臺灣博碩士論文加值系統

Paenibacillus sp. isolate BL11 是由高溫高鹼的製漿漂前黑液中 (58℃, pH 9.83) 所分離出的菌種。本研究的黑液是由台灣紙業新營廠所收集而來，其成分包含大量的半纖維素、木質素、和部分細小纖維。經由活性測試，BL11具有很強的木聚醣酶活性，並利用16S rDNA 定序鑑定出與其他菌種的親緣關係。

由Paenibacillus sp. isolate BL11 所選殖(cloning)出的木聚醣酶基因(xylX)是由1,131個核苷酸所組成，經由轉錄(transcription)及轉譯(translation)可得到大小為41kDa的木聚醣酶。分析結果顯示出選殖得到的木聚醣酶其N端胺基酸序列具有一長度為39個胺基酸的信息胜肽(signal peptide)，和木聚醣酶的分泌有關。

BL11菌種本身木聚醣酶最佳反應條件為pH 7和60℃，活性為23 IU/mg。本研究為了方便木聚醣酶的純化，將選殖出來的木聚醣酶的C端胺基酸序列接上His-tag，並在0.1 mM IPTG和 28℃培養下，大量誘導。誘導出的木聚醣酶利用與Ni-NTA agarose 的親和力，加以純化。純化後的木聚醣酶最佳反應條件仍為pH 7和60℃，而活性可高達2392 IU/mg。在pH 11的條件下，木聚醣酶活性可達517 IU/mg。

Paenibacillus sp. BL11 was isolated by screening bacterial strains from a thermo-alkaline black liquor (58°C, pH 9.83). The black liquor was collected from Hsinying Paper Mill of Taiwan Pulp and Paper Company and contained large amounts of hemicellulose, lignin and small fiber fragments. Strain BL11 exhibited strong xylanase activity and its phylogenetic relationship with other bacteria was identified by 16S rDNA sequencing. The xylanase of strain BL11 has been cloned and analyzed.

The cloned BL11 xylanase is composed of 1,131 nucleotides and can encode a protein of 41 kDa. The N-terminal of the enzyme contains a deduced signal peptide of 39 amnio acids in length. Zymographic analyses of the BL11 xylan-degrading system revealed that cloned BL11 xylanase was secreted into the extracellular medium of the bacterium.

Optimum temperature and pH for crude BL11 xylanase activity were 60°C and pH 7, respectively. The activity was 23 IU/mg. The cloned BL11 xylanase was fused to a His-tag for purification by gene manipulation. The engineered BL11 xylanase was induced with 0.1 mM of IPTG at 28°C in an E. coli host. The induced xylanase was purified with Ni-NTA agarose by bio-affinity. The optimal temperature and pH for the purified BL11 xylanase were still the same of original ones. The purified BL11 xylanase activity was 2392 IU/mg under optimal condition. At pH 11, the activity was still as high as 517 IU/mg

謝誌…………………………………………………………... Ⅰ
摘要………………………………………………………….. Ⅱ
Abstract……………………………………………………… Ⅲ
Index…………………………………………………………. Ⅳ
Table index…………………………………………………… Ⅶ
Figure index………………………...………………………... Ⅷ
ⅠIntroduction…………………….…………………………… 1
ⅡLiterature reviews………...…………………………….. 4
1. Characterization and structure of xylan……………. 4
2. Xylanolytic enzymes………………………………………. 6
3. Xylanase producing microorganisms…………………… 9
3.1 Bacterial xylanases………...…………………….... 10
3.2 Fungal xylanases and associated problems………… 11
4. Applicationsof xylanase…………………………………. 13
ⅢObjective…………………………………………………….. 15
ⅣMaterials and methods……………………………………… 16
1. Screening and isolation of bacterial strains…... 16
2. Bacterial strain identification
2.1 Preparation of bacterial chromosomal DNA........ 16
2.2 DNA electrophoresis………....………………………. 18
2.3 PCR of 16S rDNA………………………………………. 18
2.4 TA-Cloning...................................... 19
2.5 Plasmid preparation (Mini and Midi amount)...... 22
2.6 Restriction enzyme map and sub-cloning.......... 24
2.7 DNA sequencing and Blast........................ 25
3. Polysaccharide hydrolase assay
3.1 Stain assay of polysaccharide hydrolases……….. 25
3.2 SDS-PAGE and zymography.…………………………... 27
3.3 DNSA (dinitrosalicylic acid) assay……………... 29
4. Molecular cloning of xylanase gene
4.1 Restriction enzyme digestion of DNA and recovery of chromosomal DNA....................................... 31
4.2 Restriction enzyme digestion, dephosphorylation and recovery of vector DNA….............................. 33
4.3 DNA ligation……………………………………..…... 33
4.4 Electroporation and transformation.............. 35
4.5 Replica and activity assay...................... 35
4.6 Mapping, Sequencing, and Blasting............... 36
5. Overexpression of cloned xylanase
5.1 Primer design and PCR program optimization…….. 36
5.2 Cloning of PCR product in pOptima vector and recovery of insert fragment………………………....….. 36
5.3 Preparation of overexpression vector……………… 37
5.4 Ligation, electroporation, transformation and activity assay....................................... 38
6. