淡江大學覺生紀念圖書館 (TKU Library)
進階搜尋


下載電子全文限經由淡江IP使用) 
系統識別號 U0002-2007201112212800
中文論文名稱 Bacillus cereus TKU027 所生產幾丁質酶,幾丁聚醣酶及蛋白酶之純化,定性與應用
英文論文名稱 Purification and characterization of a chitinase, a chitosanase and a protease from Bacillus cereus TKU027 and their applications
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 99
學期 2
出版年 100
研究生中文姓名 劉金佩
研究生英文姓名 Chin-Pei Liu
學號 698180147
學位類別 碩士
語文別 中文
第二語文別 英文
口試日期 2011-07-12
論文頁數 100頁
口試委員 指導教授-王三郎
委員-梁慈雯
委員-王三郎
委員-王全祿
中文關鍵字 仙人掌桿菌  幾丁質酶  幾丁聚醣酶  蛋白酶 
英文關鍵字 Bacillus cereus  chitinase  chitosanase  protease 
學科別分類 學科別自然科學化學
中文摘要 菌株TKU027 係以蝦頭粉為唯一碳/氮源,篩選自台灣彰化土壤之幾丁質酶、幾丁聚醣酶及蛋白酶生產菌,經鑑定為 Bacillus cereus。幾丁質酶與蛋白酶較適生產條件為,分別於含有 1% 蝦頭粉末、0.1% K2HPO4、0.05% MgSO4.7H2O 之50 mL和100 mL 液態培養基(pH 6),於 37℃ 搖瓶(150 rpm)培養 2 天。將所得發酵液經離心、硫酸銨沉澱,以及DEAE-Sepharose與Sephacryl S-100 層析等步驟,純化出幾丁質酶CHI與幾丁聚醣酶CHS,經SDS-PAGE分別測定分子量為 65 kDa 及 63 kDa。CHI及CHS之最適反應pH、最適反應溫度、pH安定性、熱安定性分別為: pH 6、50℃、pH 5 – 8、< 40℃ 和 pH 6、60℃、pH 3 – 10、< 50℃。CHI 活性會受 Mn2+ 及PMSF 所抑制;CHS 活性則會受 Cu2+、Mn2+ 及EDTA 所抑制。
利用B. cereus TKU027 較適培養條件,將所得發酵上清液添加,分別可促進L. paracasei TKU012生長達 175% 、L. paracasei 12193 生長達144% 與 L. kefir 14011 生長達 132% 之影響。另外,利用製備自上清液的粗酵素液,水解水溶性幾丁聚醣後所得之寡醣,亦可促進 L. paracasei 12193 生長達 324% 與 L. kefir 14011 生長達 174% 之影響。此外利用PCR-DGGE技術,探討添加 B. cereus TKU027 與蝦頭粉,於淡水紅樹林土壤之菌相變化與生物降解,並於培養 5 週有最高總糖量(3511 µg/g soil)及還原糖量(1442 µg/g soil),提高總生菌數至 6 × 107 CFU/g soil。
英文摘要 The chitinase, chitosanase and protease-producing strain TKU027 was isolated from soil in Changhua of Taiwan with shrimp head powder as the sole carbon/nitrogen source and identified as Bacillus cereus. The optimized culture conditions for chitinase and protease production were found to be shaken at 37℃ for 2 days in 50 mL and 100 mL of medium containing 1% shrimp head powder (SHP) , 0.1% K2HPO4 and 0.05% MgSO4.7H2O (pH 6). A chitinase CHI and a chitosanase CHS were purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose and Sephacryl S-100. The molecular masses of CHI and CHS determined by SDS-PAGE were approximately 65 and 63 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of CHI and CHS were pH 6, 50℃, pH 5–8, <40℃ and pH 6, 60℃, pH 3–10, <50℃, respectively. The chitinase activity was inhibited by Mn2+ and PMSF. The chitosanase activity was inhibited by Cu2+, Mn2+ and EDTA.
The culture supernatant obtained from B. cereus TKU027 in the optimized culture conditions enhanced the growth of L. paracasei TKU012 most obviously up to 175%, followed by L. paracasei 12193 up to 144% and L. kefir 14011 up to 132%. The crude enzyme from B. cereus TKU027 culture supernatant hydrolyzed water soluble chitosan to produce N-acetyl chitooligosaccharides. The N-acetyl chitooligosaccharides also exhibited activity of enhancing growth for L. paracasei 12193 up to 324% and L. kefir 14011 up to 174%. In addition, B. cereus TKU027 and SHP were added in soil respectively to investigate the biodegradation of SHP and change of bacteria flora by PCR-DGGE analysis. The highest reducing sugar(1442 µg/g soil), total sugar(3511 µg/g soil)and total viable cell counts(6 × 107 CFU/g soil)were found at the 5th week incubation with B. cereus TKU027 and SHP in Tamsui mangrove river soil.
