臺灣博碩士論文加值系統

本研究自高雄金獅湖篩選出一株具幾丁聚醣酵素生產能力之根瘤菌Sinorhizobium sp. CSJ01，並探討該菌株之幾丁聚醣酵素特性與發酵條件。
在幾丁聚醣酵素特性方面，酵素之最適反應pH值與溫度分別為6.0與40℃，在穩定性方面，pH值與溫度分別在pH 3-10與20-40℃之間具有良好的穩定性。金屬離子Na+與Fe3+可促進酵素活性，但Ca2+則能抑制酵素活性。將發酵液以硫酸銨沉澱與透析處理可得到濃縮之酵素液，將濃縮之酵素液與膠態幾丁聚醣進行親和性吸附後，可得到初步精製之幾丁聚醣酵素。利用SDS-PAGE電泳檢定幾丁聚醣酵素之分子量發現，幾丁聚醣酵素可能之分子量為25、43、50或63 kDa。以幾丁聚醣酵素水解膠態幾丁聚醣，利用TLC分析水解產物發現，水解產物可能為幾丁二醣與幾丁四醣。
在Sinorhizobium sp.幾丁聚醣酵素發酵條件方面，影響酵素產量之因素包括：膠態幾丁聚醣、酵母萃出物、磷酸鉀、硫酸鎂及硫酸錳等培養基組成之濃度，培養溫度、培養液起始pH值及振盪速率。其中以膠態幾丁聚醣、酵母萃出物及培養液起始pH等三項因子對酵素產量之影響最為顯著。利用回應曲面法探討三種因子(膠態幾丁聚醣、酵母萃出物及培養液起始pH值)對幾丁聚醣酵素產量之影響，由二水準因子實驗與中心混成實驗結果發現，各因子之最適條件為：膠態幾丁聚醣0.80 %、酵母萃出物0.4 %及培養液起始pH 9.1。Sinorhizobium sp.生產幾丁聚醣酵素之最適培養條件為：膠態幾丁聚醣0.80 %、酵母萃出物0.4 %、磷酸鉀0.1%、硫酸鎂0.05%、硫酸錳0.001%、溫度30℃、振盪速率200 rpm及培養液起始pH 9.1。

A bacterial strain capable of utilizing chitosan as the sole carbon source was isolated from soil and was identified as a member of the genus Sinorhizobium sp. This strain, designed CSJ01, produced extracellular chitosan-degrading enzyme in the presence of chitosan. In this study, the fermentation condition and characterization of the chitosanase were investigated.
This crude chitosanase was stable in the alkaline side. Optimum pH was about 6, and stable pH in the incubation at 40℃ for 30 min was 3-10. Optimum temperature was around 40℃, and enzyme activity was relatively stable in the range of 20-40℃. The presence of sodium and iron ions promoted the activities; however, the enzyme activity was inhibited by calcium ion. The partial purified chitosanase was obtained by salting out with ammonium sulfate and by adsorbing with colloidal chitosan. The molecular weight was estimated to 25, 43, 50 or 63 kDa on SDS-PAGE. This chitosanase hydrolyzed colloidal 82% N-deacetylated chitosan to chitobiose and chitotetraose.
The production of Sinorhizobium sp. CSJ01 chitosanase was affected by temperature, initial pH, rotational speed, and medium composition, such as the concentrations of colloidal chitosan, yeast extract, KH2PO4, MgSO4.7H2O, MnSO4. In addition, the enzyme production was affected significantly by the initial pH of medium and the concentrations of chitosan and yeast extract. In this study, the optimal cultivation condition was investigated using response surface methodology (the factors were initial pH, the concentrations of colloidal chitosan and yeast extract). The Experimental results from 23 factorial and central composite design showed the optimal density of factors were colloidal chitosan 0.8%, yeast extract 0.4% and initial pH 9.1. The optimal condition for the production of Sinorhizobium sp. CSJ01 chitosanase was colloidal chitosan 0.8%, yeast extract 0.4%, KH2PO4 0.1%, MgSO4.7H2O 0.05%, MnSO4 0.001%, temperature 30℃, initial pH 9.1, rotational speed 200 rpm.

