學藥品對蛋白酶之影響……………………………..66
表4.5各種界面活性劑對蛋白酶之影響…………………………..67
表4.6蛋白酶之基質特異性………………………………………..68
表4.7 Bacillus subtilis蛋白酶特性比較………………………69
表4.8比較Bacillus所產酵素之N端序列分析…………………...72
表4.9本研究與其他耐界面活性劑及耐有機溶劑蛋白酶之比較..74
表4.10 Bacillus subtilis TKU007幾丁聚醣酶之純化總表…..85
表4.11各種化學藥品對幾丁聚醣酶之影響…………..….………92
表4.12各種界面活性劑對幾丁聚醣酶之影響……….……………93
表4.13各種微生物生產幾丁聚醣酶之特性比較…………….…..94
表4.14 Bacillus subtilis TKU007之發酵液對小白菜生長影響.100

1. 林易徵 (2002) 木生真菌Xylaria regalis分解Benzo(a)pyrene 酵素之分離、純化及其生化特性之探討：第13~14頁。國立陽明大學生物化學研究所碩士論文，台北。
2. 江晃榮 (1998) 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。150：19-25.
3. 洪健益 (2002) 枯草桿菌之運動性在醱酵系統的應用：第1頁。
   國立清華大學化學工程學系碩士論文，新竹。
4. 吳豐智、曾如玲 (1997) 神奇的物質-幾丁質和幾丁聚醣。化工 
   技術。5：196-201。
5. 莊榮輝 (2000) 酵素化學實驗。國立台灣大學農業化學系生物化
   學實驗室，台北。
6. 張雅敏 (2002) 黑豆種子幾丁質酶之純化與性質研究：第39-43 
   頁。私立靜宜大學食品營養研究所碩士論文，台中。
7. 張瓊瑋 (2004) Aeromonas sp. DYU-Too7與本土菌株JR1之幾丁 
   質分解酶純化與特性分析：第3頁。私立大葉大學生物產業科
   技研究所碩士論文，彰化。
8. 陳泰州 (2001) 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：第114 -115頁。國立台灣大
   學農業化學研究所碩士論文，台北。
9. 楊政國 (1999) 利用枯草菌進行蝦蟹殼去蛋白之研究：第99頁。私立大葉工學院食品工程研究所碩士論文，彰化。
10. 賴威安 (2000) Bacillus sp. P-6中蛋白酶的生產與特性分析：第37頁。國立中興大學食品科學系碩士論文，台中。
11. 蘇遠志，黃世佑 (2002) 微生物化學工程學。第372 - 374頁。
    華香園出版社。台北，台灣。
12. Adinarayana, K., Ellaiah, P. and Prasad, D. S.
Purification and  partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11. AAPS Pharm. Sci.Tech. 2003；4：56.
13. Aktuganov, G. E., Shirokov, A. V. and Melent'ev, A. I. Isolation and characterisation of chitosanase from Bacillus sp. 739 strain. Prikl. Biokhim. Mikrobiol. 2003；39：536-541.
14. Akiyama, K., Fujita, T., Kuroshima, K., Sakane, T., Yokota, A. and Takata, R. Purification and gene cloning of a chitosanase from Bacillus ehimensis EAG1. J. Biosci. Bioeng. 1999；87：383-385.
15. Anwar, A. and Saleemuddin, M. Alkaline proteases：a review.Biosource Technol. 1998；64：175-183.
16. Akuzawa, R., Tottori, A., Tsukahara, K. and Okitani, A. Purification and characterization of a cysteine proteinase from Lactococcus lactis ssp. lactis IAM 1198 . Intl. Dairy J. 1997；7：429-434.
17. Banik, R. M. and Prakash, M. Laundry detergent compatibility of the alkaline protease from Bacillus cereus. Microbiol. Res. 2004；159：135-140.
18. Beg, Q. K. and Gupta, R. Purification and characterization of an oxidation-stable, thiol-dependent serine alkaline protease from Bacillus mojavensis. Enzyme Microb. Technol. 2003；32：294-304.
19. Banerjee, U. C., Sani, R. K., Azmi, W. and Soni, R. Thermostable alkaline protease from Bacillus brevis and its characterization as a laundry detergent additive. Process Biochem. 1999；35：213-219.
20. Bradford, M. M. 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.
21. Blow, D. M., Birktoft, J. J. and Hartley, B. S. Role of buried acid group in the mechanism of action of chymotrypsin. Nature. 1969；221：337-340. 
