Penicillium janthinellum D4株篩選自越南Tay Nguyen大學之幾丁聚醣酶生

產菌。本研究探討P. janthinellum D4發酵烏賊軟骨生產幾丁聚醣酶之較適條

件，所得發酵上清液經硫酸銨沉澱、DEAE-Sepharose離子交換層析、

Macro-prep DEAE Cartridge等步驟，純化出兩種幾丁聚醣酶F1及F2。幾丁聚

醣酶F2之分子量經SDS-PAGE測量為49kDa，其最適反應溫度、最適反應

pH、熱安定性以及pH安定性分別為60℃、pH 7~9、25~40℃以及pH 10。於

抑制劑方面，幾丁聚醣酶F2的活性會受Fe2+、Cu2+、Mn2+所抑制。

The chitosanase producing strain Penicillium janthinellum D4, was 

isolated from soil in Tay Nguyen University in Vietnam. Two 

chitosanases F1 F2 were purified from the culture supernatant by 

ammonium sulfate precipitation, DEAE-Sepharose column 

chromatography and Macro-prep DEAE Cartridge. The molecular mass 

of CHS was estimated to be 49kDa by SDS-PAGE. The optimum 

temperature, optimum pH, thermal stability, and pH stability of CHS F2

were 60℃, pH 7~9, 25~40℃, and pH 10, respectively. The activity of CHS F2 was 

inhibited by Fe2+,Cu2+,and Mn2+.

目錄

中文摘要.............................................I

英文摘要............................................ II

目錄.................................................III

圖目錄...............................................VIII

表目錄...............................................XII

第一章	緒論........................................1

第二章  文獻回顧......................................2

2.1  幾丁質與幾丁聚醣.................................2

2.2  N-乙醯幾丁寡醣與幾丁寡醣.........................3

2.3  幾丁質酶與幾丁聚醣酶.............................4

第三章  材料與方法....................................7

3.1	  實驗菌株..................................7

3.2	  實驗材料..................................7

3.3	  實驗儀器..................................8

3.4  幾丁質酶活性測定.................................9

3.5  幾丁聚醣酶活性測定...............................9

3.6  較適培養條件之探討...............................10

3.6.1  培養液體積.....................................10

3.6.2  碳/氮源濃度與培養時間..........................10

3.7  酵素之分離純化...................................11

3.7.1  粗酵素液之製備.................................11

3.7.2  陰離子交換層析 (一) ...........................11

3.7.3  陰離子交換層析 (二) ...........................11

3.7.4  陽離子交換層析.................................12



3.7.5  膠體過濾層析...................................12

3.8  酵素之特性分析...................................13

3.8.1  酵素最適反應溫度...............................13

3.8.2  酵素熱安定性...................................13

3.8.3  酵素最適反應 pH................................13

3.8.4  酵素 pH安定性..................................14

3.8.5  金屬離子、化學藥品及抑制劑對酵素活性之影響.....14

3.9  還原醣量之測定...................................15

3.10  總醣量之測定 (H2SO4-Phenol 法) .................15

3.11  幾丁聚醣酶水解基質及寡醣分析....................16

3.11.1  基質之水解....................................16

3.11.2  N-乙醯幾丁寡醣之製備..........................16

3.12  MALDI-TOF-MS....................................16

第四章  結果與討論....................................17

4.1  幾丁質酶、幾丁聚醣酶生產菌之篩選.................17

4.2  酵素較適生產條件探討.............................17

4.2.1  培養液體積.....................................18

4.2.2  不同碳/氮源濃度................................18

4.3  幾丁聚醣酶之純化.................................21

4.3.1  粗酵素液之製備.................................21

4.3.2  陰離子交換樹脂層析(一).........................21

4.3.3  陰離子交換樹脂層析(二).........................22

4.3.4  陽離子交換樹脂層析.............................22

4.3.5  綜合結果.......................................23

4.4  幾丁聚醣酶分子量測定.............................24



4.4.1  SDS-PAGE.......................................24

4.5  幾丁聚醣酶之特異性分析...........................24

4.5.1  幾丁聚醣酶之最適反應溫度及熱安定性.............24

4.5.2  幾丁聚醣酶之最適pH值與酸鹼安定性...............25

4.5.3  金屬離子及化學藥品對幾丁聚醣酶活性之影響.......25

4.6  P. janthinellum D4幾丁聚醣酶之抑制劑探討.........26

4.7  還原醣與總醣含量之分析...........................28

4.7.1  利用MALDI-TOF-MS進行幾丁寡醣組成分析...........28

第五章  結論..........................................66

第六章  文獻回顧......................................67

圖目錄

圖  2.1  幾丁質、幾丁聚醣、部分乙醯化幾丁聚醣的化學結構....3

圖  4.1 (A)  培養體積對P. janthinellum D4生產幾丁聚醣酶的影響........................................................29

圖  4.1 (B)  培養體積對P. janthinellum D4生產幾丁聚醣酶的影響........................................................30

圖  4.2  烏賊軟骨粉(SPP)添加濃度對P. janthinellum D4生產幾丁聚醣酶的影響........................................................31

圖  4.3  烏賊軟骨粉(SPP)和葡萄醣(Glucose)添加濃度對P. janthinellum D4生產幾丁聚醣酶的影響.......................32

圖  4.4  烏賊軟骨粉(SPP)添加濃度對Paecilomyces lilacinus D31生產幾丁聚醣酶的影響......................................33

圖  4.5  烏賊軟骨粉(SPP)和葡萄醣(Glucose)添加濃度對Paecilomyces lilacinus D31生產幾丁聚醣酶的影響............34


圖  4.6  烏賊軟骨粉(SPP)添加濃度對Paecilomyces lilacinus D31生產幾丁質酶的影響........................................35

圖  4.7  烏賊軟骨粉(SPP)和葡萄醣(Glucose)添加濃度對Paecilomyces lilacinus D31生產幾丁質酶的影響..............36

圖  4.8  Czapek 培養基和SPP添加濃度及添加懸浮態幾丁質對Paecilomyces lilacinus D31生產幾丁聚醣酶的影響............37

圖  4.9  Czapek 培養基和SPP添加濃度及添加懸浮態幾丁質對Paecilomyces lilacinus D31生產幾丁質酶的影響..............38

圖  4.10  Czapek 培養基和烏賊軟骨粉(SPP)、蝦頭殼粉(SHP)添加濃度對Paecilomyces lilacinus D31生產幾丁聚醣酶的影響......39

圖  4.11  Czapek 培養基和烏賊軟骨粉(SPP)、蝦頭殼粉(SHP)添加濃度對Paecilomyces lilacinus D31生產幾丁質酶的影響........40

圖  4.12  Penicillium janthinellum D4幾丁聚醣酶F2之DEAE-Sepharose CL-6B層析圖譜...................................41




圖  4.13  Penicillium janthinellum D4幾丁聚醣酶F2之Macro-Prep DEAE Cartridge 層析圖................................42

圖  4.14  Penicillium janthinellum D4幾丁聚醣酶F1之CM 層析........................................................43

圖  4.15  Penicillium janthinellum D4幾丁聚醣酶F1之CM 層析........................................................44

圖  4.16  P. janthinellum D4幾丁聚醣酶F1之S-100 層析......45

圖  4.17  Penicillium janthinellum D4幾丁聚醣酶F1之CM 層析........................................................46

圖  4.18  P. janthinellum D4幾丁聚醣酶F2 SDS-PAGE之分子量分析........................................................47

圖  4.19  Penicillium janthinellum D4幾丁聚醣酶F1 SDS-PAGE之分子量分析................................................48

圖  4.20  溫度對P. janthinellum D4幾丁聚醣酶F2之影響......49

圖  4.21  酸鹼度對P. janthinellum D4幾丁聚醣酶F2之影響....50

圖  4.22  WSC經P. janthinellum D4粗酵素液F2水解不同時間所得總醣及還原醣含量..........................................51

圖  4.23  水溶性幾丁聚醣經P. janthinellum D4粗酵素液F2水解所得幾丁寡醣之MALDI-TOF-MS分析圖............................52


表目錄

表2.1   微生物來源之幾丁聚醣酶特性比較.........................................6

表3.1 DNS試劑組成...............................................................................15

表4.1  P. janthinellum D4幾丁聚醣酶F2純化總表............................56

表4.2  Penicillium janthinellum D4幾丁聚醣酶F1純化總表。...........57

表4.3  各種化學品對幾丁聚醣酶F2活性之影響...............................58

表 4.4  各種化學品對幾丁聚醣酶F2活性之影響..............................59

表  4.5   含硼化合物對P. janthinellum D4幾丁聚醣酶F2活性之抑制率..........................................................................................................60

表 4.6   含硼化合物001對P. janthinellum D4幾丁聚醣酶活性之抑制率..........................................................................................................62

表 4.7 P. janthinellum D4生產酵素之較適條件...................................63

表 4.8水溶性幾丁聚醣經Penicillium janthinellum D4粗酵素液水解不同時間之MALDI-TOF-MS幾丁寡醣組成分析..............................64,65

第六章   參考文獻

Akiyama K, Fujita T, Kuroshima KI, Sakane T, Yokota A, Takata R. 1999. Purification and gene cloning of a chitosanase from Bacillus ehimensis EAG1. Journal of Bioscience and Broengineering, 87:383-385.

Alfonos C,Martines M,Reyes F.1992. Purification and properties of two endochitosanases from Mucor rouxii implicated in its cell wall degradation. FEMS Microbiology Letters,95:187-194.

Bahar B, O,Doherty JV,Maher S, McMorrow J,Sweeney T. 2012. Chitoologosaccharide elicits acute inflammatory cytokine response through AP-1 pathway in human intestinal epithelial-like (Caco-2) cells. Molecular Immunology, 51:283-291.

Cai J,Yang J,Du Y,Fan L,Qiu Y,Li J, Kennedy JF. 2006. Purification and characterization of chitin deacetylase from Scopulariopsis brevicaulis. Carbohydrate Polymers, 65:211-217.

Cheng C,Li Y.2000. An Aspergillus chitosanase with potential for large-scale preparation of chitosan oligosaccharides .Biotechnology and Applied Biochemistry,32:197-203

Chen CT, Huang CJ, Wang YH, Chen CY. 2004. Two-step purification of Bacillus circulans chitinase A1 expressed in Escherichia coli periplasm. Protein Expression and Purification, 37:27-31.

Chen X, Xia W, Yu X. 2005. Purification and characterization of two types of chitosanase from Aspergillus sp. CJ22-326. Food Research International, 38:315-322.

Chiang CL,Chang CT, Sung HY. 2003. Purification and properties of chitosanase from a mutant of Bacillus subtilis IMR-NK1. Enzyme and Microbial Technology, 32:260-267.

Choi EH, Yang HP, Chun HS. 2012. Chitooligosaccharide ameliorates diet-induced obesity in mice and affects adipose gene expression involved in adipogenesis and inflammation. Nutrition Research, 32:218-228.

Day RB, Shibuya N, Minami E. 2003. Identification and characterization of two new members of the GRAS gene family in rice responsive to N-acetylchitooligosaccharide elicitor. Biochimica et Biophysica Acta, 1625:261-268.

Fenton D,Eveleigh D.1981. Purification and mode of action of a chitosanase from Penicillium islandicum. Journal of General Microbiology,126:151-165.

Fuguet R, Theraud M, Vey A. 2004. Production in vitro of toxic macromolecules by strains of Beauveria bassiana, and purification of a chitosanase-like protein secreted by a melanizing isolate. Comparative Biochemistry and Physiology, Part C, 138:149-161.

Gao XA, Ju WT, Jung WJ,Park RD. 2008. Purification and characterization of chitosanase from Bacillus cereus D-11. Carbohydrate Polymers, 72:513-520.

Genta FA, Blanes L,Cristofoletti PT, Lago CLD, Terra WR,Ferreira C. 2006. Purification, characterization and molecular cloning of the major chitinase from Tenebrio molitor larval midgut. Insect Biochemistry and Molecular Biology, 36:789-800.

Guo RF, Shi BS, Li DC, Ma W, Wei Q. 2008. Purification and Characterization of a novel thermostable chitinase from Thermomyces lanuginosus SY2 and cloning of its encoding gene. Agricultural Sciences in China, 7(12):1458-1465.

Ikeda M, Miyauchi K, Mochizuki A, Matsumiya M. 2009. Purification and characterization of chitinase from the stomach of silver croaker Pennahia argentatus. Protein Expression and Purification, 65:214-222.

Janek T, Lukaszewicz 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.

Jiang X, Chen D, Chen L, Yang G, Zou S. 2012. Purification, characterization, and action mode of a chitosanase from Streptomyces roseolus induced by chitin. Carbohydrate Research, 355:40-44.

Jung WJ Kuk JH, Kim KY, Jung KC, Park RD. 2006. Purification and characterization of exo-β-D-glucosaminidase from Aspergillus fumigates S-26. Protein Expression and Purification, 45:125-131.

Jung WJ,Souleimanov A, Park RD, Smith DL. 2007.Enzymatic production of N-acetylchitooligosaccharides by crude enzyme derived from Paenibacillus illioisensis KJA-424. Carbohydrate Polymers, 67:256-259.

Kageyama C,Kato K,Iyozumi H, Inagaki H, Yamaguchi A, Furuse K,Baba K.2006. Photon emission from rice cells elicited by N-acetylchitooligosaccharide are generated through phospholipid signaling in close association with the production of reactive oxygen species. Plant Physiology and Biochemistry, 44:901-909.

Khor E,Lim LY. 2003. Implantable applications of chitin and chitosan. Biomaterials, 24:2339-2349.

Kim EK, Je JY, Lee SJ, Kim YS, Hwang JW, Sung SH, Moon SH,Jeon BT, Kim SK, Jeon YJ, Park PJ. 2012. Chitooligosaccharides induce apoptosis in human myeloid leukemia HL-60 cells. Bioorganic ＆ Medicinal Chemistry Letters, 22:6136-6138.

Kim JY, Lee JK, Lee TS, Park WH. 2003. Synthesis of chitooligosaccharide derivative with quaternary ammonium group and its antimicrobial activity against Streptococcus mutans. International Journal of Biological Macromolecules, 32:23-27.

Kumar MNVR. 2000. A review of chitin and chitosan applications. Reactive ＆ Funtional Polymers, 46:1-27.

Kuroiwa T, Noguchi Y, Nakajima M, Sato S, Mukataka S, Ichikawa S. 2008. Production of chitosan oligosaccharides using chitosanase immobilized on amylase-coated magnetic nanoparticles. Process Biochemistry, 43:62-69.

Lee YG,Chung KC, Wi SG, Lee JC, Bae HJ. 2009. Purification and properties of a chitinase from Penicillium sp. LYG 0704. Protein Expression and Purification, 65:244-250.

Liang TW, Hsieh JL, Wang SL. 2012. Production and purification of a protease, a chitosanase, and chitin oligosaccharides by Bacillus cereus TKU022 fermentation. Carbohydrate Research, 362:38-46.

Li B,Zhang J, Dai F,Xia W. 2012. Purification of chitosan by using sol-gel immobilized pepsin deproteinization. Carbohydrate Polymers, 88:206-212.

Li S, Wang C, Xia W. 2008. Expression, purification and characterization of endo-type chitosanase of Aspergillus sp. CJ22-326 from Escherichia coli. Carbohydrate Research, 343:3001-3004.

Li S, Wang C, Xia W. 2009. Expression, purification, and characterization of exo-β-D-glucosaminidase of Aspergillus sp. CJ22-326 from Escherichia coil. Carbohydrate Research, 344:1046-1049.

Liu BL, Kao PM, Tzeng YM, Feng KC. 2003. Production of chitinase from Verticillium lecanii F091 using submerged fermentation. Enzyme and Microbial Technology, 33:410-415.

Liu J, Xia W. 2006. Purification and characterization of a bifunctional enzyme with chitosanase and cellulose activity from commercial cellulose. Biochemical Engineering Journal, 30:82-87.

Mario FD, Rapan`a P, Tomati U, Galli E. 2008. Chitin and chitosan from Basidiomycetes. International Journal of Biological Macromolecules, 43:8-12.

Ming M, Kuroiwa T,Ichikawa S, Seigo, Mukataka S. 2006. Production of chitosan oligosaccharides by chitosanase directly immobilized on an agar gel-coated multidisc impeller. Biochemical Engineering Journal, 28:289-294.

Ning W, Chen F, Mao B, Liu QLZ, Guo Z, He Z. 2004. N-acetylchitooligosaccharides elicit rice defense responses including hypersensitive response-like cell death, oxidative burst and denfence gene expression. Physiological and Molecular Plant Pathology, 64:263-271.

Nishimura N, Tanabe S, He DY, Yokota T, Shibuya N, Minami E. 2001. Recognition of N-acetylchitooligosaccharide elicitor by rice protoplasts. Plant Physiology Biochemistry, 39:1105-1110.

Nogawa M,Takahashi H,Kashiwagi A,Ohshima K,Okada H,Morikawa Y.1998. Purification and Characterization of Exo-β-D-Glucosaminidase from a Cellulolytic Fungus,Trichoderma reesei PC-3-7.Applied and Environmental Microbiology,64:890-895.

Okada M, Matsumura M, Shibuya N. 2001. Identification of a high-affinity binding protein for N-acetylchitooligosaccharide elicitor in the plasma membrane from rice leaf and root cells. Journal of Plant Physiology, 158:121-124.

