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研究生:古藍婷
研究生(外文):Lan-Ting Ku
論文名稱:Paenibacillus campinasensis BL11 木聚醣酶之特性與突變分析
論文名稱(外文):Characterization and mutational analysis of a xylanase from Paenibacillus campinasensis BL11
指導教授:柯淳涵柯淳涵引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:森林環境暨資源學研究所
學門:農業科學學門
學類:林業學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:63
中文關鍵詞:木聚醣&木聚醣&木聚醣&木聚醣&木聚醣&木聚醣&木聚醣&木聚醣&
外文關鍵詞:xylanasemutantpH stabilitytemperature stabilitysubstrate specificityadditivesbinding
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比較野生型木聚醣酶 pre-xylX 與兩株變異株(分別是胺基酸取代木聚醣酶 H2 (T44A) 及後段缺失木聚醣酶 L2(胺基酸序列 290 之後鹼基缺失)) 之特性。3 個木聚醣酶在 60oC 經過 8小時後在 pH 5 到 9 仍具有良好活性,另外在 pH 7 耐溫 8 小時試驗中,40oC 到 60oC 均具有良好的活性。在高溫 70oC 下,野生型 pre-xylX 和 突變株 H2 在 2 小時後仍具有 50% 的活性,而突變株 L2 的活性則不到 40%。3 個木聚醣酶對基質具有明顯的專一性,可降解 birchwood 及 oat spelt 木聚醣,但對 CMC, avicel, laminarin, β-D-Glucan, or D-(+)-cellobiose 則沒有良好的降解效果。添加物 Hg2+ 及 N-Bromosuccinimide 對 3 個木聚醣酶具有很強的抑制活性。某些金屬離子及 EDTA 對則對木聚醣酶有部分的抑制活性,其抑制的順序大小為 N- Bromosuccinimide > Hg2+ > Cu2+ > Fe3+ > Zn2+ > Fe2+ > SDS > Pb2+。野生型 pre-xylX、突變株 H2 及 突變株 L2 的 Km 值分別為 6.78 ± 0.59, 3.87 ± 0.39 及 19.23 ± 3.62 mg/mL,而 Vmax 值分別為 4953 ± 73, 11688.02 ± 504.74, 6642.27 ± 961.22 μmol/min mg。野生型 pre-xylX 和突變株 H2 具有吸附能力 (對木聚醣的相對吸附可達80%),而突變株 L2 則吸附效果不佳 (對木聚醣相對吸附量約 20%)。活性最佳的突變株 H2,能釋出較多還原糖,但吸附效果與還原糖釋出量沒有顯著的關係。另外在飽和低溫吸附試驗中,野生型 pre-xylX 對 3 種紙漿的吸附相似,對原始漿、氧漂漿及全漂漿的最大吸附量分別為115.6、115.9、及115.1 mg/g substrate。但野生型 pre-xylX 對三種漿的親和力大小則為原始漿 (51.8 L/g) > 氧漂漿 (49.3 L/g) > 全漂漿 (43.1 L/g)。在紙漿脫色試驗中,3 種木聚醣酶對釋出發色基團 (OD237 nm and OD465 nm ) 效果則相近。

Wild type pre-xylX and mutant H2 (amino acid substitution mutant, T44A) and mutant L2 (amino acid sequence deletion mutant after 270) were studied. The pH stability of three xylanases was fully stable from pH5 to 9 after 8 h of incubation. The thermal stability of three xylanases was fully stable from 40 oC to 60 oC after 8 h incubation. At 70oC after 2 h, wild type pre-xylX and mutant H2 retained about 50% of its initial activity while mutant L2 retained lower than 40%. Substrate specificity of three xylanases showed that the xylanases degraded xylans from birchwood and oat spelt, but no delectable activity on CMC, avicel, laminarin, β-D-Glucan, or D-(+)-cellobiose. Additives of Hg2+ and N- Bromosuccinimide strongly inhibited three xylanases activities. Partial inhibition was observed in the some metal ions, and EDTA. Inhibition of the xylanases was in the order of N- Bromosuccinimide > Hg2+ > Cu2+ > Fe3+ > Zn2+ > Fe2+ > Pb2+. On the other hand, 2-ME strongly promoted the xylanases activities, as over 100% residual activity. The Km value of wild type pre-xylX, mutant H2, and mutant L2 were 6.78 ± 0.59, 3.87 ± 0.39, and 19.23 ± 3.62 mg/mL, respectively while Vmax value were 4953 ± 73, 11688.02 ± 504.74, 6642.27 ± 961.22 μmol/min mg, respectively. Relative bound of mutant H2 and wild type pre-xylX were about 80% to insoluble xylan, whereas mutant L2 had no delectable binding ability (relative bound about 20%). Mutant H2 showed the highest activity among three xylanases, and it could release more