22. Chen, X., Xia, W. and Yu, X. Purification and characterization of two types of chitosanase from Aspergillus sp. CJ22-326. Food Res. Int. 2005；38：315-322.
23. Choi,Y. J., Kim, E. J., Piao, Z., Yun, Y. C. and Shin ,Y. C. Purification and characterization of chitosanase from Bacillus sp.strain KCTC 0377BP and its application for the production of chitosan oligosaccharides. Appl. Environ. Microbiol. 2004；70：4522-4531.
24. Chiang, C. L., Chang, C. T. and Sung, H. Y. Purification and properties of chitosanase from a mutant of Bacillus subtilis IMR-NK1. Enzyme Microb. Technol. 2003；32：260-267. 
25. Colomer-Pallas, A., Pereira, Y., Petit-Glatron, M. F. and Chambert, R. Calcium triggers the refolding of Bacillus subtilis chitosanase. J. Biochem. 2003；369：731-738.
26. Chang, C. T., Fan, M. H., Kuo, F. C. and Sung, H. Y. Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1. J. Agric. Food Chem. 2000；48：3210-3216.
27. Deshpande,M. V. Enzymatic degradation of chitin and its biological application. J. Sci. Ind. Res. 1986；45：273-277.
28. Eom, T. K. and Lee, K. M. Characteristics of chitosanases from Aspergillus fumigatus KB-1. Arch. Pharm. Res. 2003；26：1036-1041.
29. Ghorbel, B., Sellami-Kamoun, A. and Nasri, M. Stability studies of protease from Bacillus cereus BG1. Enzyme Microb. Technol. 2005；32：513-518. 
30. Gupta, A., Roy, I., Khare, S.K. and Gupta, M.N. Purification and characterization of a solvent stable protease from Pseudomonas aeruginosa PseA. J. Chromatogr. A. 2005；1069：155-161. 
31. Gupta, A., Roy, I., Patel, R.K., Singh, S.P., Khare, S. K. and Gupta, M. N. One-step purification and characterization of an alkaline protease from
haloalkaliphilic Bacillus sp. J. Chromatogr. A. 2005；1075：103-108. 
32. Geok, L. P., Razak, C. N. A., Rahman, R. N. Z. A., Basri, M. and Salleh, A. B. Isolation and screening of an extracellular organic solvent-tolerant protease producer. Biochem. Eng. J. 2003；13：73-77.
33. Gupta, M. N. Enzyme function in organic solvents. Eur. J. Biochem. 1992；203：25-32.
34. Ghaouth, E., Arul, A., Gremier, J. and Asselin, A. Antifungal activity of chitosan on two pathogens of strawberry fruits. Phytopathol. 1992；82：398-402.
35. Harwood, C. R. Bacillus subtilis and its relatives: molecular biological and industrial workhorses. Trends. Biotechnol. 1992 ；10：247-256.
36. Izume, M., Nagae, S., Kawagishi, H., Mitsutomi, M. and Ohtakara, A. Action pattern of Bacillus sp. 7-M chitosanase on partially  N-acetylated chitosan. Biosci. Biotechnol. Biochem.1992；56： 448-456.
37. Imoto, T. and Yagishita, K. A simple activity measurement by lysozyme. Agric. Biol. Chem. 1971；35：1154-1156.
38. Joo, H. S. and Chang, C. S. Production of an oxidant and SDS-stable alkaline protease from an alkaophilic Bacillus clausii I-52 by submerged fermentation: Feasibility as a laundry detergent additive. Enzyme Microb. Technol. 2006；38：176-183.
39. Jo, Y. Y., Jo, K. J., Jin, Y. L., Kim, K. Y., Shim, J.H., Kim, Y. W. and Park, R. D. Characterization and kinetics of 45 kDa chitosanase from Bacillus sp. P16. Biosci. Biotechnol. Biochem. 2003；67：1875-1882.
40. Jeon, Y. J., Park, P. J., and Kim, S. K. Antimicrobial effect of chitooligosaccharides produed by bioreactor. Carbohydr. Polymers. 2001；47：71-76.
41. Jones, B. L., Fontanini, D., Jarvinen, M. and Pekkarinen, A. Simplified endoproteinase assays using gelatin or azogelatin. Anal. Biochem. 1997；263：214-220.
42. Karadzic, I., Masui, A. and Fujiwara, N. Purification and characterization of a protease from Pseudomonas aeruginosa grown in cutting oil. J. Biosci. Bioeng. 2004；3：145-152. 