論文目次 中文摘要………………………………………………………Ⅰ
英文摘要………………………………………………………Ⅱ
目錄……………………………………………………………Ⅳ
圖目錄…………………………………………………………Ⅷ
表目錄………………………………………………………Ⅹ

第一章 緒論……………………………………………………1
第二章 文獻回顧……………………………………………………2
2.1 Bacillus cereus之簡介……………………………………………………2
2.2 幾丁質與幾丁聚醣……………………………………………………2
2.3 N-乙醯幾丁寡醣及幾丁寡醣……………………………………………………4
2.4 幾丁質酶與幾丁聚醣酶……………………………………………………6
2.5 蛋白酶……………………………………………………7
2.6 益生質(prebiotics)……………………………………………………8
2.7 DGGE 在環境微生物菌相分析……………………………………………………9
第三章 材料與方法……………………………………………………10
3.1 實驗菌株……………………………………………………10
3.2 實驗材料……………………………………………………10
3.3 實驗儀器……………………………………………………12
3.4 酵素生產菌株之篩選……………………………………………………12
3.5 幾丁質酶之活性測定……………………………………………………13
3.6 幾丁聚醣酶之活性測定……………………………………………………13
3.7 蛋白酶活性之測定……………………………………………………14
3.8 較適培養條件探討……………………………………………………14
3.8.1 碳/氮源濃度……………………………………………………14
3.8.2 培養體積……………………………………………………15
3.8.3 培養溫度……………………………………………………15
3.8.4 培養基 pH 值……………………………………………………15
3.8.5 培養時間……………………………………………………15
3.9 幾丁質酶及幾丁聚醣酶之分離純化……………………………………………………16
3.9.1 粗酵素液之製備……………………………………………………16
3.9.2 陰離子交換層析……………………………………………………16
3.9.3 膠體過濾層析 ……………………………………………………17
3.10 蛋白質電泳分析……………………………………………………17
3.11 酵素之特性分析……………………………………………………17
3.11.1 酵素最適反應溫度……………………………………………………17
3.11.2 酵素熱安定性……………………………………………………18
3.11.3 酵素最適反應 pH……………………………………………………18
3.11.4 酵素 pH 安定性……………………………………………………18
3.11.5 金屬離子及化學藥品對酵素活性之影響……………………………………………………19
3.11.6 界面活性劑對酵素活性之影響……………………………………………………19
3.11.7 酵素之基質特異性……………………………………………………20
3.12 N-乙醯幾丁寡醣製備……………………………………………………20
3.13 N-乙醯幾丁寡醣之組成分析……………………………………………………20
3.14 還原糖量之測定……………………………………………………21
3.15 總糖量之測定(H2SO4-Phenol法)……………………………………………………21
3.16 MALDI-TOF-MS……………………………………………………22
3.17 總生菌計數法……………………………………………………22
3.18 促進乳酸菌生長……………………………………………………22
3.18.1 經 B. cereus TKU027 發酵蝦頭粉……………………………………………………22
3.18.2 經 B. cereus TKU027 粗酵素液水解基質……………………………………………………23
3.19 DGGE(Denaturing Gradient Gel Electrophoresis)分析……………………………………………………23
第四章 結果與討論……………………………………………………25
4.1幾丁質酶與蛋白酶生產菌之篩選……………………………………………………25
4.1.1菌株TKU027之鑑定……………………………………………………25
4.2 酵素較適產生條件探討……………………………………………………27
4.2.1 碳/氮源濃度……………………………………………………27
4.2.2 培養液體積……………………………………………………27
4.2.3 通氣量……………………………………………………28
4.2.4培養溫度……………………………………………………28
4.2.5培養pH值……………………………………………………29
4.2.6較適培養條件探討結果……………………………………………………29
4.3 幾丁質酶及幾丁聚醣酶之分離純化……………………………………………………39
4.3.1 粗酵素液之製備……………………………………………………39
4.3.2 離子交換樹脂層析……………………………………………………39
4.3.3 膠體過濾層析 ……………………………………………………40
4.3.4 綜合結果……………………………………………………40
4.4 幾丁質酶與幾丁聚醣酶之分子量測定……………………………………………………41