中文摘要...............................v
英文摘要...............................vii
誌 謝...............................ix
目 錄...............................x
圖 目 錄...............................xiv
表 目 錄...............................xvi
第壹章 序論.............................................1
第貳章 文獻回顧.........................................3
2-1 幾丁類聚合物的介紹..................................3
2-1-1 幾丁質與幾丁聚醣..................................3
2-1-2 幾丁質與幾丁聚醣之應用............................6
2-1-3 幾丁寡醣..........................................6
2-1-4 幾丁聚醣之抗菌作用................................8
2-2 幾丁聚醣酵素........................................9
2-2-1 幾丁聚醣酵素的活性測定方法........................13
2-2-2 幾丁聚醣酵素之應用................................16
2-2-3 幾丁聚醣酵素生產菌之篩選..........................17
2-2-4 幾丁聚醣酵素純化方法..............................18
2-2-5 幾丁聚醣酵素之發酵條件............................19
2-2-6 幾丁聚醣酵素水解產物之分析方法....................23
2-3 回應曲面法..........................................24
2-3-1 回應曲面法之原理..................................24
2-3-2 Plackett-Burman設計...............................25
2-3-3 二水準因子設計....................................25
2-3-4 陡升路徑法........................................25
2-3-5 中心混成設計......................................26
2-3-6 回應曲面模式適切性之統計檢驗......................26
第參章 實驗材料與方法...................................29
3-1 實驗藥品與設備......................................29
3-1-1 藥品..............................................29
3-1-2 使用設備..........................................30
3-2 實驗方法............................................30
3-2-1 幾丁聚醣去乙醯度的測定............................30
3-2-2 膠態幾丁聚醣之製備................................31
3-2-3 菌株篩選、保存及鑑定..............................31
3-2-4 幾丁聚醣酵素活性分析..............................32
3-2-5 電泳檢定系統......................................34
3-2-6 粗酵素反應條件之建立..............................38
3-2-7 菌體濃度的測定....................................39
3-2-8 酵素生產用最適培養基組成的探討....................40
3-2-9 酵素生產用最適培養條件的探討......................42
3-2-10 幾丁聚醣酵素水解產物分析..........................42
3-2-11 酵素純化..........................................43
3-2-12 實驗設計..........................................44
第肆章 實驗結果與討論...................................51
4-1 幾丁聚醣酵素生產菌之篩選............................51
4-1-1 幾丁聚醣酵素生產菌之篩選與初選....................51
4-1-2 菌種鑑定..........................................51
4-2 幾丁聚醣粗酵素特性分析..............................54
4-2-1 溫度對幾丁聚醣粗酵素活性之影響....................54
4-2-2 pH值對幾丁聚醣粗酵素活性之影響....................54