43. Kim, P. I., Kang, T. H., Chung, K. J., Kim, I. S. and Chung, K. C.Purification of a constitutive chitosanase produced by Bacillus sp. MET 1299 with cloning and expression of the gene.  FEMS Microbiol. Lett. 2004；240：31-39.
44. Kazan, D., Denizci, A. A., Oner, M. N. and Erarslan, A. Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42. J. Ind. Microbiol. Biotechnol. 2003；32：335-344.
45. Kurakake, M., Yo-u, S., Nakagawa, K., Sugihara, M. and Komaki, T. Properties of chitosanase from Bacillus cereus S1. Curr. Microbiol. 2000；40：6-9.
46. Kumar, C. G. and Takagi, H. Microbial alkaline proteases：from a bioindustrial viewpoint. Biotechnol. Adv. 1999；17：561-594.
47. Kim, S. Y., Shon, D. H. and Lee, K. H. Purification and
   characteristics of two types of chitosanases form Aspergillus fumigatus KH-94. J. Microbiol. Biotechnol. 1998；8：568-574.
48. Kendra, D. F. and Hadwiger, L. A. Characterization of the smallest chitosan oligomer that is maximally antifungal to Fusarium soloni and elicits pisatin formation in Pisum. sativum. Exp. Mycol. 1984；8：398-402.
49. Kawamura, F. and Doi, R. H. Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases. J. Bacteriol. 1984；160：442-444.
50. Knorr, D. Use of chitinous polymer in food. Food Technol. 1984；1：85-89.
51. Knorr, D. Functional properties of chitin and chitosan . J. Food Sci. 1982；47：593-595.
52. Lee, Y. S., Yoo, J. S., Chung, S. Y., Lee, Y. C., Cho, Y. S. and Choi, Y. L. Cloning, purification, and characterization of chitosanase from Bacillus sp. DAU101. Appl. Microbiol. Biotechnol. 2006；accepted.
53. Li, C. H., Chen, P. Y., Chang, U. M., Kan, L. S., Fang, W. H., Tsai, K. S. and Lin, S. B. Ganoderic acid X, a lanostanoid triterpene, inhibits topoisomerases and induces apoptosis of cancer cells. Life Sci. 2005；77：252-265.
54. Lahl, W. J. and Braun, S. D. Enzymatic production of protein hydrolysates for food use. Food Technol. 1994；48：68-71.
55. Lanne, C., Boeren, S., Vos, K. and Veeger, C. Rules for optimization of biocatalysis in organic solvents. Biotechol. Bioeng.1987；XXX：81-87.
56. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970；227：680-685.
57. Mei, C. and Jiang, X. A novel surfactant- and oxidation-stable alkaline protease from Vibrio
metschnikovii DL 33–51. Process Biochem. 2005；40：2167-2172.
58. Mohamed, S.A., Fahmy, A. S., Mohamed, T. M.and Hamdy, S.M. Proteases in egg, miracidium and adult of  Fasciola gigantica. Characterization of serine and cysteine proteases from adult. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 2005；142：192-200.
59. Mane, R. R. and Bapat, M. A study of extracellular alkaline protease from Bacillus subtilis NCIM 2713. Indian. J. Exp. Biol. 2001；39：578-583.
60. Muderrizade, A., Ensari, N. Y., Aguloglu, S. and Otludil, B. Purification and characteristics of alkaline proteinase from alkalophylic Bacillus sp. Prikl. Biokhim. Mikrobiol. 2001；37：674-677.
61. Matta, H. and Punj, V. Isolation and partial characterization of a  thermostable extracellular protease of Bacillus polymyxa B-17. Internatl. J. Food Microbiol. 1998；42：139-145.
62. Mitsutomi, M., Ohtakara, A., Fukamizo, T. and Goto, S. Action pattern of Aeromonas hydrophila chitinase on partially N-acetylated chitosan. Agric. Biol. Chem. 1990；54：871-877.
63. Muzzarelli, R. A. A., Barontini, G. and Rocchetti, R. Isolation of lysozyme on chitosan. Biotch. Bioeng. 1978；20：87-92.
64. Nagano, H. and To, K. A. Purification of collagenase and specificity of its related enzyme from Bacillus subtilis FS-2. Biosci. Biotechnol. Biochem. 2000；64：181-183.
65. Omumasaba, C. A., Yoshida, N., Sekiguchi, Y., Kariya, K. and Ogawa, K. Purification and some properties of a novel chitosanase from Bacillus subtilis KH1.  J. Gen. Appl. Microbiol. 2000；46：19-27.