4.4.1 SDS-PAGE……………………………………………………41
4.4.2 幾丁質酶與幾丁聚醣酶胜肽質譜鑑定……………………………………………………41
4.4.3 綜合結果……………………………………………………41
4.5 幾丁質酶與幾丁聚醣酶之特性分析……………………………………………………49
4.5.1 幾丁質酶與幾丁聚醣酶之最適反應溫度及熱安定性……………………………………………………49
4.5.2 幾丁質酶與幾丁聚醣酶之最適反應pH及pH安定性……………………………………………………49
4.5.3 金屬離子及化學藥品對幾丁質酶與幾丁聚醣酶活性之影響……………………………………………………50
4.5.4 界面活性劑對幾丁質酶與幾丁聚醣酶活性之影響……………………………………………………51
4.5.5 幾丁質酶與幾丁聚醣酶之基質特異性……………………………………………………51
4.6 經 B. cereus TKU027 發酵蝦頭粉所得幾丁寡醣組成分析……………………………………………………59
4.7 水解基質之探討……………………………………………………59
4.7.1 還原糖與總糖含量之分析……………………………………………………59
4.7.2 利用HPLC 進行幾丁寡醣組成分析……………………………………………………60
4.7.3 利用 MALDI-TOF-MS 進行幾丁寡醣組成分析……………………………………………………60
4.8 促進乳酸菌生長……………………………………………………77
4.8.1 經 B. cereus TKU027 發酵蝦頭粉之探討……………………………………………………77
4.8.2 經 B. cereus TKU027 粗酵素液水解基質之探討……………………………………………………78
4.9 土壤中的生物降解和微生物菌相變化之探討……………………………………………………85
4.9.1 總生菌數測定……………………………………………………85
4.9.2 還原糖和總糖含量之探討……………………………………………………85
4.9.3 DGGE分析……………………………………………………86
第五章 結論……………………………………………………93
參考文獻……………………………………………………94

圖目錄
圖 2.1 幾丁質、幾丁聚醣及纖維素之結構……………………………………………………5
圖 4.1 Bacillus cereus TKU027 之顯微鏡照片……………………………………………………25
圖 4.2 16S rDNA 部分鹼基序列分析及API 試驗……………………………………………………26
圖 4.3 SHP濃度對 B. cereus TKU027幾丁質酶及蛋白酶生產之影響……………………………………………………31
圖 4.4 培養液體積對B. cereus TKU027幾丁質酶及蛋白酶生產之影響……………………………………………………32
圖 4.5 通氣量對 B. cereus TKU027幾丁質酶及蛋白酶生產之影響……………………………………………………33
圖 4.6 培養溫度對B. cereus TKU027 幾丁質酶及蛋白酶生產之影響……………………………………………………34
圖 4.7 培養液 pH 對第2天 B. cereus TKU027幾丁質酶及蛋白酶生產之影響……………………………………………………35
圖 4.8 B. cereus TKU027 於含SHP培養基生產酵素之生長曲線圖……………………………………………………36
圖 4.9 B. cereus TKU027 所生產酵素之純化分離流程圖……………………………………………………43
圖 4.10 B. cereus TKU027 幾丁質酶與幾丁聚醣酶 DEAE-Sepharose CL-6B層析圖譜……………………………………………………44
圖 4.11 B. cereus TKU027 幾丁質酶之Sephacryl S-100層析圖譜……………………………………………………45
圖 4.12 B. cereus TKU027 幾丁聚醣酶之Sephacryl S-100 層析圖譜……………………………………………………45
圖 4.13 B. cereus TKU027 幾丁質酶與幾丁聚醣酶 SDS-PAGE 之分子量分析……………………………………………………47
圖 4.14 幾丁質酶與幾丁聚醣酶之 (A) 最適反應溫度 (B) 熱安定性……………………………………………………52
圖 4.15 幾丁質酶和幾丁聚醣酶之 (A) 最適反應 pH (B) pH安定性……………………………………………………53
圖 4.16利用HPLC進行幾丁寡醣組成分析 (A) 標準品 (B) B. cereus TKU027發酵含 1% SHP…………………………………………62
圖 4.17 (A) chitin β (B) 水溶性幾丁聚醣中添加 B. cereus TKU027粗酵素液於水解不同時間的回收率、還原糖及總糖含量之變化……………………………………………………63
圖 4.18 (A) chitin β (B) 水溶性幾丁聚醣經 B. cereus TKU027 粗酵素液於水解不同時間所剩的基質粉末……………………………………………………64
圖4.19 利用HPLC進行幾丁寡醣組成分析經 B. cereus TKU027 粗酵素液水解不同時間之 chitin β,(A) 2 hr ; (B) 4 hr ; (C) 6 hr ; (D) 12 hr……………………………………………………64