Paulino AT, Simionato JI, Garcia JC, Nozaki J. 2006. Characterization of chitosan and chitin produced from silkworm chrysalides. Carbohydrate Polymers, 64:98-103.

Ratanavaraporn J, Kanokpanont S, Tabata Y, Damrongsakkul S. 2009. Growth and osteogenic differentiation of adipose-derived and bone marrow-derived stem cells on chitosan and chitooligosaccharide films. Carbohydrate Polymers, 78:873-878.

Rinaudo M. 2006. Chitin and chitosan: Properties and applications. Progress in Polymer Science, 31:603-632.

Sashiwa H, Fujishima S, Yamano N, Kawasaki N, Nakayama A,Muraki E,Sukwattanasinitt M, Pichyangkura R, Aiba SI. 2003. Enzymatic production of N-acetyl-D-glucosamine from chitin. Degradation study of N-acetylchitooligosaccharide and the effect of mixing of crude enzymes. Carbbohydrate Polymers, 51:391-395.

Shibuya N, Kaku H, Kuchitsu K, Maliarik MJ. 1993. Identification of a novel high-affinity binding site for N-acetylchitooligosaccharide elicitor in the membrane fraction from suspension-cultured rice cells. Federation of European Biochemical Societies, 329:75-78.

Somashekar D＆Joseph R. 1996. Chitosanases-properties and applications:A review. Bioresource Technology, 55:35-45.

Staufenberger T, Imhoff JF, Labes A. 2012. First crenarchaeal chitinase found in Sulfolobus tokodaii. Microbiological Research, 167:262-269.

Takai R, Hasegawa K, Kaku H, Shibuya N, Minami E. 2001. Isolation and analysis of expression mechanisms of a rice gene, EL5, which shows structural similarity to ATL family from Arabidopsis, in response to N-acetylchitooligosaccharide elicitor. Plant Science, 160:577-583.

Wang JP,Yoo JS, Kim HJ, Lee JH,Kim IH. 2009. Nutrient digestibility, blood profiles and fecal microbiota are influenced by chitooligosaccharide supplementation of growing pigs. Livestock Science, 125:298-303.

Wang J,Zhou W,Yuan H,Wang Y. 2008 a.Characterization of a novel fungal chitosanase Csn2 from Gongronella sp. JG. Carbohydrate Research,343:2583-2588.

Wang SL, Chen SJ, Wang CL. 2008 b. 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, Liuo JY, Liang TW, Liu KC. 2009. Conversion of squid pen by using Serratia sp. TKU020 fermentation for the production of enzymes antioxidant, and N-acetylchitooligosaccharides. Process Biochemistry, 44:854-861.

Wang SL, Peng JH,Liang TW, Liu KC. 2008 c. Purification and characterization of a chitosanase from Serratia marcescens TKU011. Carbohydrate Research, 343:1316-1323.

Wang SL, Yeh PY. 2008 d. Purification and characterization of a chitosanase from a nattokinase producing strain Bacillus subtilis TKU007. Process Biochemistry, 43:132-138.

Wang X, Ding X, Gopalakrishnan B, Morgan TD, Johnson L, White FF,Muthukrishnan S, Kramer KJ. 1996. Characterization of a 46 kDa insect chitinase from transgenic tobacco. Insect Biochemistry and Molecular Biology, 26:1055-1064.

Wang Z, Zheng L, Yang S, Niu R,Chu E, Lin X. 2007. N-acetylchitooligosaccharide is a potent angiogenic inhibitor both in vivo and in vitro. Biochemical and Biophysical Research Commnications, 357:26-31.

Yamamoto Y, Fukunaga Y, Aoyagi H, Tanaka H. 1995. Purification and characteristics of chitibase secreted by cultured Wasabia japonica cells. Journal of Fermentation and Bioengineering, 80:148-152. 

Zhang J, Kopparapu NK, Yan Q, Yang S, Jiang Z. 2013. Purification and characterization of a novel chitinase from persimmon (Diospyros kaki) with antifungal activity. Food Chemistry, 138:1225-1232.

Zhang X,Dai A,Zhang X,Kuroiwa K,Kodaira R,Shimosaka M,Okazaki M. 2000.Purification and characterization of chitosanase and exo-β-D-glucosaminidase from koji mold,Aspergillus oryzae IAM2660. Bioscience Biotechnology and Biochemistry,64:1896-1902.

Zhou W,Yuan H,Wang J,Yao J. 2008.Production,purification and characterization of chitosanase produced by Gongronella sp. JG.Letters in Applied Microbiology,46:49-54.