4-2-3 基質濃度對幾丁聚醣粗酵素活性之影響................54
4-2-4 反應時間對幾丁聚醣粗酵素活性分析之影響............57
4-2-5 金屬離子對幾丁聚醣粗酵素活性之影響................57
4-2-6 溫度對幾丁聚醣粗酵素穩定性之影響..................57
4-2-7 pH值對幾丁聚醣粗酵素穩定性之影響..................62
4-3 幾丁聚醣酵素發酵條件之探討..........................62
4-3-1 菌株生長曲線與酵素活性的關係......................62
4-3-2 碳源種類對酵素產量之影響..........................62
4-3-3 膠態幾丁聚醣濃度對酵素產量之影響..................66
4-3-4 氮源種類對酵素產量之影響..........................66
4-3-5 酵母萃出物濃度對酵素產量之影響....................69
4-3-6 不同鹽類對酵素產量之影響..........................69
4-3-7 微量金屬對酵素產量之影響..........................69
4-3-8 溫度對酵素產量之影響..............................72
4-3-9 起始pH值對酵素產量之影響..........................72
4-3-10 振盪速率對酵素產量之影響..........................76
4-3-11 幾丁聚醣粗酵素水解產物之分析......................76
4-4 酵素純化............................................76
4-4-1 硫酸銨沉澱之電泳檢定..............................76
4-4-2 膠態幾丁聚醣之親和性吸附作用......................79
4-5 實驗設計............................................82
4-5-1 前置實驗..........................................82
4-5-2 二水準因子實驗設計結果............................82
4-5-3 陡升路徑實驗設計結果..............................84
4-5-4 中心混成設計實驗結果..............................84
第五章 結論.............................................94
5-1 結論................................................94
5-2 展望................................................95
參考文獻.................................................96
圖目錄
圖2.1 幾丁質、幾丁聚醣及纖維素構造的相似性.............4
圖2.2 幾丁質及幾丁聚醣之製備流程.......................5
圖2.3 幾丁質與幾丁聚醣酵素之水解路徑...................12
圖4.1 菌落型態與其產生之透明環.........................52
圖4.2 菌株型態.........................................53
圖4.3 溫度對幾丁聚醣酵素活性之影響.....................55
圖4.4 pH值對幾丁聚醣酵素活性之影響.....................56
圖4.5 基質濃度對幾丁聚醣酵素活性之影響.................58
圖4.6 反應時間對於產物生成速率之影響...................59
圖4.7 金屬離子對幾丁聚醣粗酵素活性之影響...............60
圖4.8 溫度對幾丁聚醣酵素穩定性之影響...................61
圖4.9 pH值對幾丁聚醣酵素穩定性之影響...................63
圖4.10 菌株生長曲線與酵素活性對時間的關係...............64
圖4.11 碳源對幾丁聚醣酵素產量之影響.....................65
圖4.12 幾丁聚醣濃度對幾丁聚醣酵素產量之影響.............67
圖4.13 氮源對幾丁聚醣酵素產量之影響.....................68
圖4.14 酵母萃出物濃度對幾丁聚醣酵素產量之影響...........70
圖4.15 鹽類對幾丁聚醣酵素產量之影響.....................71
圖4.16 微量金屬對幾丁聚醣酵素產量之影響.................73
圖4.17 培養溫度對幾丁聚醣酵素產量之影響.................74
圖4.18 起始pH值對幾丁聚醣酵素產量之影響.................75
圖4.19 振盪速率對幾丁聚醣酵素產量之影響.................77
圖4.20 幾丁聚醣酵素水解膠態幾丁聚醣所得產物.............78
圖4.21 以SDS-PAGE檢定不同硫酸銨濃度下沉澱出之蛋白質.....80
圖4.22 以SDS-PAGE檢定膠態幾丁聚醣之親和性吸附結果.......81
圖4.23 酵母萃出物與培養液起始pH值對幾丁聚醣酵素產量