reducing sugar than the others. There was not specific relationship between binding ability and hydrolysis. In enzyme adsorption study under 4oC, wild type pre-xylX showed similar adsorption ability among three different pulps. Maximum adsorption capacity onto control pulp, oxygen bleached pulp, and fully bleached pulp were 115.6, 115.9, and 115.1 mg/g substrate, respectively. But affinity of wild type pre-xylX was observed in the order of control pulp (51.8 L/g) > oxygen bleached pulp (49.3 L/g) > fully bleached pulp (43.1 L/g). Wild type pre-xylX, mutant H2 and mutant L2 had similar abilities in releasing chromophores (OD237 nm and OD465 nm).

Index
Page
摘要……………………………………………………………. i
Abstract…………………………………………………….... ii
Index…………………………………………………………… iv
Table index………………………………………………....... vi
Figure index………………………………………………..... vii
I Introduction………………………………………………….... 1
II Literature reviews…………………………………………… 4
2.1 Characterization and structure of xylan…………………… 4
2.2 Xylanse enzyme system…………………………………… 5
2.3 Xylanase producing microorganisms……………………... 6
2.3.1 Bacterial xylanases…………………………………...... 6
2.3.2 Fungal xylanases and associated problems……………. 8
2.4 Mutant of bacterial xylanase………………………………. 10
2.5 Paenibacillus sp. isolate BL11……………………………. 15
2.6 Mutants H2 and mutant L2………………………………. 17
2.7 Xylanase-pretreatment…………………………………….. 22
III Materials and methods………………………………………. 24
3.1 Bacterial strains and materials…………………………… 24
3.2 Fermentation production and purification of the xylanases... 24
3.3 Effect of pH and temperature on the xylanases stabilities…. 25
3.4 Substrate of specificity…………………………………....... 25
3.5 Effect of additives on the xylanases activities…………....... 26
3.6 Kinetic parameters……………………………………...... 26
3.7 Xylanases binding and adsorption study…………………… 27
3.7.1 The xylanases binding………...……………...…………. 27
3.7.2 The xylanases adsorption……...………………...……… 27
3.8 Color removal from a eucalypt pulp……………………….. 28
IV Results and discussion………………………………………. 29
4.1 Fermentation production and purification of the xylanases... 29
4.2 Effect of pH and temperature on the xylanses stabilities....... 32
4.3 Substrate specificity…………...…………………………… 35
4.4 Effect of additives on the xylanases activities...…………… 36
4.5 Kinetic parameters…..……………………………………... 38
4.6.1 The xylanases binding……………………………………. 42
4.6.2 The xylanases adsorption………………………………… 49
4.7 Color removal……………………………………………… 51
V Conclusion…………………………………………………….. 54
VI References………………………………………………….….. 56


Arase, A., Yomoa, T., Urabe, I., Hata, Y., Katsube, Y., Okada, H., 1993. Stabilization of xylanase by random mutagenesis. Federation of European Biochemical Societies Letters 316, 123 - 127.
Bailey, M. J., Buchert, J., Viikari, L., 1993. Effect of pH on production of xylanase by Trichoderma reesei on xylan and cellulose-based media. Applied Microbiology and Biotechnology 40, 223- 233.
Balakrishnan, H., Srinivasan, M. C., Rele, M. V., Chaundhari, K., Chandwadkar, A. J., 2000. Effect of synthetic zeolites on xylanase production from an alkalophilic Bacillus sp. Current Science 79, 95 - 99.