圖4.20 利用HPLC進行幾丁寡醣組成分析經 B. cereus TKU027 粗酵素液水解不同時間之 chitin β,(A) 24 hr;(B) 48 hr;(C) 72 hr……………………………………………………66
圖4.21 利用HPLC進行幾丁寡醣組成分析經 B. cereus TKU027 粗酵素液 水解不同時間之水溶性幾丁聚醣 (A) 2 h ; (B) 4 h; (C) 6 h ; (D) 12 h……………………………………………………67
圖4.22 利用HPLC進行幾丁寡醣組成分析經 B. cereus TKU027 粗酵素液水解不同時間之水溶性幾丁聚醣 (A) 24 h ; (B) 48 h ; (C) 72 h ……………………………………………………68
圖 4.23 chitin β 經 B. cereus TKU027 粗酵素液水解所得幾丁寡 MALDI-TOF-MS 分析圖……………………………………………………69
圖 4.24 水溶性幾丁聚醣經 B. cereus TKU027 粗酵素液水解所得幾丁寡 醣之MALDI-TOF-MS 分析圖……………………………………………………72
圖 4.25 B. cereus TKU027 發酵蝦頭粉末所得上清液及幾丁寡醣對於 (A)……………………………………………………L. paracasei TKU010 (B) L. paracasei TKU012 生長之影響……………………………………………………80
圖 4.26 B. cereus TKU027 發酵蝦頭粉末所得上清液及幾丁寡醣對於 (A) L. paracasei 12193 (B) L. kefir 14011 生長之影響……………………………………………………81
圖 4.27 (A) CHI β (B) WSC經 B. cereus TKU027 粗酵素液水解不同時間所得幾丁寡醣添加於 L. paracasei 12193 生長之影響……………………………………………………82
圖 4.28 (A) CHI β (B) WSC經 B. cereus TKU027 粗酵素液水解不同時間所得幾丁寡醣添加於 L. kefir 14011 生長之影響……………………………………………………83
圖 4.29 四種樣品於八週培養之總生菌數……………………………………………………88
圖 4.30 四種樣品於八週培養之總糖含量變化……………………………………………………89
圖 4.31 四種樣品於八週培養之還原糖含量變化……………………………………………………89
圖 4.32 16S rDNA 片段聚合酶鏈鎖反應-變性梯度凝膠電泳圖……………………………………………………90

表目錄
表 2.1 幾丁質與幾丁聚醣之應用……………………………………………………5
表 2.2 土壤中幾丁質分解酵素生產菌之碳/氮源……………………………………………………7
表 3.1 DNS 試劑組成……………………………………………………21
表 4.1 B. cereus TKU027 生產酵素之較適條件……………………………………………………37
表4.2 以含幾丁質水產廢棄物為幾丁質酶/蛋白酶生產菌唯一碳/氮源之較適生產條件比較……………………………………………………38
表 4.3 B. cereus TKU027 幾丁質酶之純化總表……………………………………………………46
表 4.4 B. cereus TKU027 幾丁聚醣酶之純化總表……………………………………………………46
表 4.5 B. cereus TKU027 幾丁質酶和幾丁聚醣酶胜肽質譜鑑定結果……………………………………………………48
表 4.6 金屬離子及化學藥品對幾丁質酶及幾丁聚醣酶活性之影響……………………………………………………54
表 4.7 界面活性劑對幾丁質酶及幾丁聚醣酶活性之影響……………………………………………………54
表 4.8 B. cereus TKU027 幾丁質酶及幾丁聚醣酶之基質特異性……………………………………………………55
表 4.9 微生物來源之幾丁質酶與幾丁聚醣酶特性比較……………………………………………………56
表 4.10 不同發酵上清液所得幾丁寡醣之組成……………………………………………………61
表 4.11 chitin β 經 B. cereus TKU027 粗酵素液水解不同時間之MALDI- TOF-MS 幾丁寡醣組成分析……………………………………………………75
表 4.12 水溶性幾丁聚醣經 B. cereus TKU027 粗酵素液水解不同時間之 MALDI-TOF-MS 幾丁寡醣組成分析……………………………………………………76
表 4.13 B. cereus TKU027 發酵蝦頭粉末所得上清液及幾丁寡醣對於乳酸菌生長(12 與 48小時)之影響……………………………………………………79
表 4.14 CHI β和WSC經 B. cereus TKU027 粗酵素液水解不同時間所得幾丁寡醣添加於乳酸菌生長(12小時)之影響……………………………………………………84
表 4.15 CHI β和WSC經 B. cereus TKU027 粗酵素液水解不同時間所得幾丁寡醣添加於乳酸菌生長(48小時)之影響……………………………………………………84
表 4.16 四種樣品於八週培養之總生菌數……… …………………………88
參考文獻 Aiba S (1994) Preparation of N-acetylchitooligosaccharides by hydrolysis of chitosan with chitinase followed by N-acetylation. Carbohydrate Research, 265:323-328.