之影響(回應曲面圖)...............................88
圖4.24 酵母萃出物與培養液起始pH值對幾丁聚醣酵素產量
之影響(等高線圖).................................89
圖4.25 膠態幾丁聚醣與培養液起始pH值對幾丁聚醣酵素產量
之影響(回應曲面圖)...............................90
圖4.26 膠態幾丁聚醣與培養液起始pH值對幾丁聚醣酵素產量
之影響(等高線圖).................................91
圖4.27 膠態幾丁聚醣與酵母萃出物對幾丁聚醣酵素產量
之影響(回應曲面圖)...............................92
圖4.28 膠態幾丁聚醣與酵母萃出物對幾丁聚醣酵素產量
之影響(等高線圖).................................93
表目錄
表2.1 幾丁質與幾丁聚醣之應用範圍.......................7
表2.2 不同微生物來源之幾丁聚醣酵素.....................10
表2.3 幾丁聚醣酵素之活化劑與抑制劑.....................14
表2.4 不同菌株之幾丁聚醣酵素之作用型式.................15
表2.5 各種幾丁聚醣酵素生產菌之培養基組成與培養條件.....20
表3.1 篩菌培養基.......................................33
表3.2 LB培養基.........................................33
表3.3 SDS-PAGE 膠體溶液的配置..........................37
表3.4 23因子設計之控制因子與水準.......................45
表3.5 23因子實驗設計表.................................46
表3.6 陡升路徑實驗設計表...............................47
表3.7 中心混成自變數變級...............................49
表3.8 中心混成實驗設計表...............................50
表4.1 幾丁聚醣酵素產量之23因子實驗結果.................83
表4.2 幾丁聚醣酵素產量之陡升路徑實驗結果...............85
表4.3 幾丁聚醣酵素產量之中心混成實驗結果...............86

1. 山口壽。幾丁聚醣的抗菌性：對鹽漬生菜之保存效果，食品と開發，21(8)：20-24(1988)。
2. 王三郎編著。水產資源利用學。高立圖書公司，台北(1996)。
3. 王三郎編著。生物技術。高立圖書公司，台北(2000)。
4. 王三郎編著。應用微生物學。高立圖書公司，台北(1997)。
5. 內田泰。キチン，キトサンの抗菌性，特集これかろ天然抗菌(1988)。
6. 李昌憲、洪哲潁、熊光濱。利用回應曲面法進行以Streptococcus faecalis生產酪胺酸脫羧之培養基最適化研究。中國農業化學會誌，30(2)：264-272 (1992)。
7. 李建良。液態培養生產冬蟲夏草菌絲體與冬蟲夏草多醣之研究。國立交通大學生物科技研究所碩士論文，新竹(2000)。
8. 李根永、李孟修。Corynebacterium glutamicum在高濃度鹽份培養基脯胺酸發酵之研究。中國農業化學會誌，36(1)：57-64 (1998)。
9. 阮進惠，林翰良，羅淑珍。幾丁聚醣水解物之連續式生產及其抑菌作用。中國農業化學會誌，35(6)：596-611 (1997)。
10. 周聰麟。Aspergillus clavatus NTU-FC-7之幾丁聚醣純化與酵素特性之探討。國立台灣大學農業化學研究所碩士論文，台北 (2000)。
11. 洪哲潁、陳國誠。回應曲面實驗設計法在微生物酵素生產上之應用。化工專論，39(2)：3-17 (1992)。
12. 洪啟章。Bacillus cereus NTU-FC-4菌株之幾丁質酵素及幾丁聚醣酵素之研究。國立台灣大學農業化學研究所碩士論文，台北 (1994)。
13. 財團法人生物技術開發中心。新生技產品：幾丁質、幾丁聚醣(甲殼類)產業現況與展望。財團法人技術開發中心，台北市(1996)。
14. 宮尾茂雄。機能性を有する保存性向上物質の漬物への利用。食品と科學，3：83 (1998).
15. 張珍田。幾丁質及幾丁聚醣之研究。生命科學簡訊，14(3): 3-8(2000)。
16. 袁國芳。幾丁質/幾丁聚醣在膳食與醫療之助益及潛在問題。食品工業，32(4)：1-8 (2000)。
17. 袁國芳，幾丁質與幾丁聚醣在食品工業上之應用。食品工業，31(10)：19-23(1999)。
18. 陳文章、劉光哲。回應曲面實驗設計法於嗜鹼性Bacillus sp.生產環狀糊精葡萄糖苷轉移培養基之最適化應用。大同學報，23：294-298(1993)。
19. 陳美惠、莊淑惠、吳志律。幾丁聚醣的物化特性。食品工業，31(10)：1-6(2000)。
20. 陳美惠。幾丁聚醣之抑菌作用。食品工業，31(10)：29-33 (2000)。
21. 陳重儒。以冬蟲夏草浸液醱酵生產胞外多醣體之最適化研究。大葉大學食品工程學系研究所碩士論文，彰化(2001)。
22. 陳彥霖。幾丁質及幾丁聚醣在紡織工業之應用。食品工業，32(4)：66-73(2000)。
23. 陳樹人。以Acinetobacter radioresistens生產耐鹼性脂肪酵素-醱酵生理之探討。國立成功大學化學工程學系博士論文，台南(1999)。
24. 梁舜欣。N-乙醯幾丁寡醣之製備。國立台灣大學農業化學研究所碩士論文，台北 (1990)。
25. 張薏苓。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，台中(1999)。
26. 黃淑貞。發酵培養生產色胺酸之研究。大葉大學食品工程系研究所碩士論文，彰化(2001)。
27. 黃德琳。黴菌中幾丁聚醣酵素之篩選、純化與性質的研究。國立交通大學應用化學研究所碩士論文，新竹 (2000)。
28. 楊世民、林讚峰。簡介利用回應曲面實驗設計法決定工業微生物的最佳培養基。製酒科技專論彙編，16: 135-150 (1994)。
29. 鄭茜如。以Aspergillus glavus NTU-FC-8生產幾丁聚醣之研究。國立台灣大學農業化學研究所碩士論文，台北(2000)。
30. 蔡震壽。我國漁業廢棄物質量基線資料調查與處理措施規劃。九十年度農委會科技計畫期末摘要報告，農委會(2001)。
31. 潘結昌。利用反應曲面法尋求以Aspergillus terreus生產lovastatin之培養基最適化研究。朝陽大學應用化學系研究所碩士論文，台中(2000)。
32. 賴淑琪。水產廢棄物蝦、蟹外殼之高度利用。食品工業，11(4)：23-28 (1979)。
33. 賴進此。幾丁類物質在生物技術上之應用。食品工業，32(4)：52-65(2000)。
34. 戴瑞益、柯順隆。化學分析試藥配製法，正文書局，台北。
35. 謝魁鵬、魏耀揮。最新生物化學實驗，藝軒圖書出版社，台北。
36. 蘇仲卿、張珍田及莊榮輝。利用親和層析法分離豬內胰臟蛋白質水解酵素trypsin和chymotrypsin之中間規模試驗。中國農業化學會誌，19(3)：218-225 (1981)。
37. Akiyama, K., Fujita, T., Kuroshima, K.I., Sakane, T., Yokota, A. and Takata, R., Purification and gene cloning of a chitosanase from Bacillus ehimensis EAG1, J. Biosci. Bioeng., 87(3): 383-385 (1999).
38. Alfonso, C., Martinez, M. J., and Reyes, F., Purification and properties of two endo-chitosanase from Mucor rouxii implicated in its cell wall degradation, FEMS Microbiol. Lett., 95: 187-194 (1992).
39. Allan, C.R. and Hadwiger, L.A., The fungicidal effect of chitosan on fungi of varying cell wall composition, Experimental Mycology, 3: 285-287 (1979).
40. Ando, A., Noguchi, K., Yanagi, M., Shinoyama, H., Kagawa, Y., Hirata, H., Yabuki, M. and Fujii T., Primary structure of chitosanase produced by Bacillus circulans MH-K1, J. Gen. Appl. Microbiol., 38: 135-144 (1992).
41. Aruchami, M., Goeri, N. and Sundra, Rajulu G., Chitin deacetylase in invertebrates, In chitin in nature and technology, edit by Muzzarelli, R., Jeuniaux, C. and Gooday, G.W., Plenum Press, New York and Landon, 263-267 (1987).
42. Boller, T., Gehri, A., Mauch, F. and Vogeli, U., Chitinase in bean leaves: induction by ethylene, purification, properties and possible function, Planta, 157: 22-31 (1983).
43. Boucher, I., Dupuy, A. , Vidal, P., Neugebauer, W. A., and Brzezinski, R., Purification and characterization of a chitosanase from Streptomyces N174, Appl. Microbiol. Biotechnol., 38: 188-193 (1992).
44. Collinge, D.B., Kargh, K.M., Mikkelsen, J.D., Nielsen, K.K., Rasmussen, U. and Vad, K., Plant chitinase, Plant J., 3: 31-40 (1993).
45. Davis, B. and Eveleigh, D.E., Chitosanase: occaurrence, production and immobilization, In Chitin, Chitosan and Related Enzymes, edited by Zikakis, J.P., Academic Press Orlando,161-179 (1984).