66. Ogino, H., Watanabe, F., Yamada, M., Nakagawa, S., Hirose, T., Noguchi, A., Yasuda, M. and Ishikawa, H. Purification and characterization of organic solvent-stable protease from organic solvent-tolerant Pseudomonas aeruginosa PST-01. J. Biosci.Bioeng. 1999；87：61-68.
67. Ogino, H., Yamada, M., Watanabe, F., Ichinose, H., Yasuda, M. and Ishikawa, H. Peptide synthesis catalyzed by organic solvent-stable protease from Pseudomonas aeruginosa PST-01 in monophasic aqueous-organic solvent systems.  J. Biosci. Bioeng. 1999；88：513-518.
68. Okajima, S., Ando, A., Shinoyama, H. and Fujii, T. Purification and characterization of an extracellular chitosanase produced by Amycolatopsis sp. CsO-2. J. Ferment. Bioeng. 1994；77：617-620.
69. Ohtakara, A., Matsunaga, H. and Mitsutomi, M. Action pattern of Streptomyces grisous chitinase on partially N-acetylated chitosan. Agric. Biol. Chem. 1990；54：3191-3199. 
70. Ohtakara, A. and Izume, M. Preparation of D-glucosanmine oligosaccharide by enzymatic hydrolysis of chitosan. J. Agric. Food Chem.1987；51：1189-1191.
71. Peng, Y., Huang, Q., Zhang, R. H. and Zhang, Y. Z. Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chinese soybean food. Comp. Biochem. Physiol. B. 2003；134：45-52. 
72. Park, J. K., Shimono, K., Ochiai, N., Shigeru, K., Kurita, M., Ohta, Y., Tanaka, K., Matsuda, H. and Kawamukai, M. Purification, characterization, and gene analysis of a chitosanase (ChoA) from Matsuebacter chitosanotabidus 3001. J. Bacteriol. 1999； 181：6642-6649.
73. Phadatare, S. U., Deshpande, V. V. and Srinivasan, M. C. High activity alkaline protease from Conidiobolus coronatus (NCL86.8.20)：Enzyme production and compatibility with commercial Detergents. Enzyme Microb. Technol. 1993；15：72-76.
74. Pantaleone, D., Yalpani, M. and Scollar, M. Unusual susceptibility of chitosan to enzyme hydrosis. Carbohydr. Polymers. 1992；237：325-332.
75. Pelletier, A. and Sygusch, J. Purification and characterization of three chitosanase activities from Bacillus megaterium P1. Appl. Environ. Microbiol. 1990；56：844-848.
76. Priest, F. G. Extracellular enzyme synthesis in the genus Bacillus. Bacteriol. Rev. 1977；41：711-753.
77. Rawlings, N. D. and Barrett, A. J. Evolutionary families of peptidases. Biochem. J. 1993；290：205-218.
78. Setyorini, E., Takenaka, S., Murakami, S. and Aoki, K. Purification and characterization of two novel halotolerant extracellular proteases from Bacillus subtilis strain FP-133. Biosci. Biotechnol. Biochem. 2006；70：433-440.
79. Sushil, K., Neeru, S., Sharma, M. R., Saharan, and Randhir, S. Extracellular acid protease from Rhizopus oryzae：purification and characterization. Process Biochem. 2005；40：1701-1705.
80. Suzuki T. and Tahara Y. Characterization of the Bacillus subtilis ywtD gene, whose product is involved in gamma-polyglutamic acid degradation. J. Bacteriol. 2003；185：2379-2382.
81. Surti, A. M. and Bapat, M. M. Isolation and purification of an extracellular protease from a new strain of Bacillus subtilis, viz. NCIM 2711. Indian. J. Exp. Biol. 2003；41：614-619.
82. Singh, J., Batra, N. and Sobti, R. C. Serine alkaline protease from a newly isolated Bacillus sp. SSR1. Process Biochem. 2001；36：781-785.
83. Suh, H. J. and Lee, H. K. Characterization of a keratinolytic serine protease from Bacillus subtilis KS-1. J. Protein Chem. 2001；20：165-169.
84. Sierecka, J. K. Purification and partial characterization of neutral protease from a virulent strain of Bacillus cereus. Inter. J. Biochem. Cell Biol. 1998；30：579-595.
85. Sergeeva, M. V., Paradkar, V. M. and Dordick, J. S. Peptide synthesis using proteases dissolved in organic solvents. Enzyme Microb. Technol. 1997；20：623-628.