Balajrishnan, H., Satyanarayana, L., Gaikward, S. M., Suresh, C. G., 2006, Structural and active site modification studies implicate Glu, Trp, and Arg in the activity of xylanase from alkalophilic Bacillus sp. (NCL 87-6-10). Enzyme and microbial Technology 39, 67 - 73.
Beg, Q. K., Kapoor, M., Mahajan, L., Hoondal, G. S., 2001. Microbial xylanases and their industrial applications: a review. Applied Microbiology and Biotechnology 56, 326-38.
Biely, P., 1985. Microbial xylanolytic systems. Trends in Biotechnology 3, 286- 301.
Bi, R., Sun, X., Ren, S., 2000. The study on xylanase fermentation by Aspergillus niger sp. Industrial Microbiology 30, 53-59.
Boussaid, A., Saddler, J.N., 1999. Adsorption and activity profiles of cellulases during the hydrolysis of two Douglas fir pulps. Enzyme Microbial Technology 24, 138-143.
Chang, P., Tsai, W. S., Tsia, C. L., Tseng, M. J., 2004. Cloning and characterization of two thermostable xylanases from an alkaliphilic Bacillus firmus. Biochemical and Biophysical Research Communications 319, 1017 - 1025.
Chauthaiwale, J., Rao, M., 1994. Chemical modification of xylanase from alkalothermophilic Bacillus species : evidence for essential carboxyl group. Biochimica et Biophysica Acta 1204, 164 - 168.
Chen, T. H., 2006. Improvement of thermostability of endo-1,4-β-xylanase by error-prone PCR. Institute of Biotechnology, Taiwan, National Tsing Hua University. Master Thesis.
Coates, J. D., Michaelidou, U., Bruce, R. A., O’Connor, S. M., Crespi, J. N., Achenbach, L. A., 1999. Ubiquity and diversity of dissimilatory (per)chlorate-reducing bacteria. Applied and Environment Microbiology 65, 5234 - 5241.
Collins, T., Gerday, C., Feller, G., 2005. Xylanases, xylanase families and extremophilic xylanases. Federation of European Microbiological Societies Microbiology Reviews 29, 3 - 23.
Coughlan, M. P., Hazlewood, G. P., 1993. β-1,4-D-xylan degrading enzyme systems, biochemistry, molecular biology and applications. Biotechnology and Applied Biochemistry 17, 259 - 289.
Dahlberg, L., Holst, O., Kristjansson, J., 1993. Thermostable xylanolytic enzymes from Rhodothermus marinus frown on xylan. Applied Microbiology and Biotechnology 40, 63 - 68.
Davoodi, J., Wakarchuk, W. W., Carey, P. R., Surewicz, W. K., 2007. Mechanism of stabilization of Bacillus circulans xylanase upon the introduction of disulfide bonds. Biophysical Chemistry 125, 453 - 461.
Deutscher, M. P., 1990. Maintaining protein stability. In : Deutscher, M. P., editor. Guild to protein purification. Methods in Enzymology 182, 83 - 89.
Dhillon, A., Gupta, J. K., Jauhari, B. M., Khanna, S., 2000. A cellulase-poor, thermostable, alkalitolerant xylanase produced by Bacillus circulans AB 16 grown on rice straw and its application in biobleaching of eucalyptus pulp. Bioresource Technology 73, 273 - 277.
Dumon, C. A., Varvak, M. A., Wall, J. E., Flint, R. J., Lewis, J. H., Lakey, C., Morland, P., Luginbuhl, S., Healey, T., Todaro, G., Desantis, M., Sun, L., Parra-Gessert, X. Q., Tan, D., Weiner, P., Gilbert, H. J., 2008. Engineering hyperthermostability into a GH11 xylanase is mediated by subtle changes to protein structure. Journal of Biological Chemistry 283, 22557 - 22564.
Ferreira, L. M. A., Durrant, A. J., Hall, J., Hazlewood, G. P., Gilbert, H. J., 1990. Spatial separation of protein domains is not necessary for catalytic activity orsubstrate binding in a xylanase. Biochemical Journal 269, 261- 264.