46. Dumas-Gaudot, E., Grenier, J., Furlan, V. and Asselin, A., Chitinase, chitosanase, and β-1,3 glucanase activities in Allium and Pisum roots colonized by Glomus species, Plant Science, 84(1): 17-24 (1992).
47. Errington, N., Harding, S.E., Varum, K.M. and Illum, L., Hydrodyanic characterization of chitosans varying in degree of acetylation, Int. J. Biol. Macromol., 15: 113-117 (1993).
48. Fenton, D. M., and Elveleigh, D. E., Purification and mode of action of a chitosanase from Penicillium islandicum, Journal of General Microbiology, 126: 151-165 (1981).
49. Flach, J., Pilet, P.E. and Jolles, P., What’s new in chitinase research? Experientia, 48: 701-716 (1992).
50. Fukamizo, T., Ohkawa, T., Ikeda, Y., Goto, S., Specificity of chitosanase from Bacillus pumilus, Biochimica et Biophysica Acta., 1205: 183-188 (1994).
51. Gowri, N., Aruchami, M. and Sundra Rajulu G., Natural deacetylation of the cuticle in Sacculina rotundan, In: chitin in nature and techology, edit by Muzzarelli, R., Jeuniaux, C. and Gooday, G.W., Plenum Press, New York and Landon, 266-268 (1987).
52. Imoto, T. and Yagishita, K., A simple activity measurement of lysozyme, Agric. Biol. Chem., 35(7): 1154-1156 (1971).
53. Jeuniaus, C., In methods in Enzymology, Academic Press, New York, 8: 644-654 (1966).
54. Knorr, D., Dye binding properties of chitin and chitosan,J. Food Sci., 48:36-37 (1983).
55. Knorr, D., Use of chitinous polymers in food, A challenge for food research and development, Food Technology., 38: 85-95 (1984).
56. Koga, D., Isogai, A., Sakuda, S., Matsumoto, S., Suzuki, A., Kimura, S. and Ide, A., Specific inhibition of Bombyx mori chitinase by allosamidin, Agric. Biol. Chem., 51(2): 471-476 (1987).
57. Koga, D., Mizuki, K., Ide, A., Kono, M., Matsui, T. and Shimizu, C., Kinetics of a chitinase from a prawn Penaeus japonicus, Agric. Biol. Chem., 54(10): 2505-2515 (1990).
58. Koga, D., Nakashima, M., Matsukura, T., Kimura, S. and Ide, A., Purification and properties of β-N-acetyl-D-glucosaminidase from alimentary canal of the silkworm Bombyx mori, Agric. Biol. Chem., 50(9): 2357-2368 (1986).
59. Koga, D., Tsukamoto, T., Sueshige, N., Usumi, T. and Ide, A., Kinetics of chitinase from yam, Dioscorea opposita THUNB, Agric. Biol. Chem., 53(12): 3121-3126 (1989).
60. Kurakake, M., Shou, Yo-u, Nakagawa, K., Sugihara, M. and Komaki, T., Properties of chitosanase from Bacillus cereus S1, Current Microbiology, 40: 6-9 (2000).
61. Leuba, J.L. and Stossel, P., Chitosan and other polyamines: antifungal activity and interaction with biology membranes, In: chitin in nature and technology, edit by Muzzarelli, R., Jenuiaux, C. and Goody, G.W., Plenum Press, New York, 215-222 (1986).
62. Li, Y.K. and Cheng, C.Y., An Aspergillus chitosanase with potential for large-scale preparation of chitosan oligosaccharides, Biotechnol. Appl. Biochem., 32: 197-203 (2000).
63. Molano, J., Puran, A. and Cabib, E., A rapid and sensitive assay for chitinase using tritiated chitin, Analytical Biochemistry, 83: 648-656 (1977).
64. Nogawa, M., Takahashi, H., Kashiwagi, A., Ohshima, K., Okada, H.and Morikawa, Y., Purification and characterization of exo-β-D-glucosaminidase from a cellulolytic fungus Trichoderma reesei PC-3-7, Appl. Environ. Microbiol., 64(3): 890-895 (1998).
65. Ohtakara, A. and Izume, M., Preparation of D-glucosamine oligosaccharide by enzymatic hydrolysis of chitosan, Agric. Biol. Chem., 51(4): 1189-1191 (1987).
66. Okajima, S., Kinouchi, T., Mikami, Y., and Ando, A., Purification and some properties of a chitosanase of Nocardioides sp., J. Gen. Appl. Microbiol., 41: 351-357 (1995).
67. Otakara, A., Mitsutomi, M. and Uchida, Y., Purification and some properties of chitinase from Vibrio sp., J. Ferment. Technol., 57(3): 169-177 (1979).
68. Pelletier, A. and Sygusch, J., Purification and characterization of three chitosanase activities from Bacillus megaterium P1, Appl. Environ. Microbiol., 56(4): 844-848 (1990).
69. Powing, R.F. and Irzykiewicz, H., Separation of chitin oligosaccharides by thin-layer chromatography, J. chromatogr., 29: 115-119 (1967.)
70. Razdan, A., and Petterson, D., Effect of chitin and chitosan on nutrient digestibility and plasma lipid concentration in broiler chicken, British Journal of Nutrition., 72: 277-288 (1994).
71. Sakai, K., Katsumi, R., Isobe, A. and Nanjo, F., Purification and hydrolytic action of a chitosanase from Nocardia orientalis, Biochimica et. Biophysica Acta, 1079: 65-72 (1991).
72. Seino, H., Tsukuda, K. and Shimasue, Y., Properties and action pattern of a chitosanase from Bacillus sp. PI-7S, Agric. Biol. Chem., 55(9): 2421-2423 (1991).
73. Shimosaka, M., Nogawa, M., Wang, X.Y., Kumehara, M. and Okazaki, M., Production of two chitosanase from a chitosan- assimilating bacterium Acinetobacter sp. strain CHB101, Appl. Environ. Microbiol., 61(2): 438-442 (1995).
74. Somashekar, D. and Joseph, R., A new spectrophotometric method of assay for chitosanase based on calcofluor white dye binding, Carbohvdrate Polymers, 34: 343-346 (1997).
75. Somashekar, D. and Joseph, R., Chitosanase-properties and applications: a review, Bioresource Technology, 55: 35-45 (1996).
76. Somashekar, D. and Joseph, R., Partial purification and properties of a novel chitosanase secreted by Rhodotorula gracilis, Letters Applied Microbiology, 14: 1-4 (1992).
77. Tominaga, Y., and Tsujisaka, Y., Purification and some enzymatic properties of the chitosanse from Bacillus R-4 which lyses Rhizopus cell wall, Biochim. Bicphys. Acta., 410:145-151 (1975).
78. Tsai, G.J., Su, W.H. and Chen, H.C., Antimicrobial activity of shrimp chitin and chitosan against Escherichia coli, J. Food Prot., 62: 239-248 (1999).
79. Uchida, Y., Tateishi, K., Shida, O., Kodowaki, K., Purification and enzymic properties of chitosanases from Bacillus licheniforis UTK and their application, In Adv., Chitin Chitosan, edited by Brine, C.J., Bandforf, P.A., Zikakis, J.P., Elsevier, London, 282-291 (1992).
80. Yamasaki, Y., Fukumoto, I., Kumagai, N., Ohta, Y., Nakagawa, T., Kawamukai, M. and Matsuda, H., Continuous chitosan hydrolyzate production by immobilized chitosanolytic enzyme from Enterobacter sp. G-1, Biosci. Biotech. Biochem., 56(10): 1546-1551 (1992).
81. Yoshihara, K., Hosokawa, J., Kubo, T., Nishiyama, M. and Koba, Y., Purification and properties of a chitosanase from Pseudomonas sp. H-14, Biosci. Biotech. Biochem., 56(6): 972-973 (1992).