Aiba SI (1992) Studies on chitosan: 4. Lysozymic hydrolysis of partially N-acetylated chitosans. International Journal of Biological Macromolecules, 14:225-228.
Banik RM and Prakash M (2004) Laundry detergent compatibility of the alkaline protease from Bacillus cereus. Microbiological Research, 159:135-140.
Bernfeld P (1955) Amylase, α and β. Methods in Enzymology, 1:149-158.
Boller T, Gehri A, Mauch F, and Vögeli U (1983) Chitinase in bean leaves: induction by ethylene, purification, properties, and possible function. Planta, 157:22-31.
Chang WT, Chen YC, and Jao CL (2007) Antifungal activity and enhancement of plant growth by Bacillus cereus grown on shellfish chitin wastes. Bioresource Technology, 98:1224-1230.
Chen H, Liu LJ, Zhu JJ, Xu B, and Li R (2010a) Effect of soybean oligosaccharides on blood lipid, glucose levels and antioxidant enzymes activity in high fat rats. Food Chemistry, 119:1633-1636.
Chen WC, Tseng WN, Hsieh JL, Wang YS, and Wang SL (2010b) Biodegradation and microbial community changes upon shrimp shell wastes amended in mangrove river sediment. Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 45:473-477.
Chiang CL, Chang CT, and Sung HY (2003) Purification and properties of chitosanase from a mutant of Bacillus subtilis IMR-NK1. Enzyme and Microbial Technology, 32:260-267.
Choi YJ, Kim EJ, Piao Z, Yun YC, and Shin YC (2004) Purification and characterization of chitosanase from Bacillus sp. strain KCTC 0377BP and its application for the production of chitosan oligosaccharides. Applied and Environmental Microbiology, 70:4522-4531.
Chui VWD, Mok KW, Ng CY, Luong BP, and Ma KK (1996) Removal and recovery cooper(II), chromium(III), and nickel(II) from solution using crude shrimp chitin packed in small columns. Environment International, 22:463-468.
Cohen-Kupiec R and Chet I (1998) The molecular biology of chitin digestion. Current Opinion in Biotechnology, 9:270-277.
Deshpande MV (1986) Enzymatic degradation of chitin and its biological application. Journal of Scientific and Industrial Research, 45:273-277.
Dubois M, Gilles KA, Hamilton JK, Rebers PA, and Smith F (1956) Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28:350-356.
Ekowati C, Hariyadi P, Witarto AB, Hwang JK, and Suhartono MT (2006) Biochemical characteristics of chitosanase from the Indonesian Bacillus licheniformis MB-2. Molecular Biotechnology, 33:93-102.
Fischer SG and Lerman LS (1979) Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis. Cell, 16:191-200.
Gao XA, Ju WT, Jung WJ, and Park RD (2008) Purification and characterization of chitosanase from Bacillus cereus D-11. Carbohydrate Polymers, 72:513-520.
Ghaouth AE, Arul J, Grenier J, and Asselin A (1992) Effect of chitosan and other polyions on chitin deacetylase in Rhizopus stolonifer. Experimental Mycology, 16:173-177.
Harish Prashanth KV and Tharanathan RN (2007) Chitin/chitosan: modifications and their unlimited application potential—an overview. Trends in Food Science & Technology, 18:117-131.
He H, Silo-Suh LA, Handelsman J, and Clardy J (1994) Zwittermicin A, an antifungal and plant protection agent from Bacillus cereus. Tetrahedron Letters, 35:2499-2502.
Hirano S (1999) Chitin and chitosan as novel biotechnological materials. Polymer International, 48:732-734.