Georis, J., Giannotta, F., Buyl, E. D., Granier, B., Frere, J. M., 2000. Purification and properties of three endo-β-1,4-xylanases produced by Streptomyces sp. strain S38 which differ in their ability to enhance the bleaching of Kraft pulps. Enzyme and Microbial Technology 26, 178 - 186.
Gerber, P.J., Heitmann, J.A., Joyce, T.W., Buchert, J., Siika-aho, M., 1999. Adsorption of hemicellulases onto bleached Kraft fibers. Journal of Biotechnology 67, 67 - 75.
Gomes, I., Gomes, W., Steiner, W., Esterbauer, H., 1992. Production of cellulase and xylanase by a wild strain of Trichoderma viride. Applied Microbiology and Biotechnology 36, 701 - 709.
Gomes, J., Purkarthofer, H., Hayn, M., Sinner, J., Steiner, W., 1993. Production of a high level of cellulase-free xylanase by the thermophilic fungus Thermomyces lanuginosus in labatory and pilot scales using lignocellulosic materials. Applied Microbiology and Biotechnology 39, 700 - 713.
Grabski, A. C., Jeffries, T. W., 1991. Production, purification and characterization of β-(1-4)-Endoxylanase of Streptomyces roseiscleroticus. Applied and Environmental Microbiology 57, 987 - 991.
Gupta, S., Bhushan, B., Hoondal, G. S., 2000. Isolation, purification and characterization of xylanase from Staphylococcus sp. SG - 13 and its application in biobleaching of kraft pulp. Journal of Applied Microbiology 88, 325 - 334.
Haarhoff, J., Moes, C. J., Cerff, C., Wyk, W. J. V., Gerischer, G., Janse, B. J. H., 1999. Characterization and biobleaching effect of hemicellulases produced by thermophilic fungi. Biotechnology Letters 21, 415 - 420.
Hreggvidsson, G. O., Kaiste, E., Holst, O., Eggertsson, G., Palsdottir, A., Kristjansson, J. K., 1996. An extremely thermostable cellulase from the themophilic eubacterium Rhodothermus marinus. Applied and Environmental Micobiology 62, 3047 - 3049.
Hwang, I. T., Lim, H. K., Song, H. Y., Cho, S. J., Chang, J. S., Park, N. J., 2010. Cloning and characterization of a xylanase, KRICT PX1 from the strain Paenibacillus sp. HPL-001. Biotechnology Advances doi : 10.1016/j.biotec-
hadv.2010.05.007.
Irwin, D., Jung, E. D., Wilson, D. B., 1994. Characterization and sequence of a Thermomonospora fusca xylanase. Applied and Environmental Microbiology 60, 763 - 770.
Ihsanawati, T., Kumasaka, T., Kaneko, C., Morokuma, R., Yatsunami, T., Sato, S., Tanaka, N., 2005. Structure basis of the substite and the highly thermal stability of xylanase 10B from Thermotoga maritime MSB8. Proteins Structure Function and Bioinformatics 61, 999 - 1009.
Jeong, M. Y., Kim, S., Yun, C. W., Choi, Y. J., Cho, S. G., 2007. Engineering a de novo internal disulfide bridge to improve the thermal stability of xylanase from Bacillus stearothermophilus No. 236. Journal of Biotechnology 127, 300 - 309.
Joo, J. C., Pohkrel, S., Pack, S. P., Yoo, Y. J., 2010. Thermostabilization of Bacillus circulans xylanase via computational design of a flexible surface cavity. Journal of Biotechnology 146, 31 - 39.
Ker, Q. U., 2009. Coned and overexpression of Paenibacillus sp. isolate BL11 xylanase and mutants. Department of Forestry and Resource Conservation, Taiwan, National Taiwan University. Master Thesis.
Khasin, A., Alchanati, I., Shoham, Y., 1993. Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6. Applied and Environmental Microbiology 59, 1725 - 1730.
Khandeparkar, R., Bhosle, N. B., 2007. Application of thermoalkalophilic xylanase from Arthrobacter sp. MTCC 5214 in biobleaching of kraft pulp. Bioresource Technology 98: 899 - 891.