86. Sakai,K., Katsumi,R., Lsobe, A. and Nanjo, F. Purification and hydrolytic action of a chitosanase from Nocardia orientalis. Biochim. Biophys. Acta. 1991；1079：65-72.
87. Shimogaki, H., Takeuchi, K., Nishino, T., Ohdera, M., Kudo, T., Ohba, K., Iwama, M. and Irie, M. Purification and properties of a novel surface-active agent- and alkaline-resistant protease from Bacillus sp. Y. Agric. Biol. Chem. 1991；55：2251-2258.
88. Saito, M. Tabeta, R. and Oogawa, K. 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.1987：20-24.
89. Suzuki, K., Tokro, A. and Okawa, A. Enhancing effects of N-acetyl-chito-oligosaccharides on the active oxygengenerating and microbicidal activities of peritoneal exudatecells in mice. Chem. Pharm. Bull. 1985；33：886-888.
90. Sannan, T., Kurrta, K. and Lwakura, Y. Studies on chitin 2.Effect of deacetylation on solubility. Macromol. Chem. 1976；177：3589-3592.
91. Tokoro, A., Kobayashi, M., Tatewaki, N., Suzuki, K.,Okawa, Y., Mikami, T., Suzuki, S. and Suzuki, M. Protective effects of N-acetylated chitohexaose on Listeria monocytogens infection in mice. Microbiol. Immunol. 1989；33：357-367. 
92. Tokor, A., Suzuki, K. and Suzuki, S. Growth-inhibitory effect of N-acetylchitohexaose and chitohexaose against Meth-Asolid tumor. Chem. Pharm. Bull. 1988；36：784-790. 
93. Toei, K. and Kohara, T. A conductometric method for colloid titrations. Analytica. Chimica. Acta. 1976；83：59-65.
94. Tominaga, Y. and Tsujisaka, Y. Purification and some enzymatic properties of the chitosanase from Bacillus R-4 which lyses Rhizopus cell walls. Biochim. Biophys. Acta. 1975；410：145-155.
95. Todd, E.W. Quantitative studies on the total plasmin and trypsin inhibitor of human blood serum. J. Exp. Med. 1949；39：295-308.
96. Wang, S. L and Yeh, P. Y. Production of a surfactant- and solvent-stable alkaliphilic protease by bioconversion of shrimp shell wastes fermented by Bacillus subtilis TKU007. Process Biochem. 2006；41：1545-1552.
97. Wang, S. L., Kao, T. Y., Wang, C. L., Yen, Y. H., Chern, M. K. and Chen, Y. H. 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. 2006；39：724-731.
98. Yoo, J. J., Lee, Y. S., Song, C. Y. and Kim, B. S. Purification and characterization of a 43-kilodalton extracellular serine proteinase 
   from Cryptococcus neoformans. J. Clin. Microbiol. 2004；42：722-726.
99. Yang, J. K., Shih, I. L., Tzeng, Y. M. and Wang, S. L. Production and purification of protease from a Bacillus subtilis that can deproteinize crustacean wastes. Enzyme Microb. Technol. 2000；26：406-413.
100. Yasuda, M., Aoyama, M., Sakaguchi, M., Nakachi, K. and Kobamoto, N. Purification and characterization of a soybean-milk-coagulating enzyme from Bacillus pumilus TYO-67. Appl.Microbiol. Biotechnol. 1999；51：474-479.
101. Yalpani, M. and Pantaleone, D. An examination of the unusual susceptibilities of aminoglycans to enzymatic hydeolysos. Carbohydr. Res. 1994；256：159-175.
102. Yamasaki, Y., Hayashi, I., Ohta, Y., Nakagawa, T., Kawamakai, M. and Matsuda, H. Purification and mode of action of chitosanolytic enzyme from Enterobacter sp. G-1. Biosci. Biotechnol. Biochem. 1993；57：444-449.
103. Zhu, X. F., Wu, X. Y. and Dai, Y. Fermentation conditions and properties of a chitosanase from Acinetobacter sp. C-17. Biosci. Biotechnol. Biochem. 2003；67：284-290.
104. Zhang, X. Y., Dai, A. L., Zhang, X. K., Kuroiwa, K., Kodaira, R., Shimosaka, M. and Okazaki, M. Purification and characterization of chitosanase and exo-beta-D-glucosaminidase from a Koji mold, Aspergillus oryzae IAM2660. Biosci. Biotechnol. Biochem. 2000；64：1896-1902.