Imoto T and Yagishita K (1971) A simple activity measurement by lysozyme. Agricultural and Biological Chemistry, 35:1154-1156.
Janek T, Łukaszewicz M, Rezanka T, and Krasowska A (2010) Isolation and characterization of two new lipopeptide biosurfactants produced by Pseudomonas fluorescens BD5 isolated from water from the Arctic Archipelago of Svalbard. Bioresource Technology, 101:6118-6123.
Jeon YJ, Park PJ, and Kim SK (2001) Antimicrobial effect of chitooligosaccharides produced by bioreactor. Carbohydrate Polymers, 44:71-76.
Kim PI, Kang TH, Chung KJ, Kim IS, and Chung KC (2004) Purification of a constitutive chitosanase produced by Bacillus sp. MET 1299 with cloning and expression of the gene. FEMS Microbiology Letters, 240:31-39.
Knorr D (1984a) Use of chitinous polymers in food. Food Technology, 1:85-89.
Knorr D (1984b) Use of chitinous polymers in food-a challenge for food research and development. Food Technology, 38: 85-97.
Koga D, Tsukamoto T, Sueshige N, Usumi T, and Ide A (1989) Kinetics of chitinase from yam, Dioscorea opposite thunb. Agricultural and Biological Chemistry, 53:3121-3126.
Kumar MNVR (2000) A review of chitin and chitosan applications. Reactive and Functional Polymers, 46:1-27.
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227:680-685
Lee YS, Yoo JS, Chung SY, Lee YC, Cho, YS, and Choi YL (2006) Cloning, purification, and characterization of chitosanase from Bacillus sp. DAU101. Applied Microbiology and Biotechnology, 73:113-121.
Lee YS, Park I H, Yoo JS, Chung SY, Lee YC, Cho YS, Ahn SC, Kim CM, and Choi YL (2007) Cloning, purification, and characterization of chitinase from Bacillus sp. DAU101. Bioresource Technology, 98:2734-2741.
Li YC, Sun XJ, Bi Y, Ge YH, and Wang Y (2009) Antifungal Activity of Chitosan on Fusarium sulphureum in Relation to Dry Rot of Potato Tuber. Agricultural Sciences in China, 8:597-604.
Liang TW, Chen YJ, Yen YH, and Wang SL (2007) The antitumor activity of the hydrolysates of chitinous materials hydrolyzed by crude enzyme from Bacillus amyloliquefaciens V656. Process Biochemistry, 42:527-534.
Liang TW, Wu YY, Huang TY, Wang CY, Yen YH, Liu CP, Chen YC, and Wang SL (2010a) Conversion of squid pen by a novel strain Lactobacillus paracasei subsp. paracasei TKU010, and its application in antimicrobial and antioxidants activity. Journal of General and Applied Microbiology, 56:481-489.
Liang TW, Kuo YH, Wu PC, Wang CL, Dzung NA, and Wang SL (2010b) Purification and Characterization of a Chitosanase and a Protease by Conversion of Shrimp Shell Wastes Fermented by Serratia Marcescens Subsp. Sakuensis TKU019. Journal of the Chinese Chemical Society, 57:857-863.
Ma C, Ni X, Chi Z, Ma L, and Gao L (2007) Purification and characterization of an alkaline protease from the marine yeast Aureobasidium pullulans for bioactive peptide production from different sources. Marine Biotechnology, 9:343-351.
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31:426-428.
Milner JL, Silo-Suh L, Lee JC, He H, Clardy J, and Handelsman J (1996) Production of kanosamine by Bacillus cereus UW85. Applied and Environmental Microbiology, 62:3061-3065.
Muzzarelli RAA and Rocchetti R (1985) Determination of the degree of acetylation of chitosans by first derivative ultraviolet spectrophotometry. Carbohydrate Polymers, 5:461-472.
Neri DFM, Balcão VM, Costa RS, Rocha ICAP, Ferreira EMFC, Torres DPM, Rodrigues LRM, Jr LBC, and Teixeira JA (2009) Galacto-oligosaccharides production during lactose hydrolysis by free Aspergillus oryzae β-galactosidase and immobilized on magnetic polysiloxane-polyvinyl alcohol. Food Chemistry, 115:92-99.
Ohtakara A, Matsunaga H, and Mitsutomi M (1990) Action pattern of Streptomyces griseus chitinase on partially N-acetylated chitosan. Agricultural and Biological Chemistry, 54:3191-3199.