Kim, H., S., Pokhrel, S., Yoo, Y. J., 2008. Mutation of non-conserved amino acids surrounding catalytic site to shift pH optimum of Bacillus circulans xylanase. Journal of Molecular Catalysis B: Enzymatic 55, 30 - 136.
Ko, C. H., Chen, W. L., Tsai, C. H., Jane, W. N., Liu, C. C., Tu, J., 2007. Paenibacillus campinasensis BL11 : A wood material-utilizing bacterial stain isolated from black liquor. Bioresource Technology 98, 2727 -2733.
Kumar, R., Wyman, C.E., 2009. Cellulase adsorption and relationship to features of corn stover solids produced by leading pretreatments. Biotechnology and Bioengineer 103, 252 - 67.
Kuno, A., Shimizu, D., Kaneko, S., Hasegawa, T., Gama, Y., Hayashi, K., Kusakabe, I., Taira, K., 1999. Significant enhancement in the binding of p-nitrophenyl-β-D-xylobioside by the E128H mutant F/10 xylanase from Streptomyces olivaceoviridis E-86. Federation of European Biochemical Societies Letters 450, 299 - 305.
Kulkarni, N. J. C., Shebdue, A., Rao, M., 1999. Molecular and biotechnological aspects of xylanases. Federation of European Microbiological Societies Microbiology Reviews 23, 411 - 456.
Liu, X., Qu, Y., You, F., Liu, Y., 2002. Studies on the key amino acid residues responsible for the alkali-tolerance of the xylanase by site-directed or random mutagenesis. Journal of Molecular Catalysis B: Enzymatic 18, 307 - 313.
Luthi, E., Jasmat, N. B., Bergquist, P. L., 1990. Xylanase from the extremely thermophilic bacterium Caldocellum saccharolyticum : overexpression of the gene in Escherichia coli and characterization of the gene product. Applied and Environmental Microbiology 36, 2677 - 2681.
Lundgren, K. R., Bergkvist L., Hogman, S., Joves, H., Eriksson, G., Bartfai, T., Laan, J. V. D., Rosenberg, E., Shoham, Y., 1994. TCF Mill Trial on softwood pulp with Korsnas thermostable and alkaline stable xylanases T6. Federation of European Microbiological Societies Microbiology Reviews 13, 365 - 371.
MacLeod, A. M., Tull, D., Rupitz, K., Warren, R. A. J., Wither, S. G., 1996. Mechanistic consequences of mutation of active site carboxylates in a retaining beta-1,4-glycanase from Cellulomonas fini. Biochemistry 35, 13165 - 13172.
Maheswari, M. U., Chandra, T. S., 2000. Production and potential applications of a xylanase from a new strain of Streptomyces roseiscleroticus. World Journal of Microbiology and Biotechnology 16, 257 - 263.
Mamo, G., Hatti-Kaul, R., Mattiasson, B., 2006. A thermostable alkaline active endo-β-1-4-xylanase from Bacillus halodurans S7 : Purification and characterization. Enzyme and Microbial Technology 39, 1492 - 1498.
Mamo, G., Thunnissen, M., Hatti-Kaul, R., Mattiasson, B., 2009. An alkaline active xylanase: Insights into mechanisms of high pH catalytic adaptation. Biochimie 91, 1187 - 1196.
Mangala, S. L., Kittur, F. S., Nishimoto, M., Sakka, K., Ohmiya, K., Kitaoka, M., Hayashi, K., 2003. Fusion of family VI cellulose binding domains to Bacillus halodurans xylanase increases its catalytic activity and substrate-binding capacity to insoluble xylan. Journal of Molecular Catalysis B : Enzymatic 21, 221 - 230.
Mathrani, I. M., Ahring, B. K., 1992. Thermophilic and alkalophilic xylanases from several Diclyoglomus isolates. Federation of European Microbiological Societies Microbiology Letters 38, 23 - 31.
Miller, G. L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Annual Chemistry 31, 426 - 428.
Nagarathnamma, R., Bajpai, P., 1999. Decolorization and detoxification of extraction-stage effluent from chlorine bleaching of kraft pulp by Rhizopusoryzae. Applied and Environment Microbiology 65, 1078 - 1082.