Reginster JY, Deroisy R, Rovati LC, Lee RL, Lejeune E, Bruyere O, Giacovelli G, Henrotin Y, Dacre JE, and Gossett C (2001) Long-term effects of glucosamine sulphate on osteoarthritis progression: a randomised, placebo-controlled clinical trial. Lancet, 357:251-256.
Rollins DM and Colwell RR (1986) Viable but nonculturable stage of Campylobacter jejuni and its role in survival in the natural aquatic environment. Applied and Environmental Microbiology, 52:531-538.
Sakai K, Yokota A, Kurokawa H, Wakayama M, and Moriguchi M (1998) Purification and characterization of three thermostable endochitinases of a noble Bacillus Strain, MH-1, isolated from chitin-containing compost. Applied and Environmental Microbiology, 64:3397-3402.
Schallmey M, Singh A, and Ward OP (2004) Developments in the use of Bacillus species for industrial production. Canadian Journal of Microbiology, 50: 1-17.
Shahidi F, Arachchi JKV, and Jeon YJ (1999) Food applications of chitin and chitosans. Trends in Food Science and Technology, 10:37-51.
Shikha, Sharan A, and Darmwal NS (2007) Improved production of alkaline protease from a mutant of alkalophilic Bacillus pantotheneticus using molasses as a substrate. Bioresource Technology, 98:881-885.
Steer T, Carpenter H, Tuohy K, and Gibson GR (2000) Perspectives on the role of the human gut microbiota and its modulation by pro- and prebiotics. Nutrition Research Reviews, 13:229-254.
Su C, Wang D, Yao L, and Yu Z (2006) Purification, characterization, and gene cloning of a chitosanase from Bacillus species strain S65. Journal of Agricultural and Food Chemistry, 54:4208-4214.
Suetsuna K (2000) Antioxidant peptides from the protease digest of prawn (Penaeus japonicus) muscle. Marine Biotechnology, 2:5-10.
Suzuki K, Mikami T, Okawa Y, Tokoro A, Suzuki S, and Suzuki M (1986) Antitumor effect of hexa-N-acetylchitohexaose and chitohexaose. Carbohydrate Research, 151:403-408.
Thompson SE, Smith M, Wilkinson MC, and Peek K (2001) Identification and Characterization of a Chitinase Antigen from Pseudomonas aeruginosa Strain 385. Applied and Environmental Microbiology, 67:4001-4008.
Todd EW (1949) Quantitative studies on the total plasmin and the trypsin inhibitor of human blood serum. Journal of Experimental Medicine, 39:295-308.
Toharisman A, Suhartono MT, Spindler-Barth M, Hwang JK, and Pyun YR (2005) Purification and characterization of a thermostable chitinase from Bacillus licheniformis Mb-2. World Journal of Microbiology & Biotechnology, 21:733-738.
Wang SL and Chio SH (1998) Deproteinization of shrimp and crab shell with the protease of Pseudomonas aeruginosa K-187. Enzyme and Microbial Technology, 22:629-633.
Wang SL and Yeh PY (2006) Production of a surfactant- and solvent-stable alkaliphilic protease by bioconversion of shrimp shell wastes fermented by Bacillus subtilis TKU007. Process Biochemistry, 41:1545-1552.
Wang SL, Kao TY, Wang CL, Yen YH, Chern KM, and 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 and Microbial Technology, 39:724-731.
Wang SL, Lin HT, Liang TW, Chen YJ, Yen YH, and Guo SP (2008a) Reclamation of chitinous materials by bromelain for the preparation of antitumor and antifungal materials. Bioresource Technology, 99:4386-4393.
Wang SL, Chen SJ, and Wang CL (2008b) Purification and characterization of chitinases and chitosanases from a new species strain Pseudomonas sp. TKU015 using shrimp shells as a substrate. Carbohydrate Research, 343:1171-1179.
Wang SL, Huang TY, Wang CY, Liang TW, Yen YH, and Sakata Y (2008c) Bioconversion of squid pen by Lactobacillus paracasei subsp. paracasei TKU010 for the production of proteases and lettuce enhancing biofertilizers. Bioresource Technology, 99:5436-5443.
Wang SL, Wang CY, and Huang TY (2008d). Microbial reclamation of squid pen for the production of a novel extracellular serine protease by Lactobacillus paracasei subsp. paracasei TKU012. Bioresource Technology, 99:3411-3417.
Wang SL, Peng JH, Liang TW, and Liu KC (2008e) Purification and characterization of a chitosanase from Serratia marcescens TKU011. Carbohydrate Research, 343:1316–1323.
Wang SL and Yeh PY (2008) Purification and characterization of a chitosanase from a nattokinase producing strain Bacillus subtilis TKU007. Process Biochemistry, 43:132-138.
Wang SL, Chen TR, Liang TW, and Wu PC (2009a) Conversion and degradation of shellfish wastes by Bacillus cereus TKU018 fermentation for the production of chitosanases and bioactive materials. Biochemical Engineering Journal, 48:111-117.
Wang SL, Chao CH, Liang TW, and Chen CC (2009b) Purification and characterization of protease and chitinase from Bacillus cereus TKU006 and conversion of marine wastes by these enzymes. Marine Biotechnology, 11:334-344.
Wang SL, Lin CL, Liang TW, Liu KC, and Kuo YH (2009c) Conversion of squid pen by Serratia ureilytica for the production of enzymes and antioxidants. Bioresource Technology, 100:316-323.
Wang SL, Liou JY, Liang TW, and Liu KC (2009d) Conversion of squid pen by using Serratia sp. TKU020 fermentation for the production of enzymes, antioxidants, and N-acetyl chitooligosaccharides. Process Biochemistry, 44:854-861.
Wang SL, Wu PC, and Liang TW (2009e) Utilization of squid pen for the efficient production of chitosanase and antioxidants through prolonged autoclave treatment. Carbohydrate Research, 344:979-984.
Wang SL, Li JY, Liang TW, Hsieh JL, and Tseng WN (2010a) Conversion of shrimp shell by using Serratia sp. TKU017 fermentation for the production of enzymes and antioxidants. Journal of Microbiology and Biotechnology, 20:117-126.
Wang SL, Chang TJ, and Liang TW (2010b) Conversion and degradation of shellfish wastes by Serratia sp. TKU016 fermentation for the production of enzymes and bioactive materials. Biodegradation, 21:321-333.
Wang SL, Lin BS, Liang TW, Wang CL, Wu PC, and Liu JR (2010c) Purification and characterization of chitinase from a new species strain, Pseudomonas sp. TKU008. Journal of Microbiology and Biotechnology, 20:1001-1005.
Wang SL, Hsu WH, and Liang TW (2010d) Conversion of squid pen by Pseudomonas aeruginosa K187 fermentation for the production of N-acetyl chitooligosaccharides and biofertilizers. Carbohydrate Research, 345:880-885.
Wang SL, Liang TW, and Yen YH (2011a) Bioconversion of chitin-containing wastes for the production of enzymes and bioactive materials. Carbohydrate Polymers, 84:732-742.
Wang SL, Tseng WN, and Liang TW (2011b) Biodegradation of shellfish wastes and production of chitosanases by a squid pen-assimilating bacterium, Acinetobacter calcoaceticus TKU024. Biodegradation, in press.
Wen CM, Tseng CS, Cheng CY and, Li YK (2002) Purification, characterization and cloning of a chitinase from Bacillus sp. NCTU2. Biotechnology and Applied Biochemistry, 35:213-219.
Wiwat C, Siwayaprahm P, and Bhumiratana A (1999) Purification and characterization of chitinase from Bacillus circulans No.4.1. Current Microbiology, 39:134-140.
Wu Y, Wang Y, Luo G, and Dai Y (2009) In situ preparation of magnetic Fe3O4-chitosan nanoparticles for lipase immobilization by cross-linking and oxidation in aqueous solution. Bioresource Technology, 100:3459-3464.
Xu Q, Chao YL, and Wan QB (2009) Health benefit application of functional oligosaccharides. Carbohydrate Polymers, 77:435-441.
Yuli PE, Suhartono MT, Rukayadi Y, Hwang JK, and Pyun YR (2004) Characteristics of thermostable chitinase enzymes from the indonesian Bacillus sp.13.26. Enzyme and Microbial Technology, 35:147-153.
Ziemer CJ and Gibson GR (1998) An overview of probiotics, prebiotics and synbiotics in the functional food concept: Perspectives and future strategies. International Dairy Journal, 8:473-479.

論文使用權限
  • 同意紙本無償授權給館內讀者為學術之目的重製使用,於2013-07-20公開。
  • 同意授權瀏覽/列印電子全文服務,於2013-07-20起公開。


  • 若您有任何疑問,請與我們聯絡!
    圖書館: 請來電 (02)2621-5656 轉 2281 或 來信