Patel, R. N., Grabski, A. C., Jeffries, T. W., 1993. Chromophore release from kraft pulp by purified streptomyces roseiscleroticus xylanases. Applied Microbiology and Biotechnology 39, 405 - 412.
Pokhrel, D., Viraraghavan, T., 2004. Treatment of pulp and paper mill waste water - a review. Science of the Total Environment 333, 37 - 58.
Ryu, K., Kim, Y., 1998. Adsorption of a xylanase purified from Pulpzyme HC onto alkali-lignin and crystalline cellulose. Biotechnology Letters 20, 987 - 990.
Sá-Pereira, P., Mesquita, A., Duarte, J., Barros, M. R. A., Costa-Ferreira, M., 2002. Rapid production of thermostable cellulase-free xylanase by a strain of Bacillus subtilis and its properties. Enzyme and Microbial Technology 30, 924 - 933.
Shoham, Y., Schwartz, Z., Khasin, A., Gat, O., Zosim, Z., Rosenberg, E., 1992. Delignification of wood pulp by a thermostable xylanase from Bacillus stearothermophilus strain T - 6. Biodegradation 3, 207 - 218.
Singh, S., Pillay, B., Dilsook, V., Prior, B. A., 2000. Production and properties of hemicellulase by a Thermomyces lanuginosus strain. Journal of Applied Microbiology 88, 975 - 982.
Srinivasan, M. C., Rele, M. V., 1999. Microbial xylanases for pulp and paper industry. Current Science 77, 137 - 142.
Steiner, W., Lafferty, R. M., Gomes, I., Esterbauer, H., 1987. Studies on a wild strain of Schizophyllum commune : cellulase and xylanase production and formation of the extracellular polysaccharide Schizophyllan. Biotechnology and Bioengineering 30, 169 - 179.
Stepanova, L. I., Lindstrom-Seppa, P., Hanninen, O. O. P., Kotelevtsev, S. V., Glaser, V. M., Novikov, C. N., 2000. Lake Baikal: biomonitoring of pulp and paper mill waste water. Aquatic Ecosystem Health and Management 3, 259 - 269.
Subramaniyan, S., Prema, P., 2000. Cellulase free xylanases from Bacillus and other microorganisms. Federation of European Microbiological Societies Microbiology Letters 183, 1 - 7.
Techapun, C., Poosaran, N., Watanabe, M., Sasaki, K., 2003. Thermostable and alkaline-tolerant microbial cellulase-free xylanases produced from agricultural wastes and the properties required for use in pulp bleaching bioprocesses: a review. Process Biochemistry 38, 1327-1340.
Tenkanen, M., Buchert, J., Viikari, L., 1995. Binding of hemicellulases on isolated polysaccharide substrates. Enzyme Microbial Technology 17, 499 – 505.
Tsai, C. H., 2005. Molecular cloning, overexpression and purification of Paenibacillus sp. isolate BL11 xylanase from black liquor. Department of Forestry and Resource Conservation, Taiwan, National Taiwan University. Master Thesis.
Turkiewicz, M., Kalinowska, H., Zielinska, M., Bielecki, S., 2000. Purification and characterization of two endo-1,4-β-xylanases from Antarctic Krill, Euphausia superb Dana. Comp. Biochemistry and Physiology 127, Part B, 325 - 335.
Vicuna, R., Escobar, F., Osses, M., Jara, A., 1997. Bleaching of eucalyptus kraft pulp with commercial xylanases. Biotechnology Letters 19, 575 - 578.
Viikari, I., Kantelinen, A., Sundquist, J., Linko, M., 1994. Xylanases in bleaching : from an idea to the industry. Federation of European Microbiological Societies Microbiology Review 13, 335 - 350.
Ximenes, F. A., Sousa, M. V., Puls, J., Silva, F. G., Filho, E. X. F., 1999. Purification and characterization of a low-molecular-weight xylanase produced by Acrophialophora nainiana. Current Microbiology 38, 18 - 21.
Yang, J. H., Park, J. Y., Kim, S. H., Yoo, Y. J., 2008. Shifting pH optimum of Bacillus circulans xylanase based on molecular modeling. Journal of Biotechnology 133, 294 - 300.


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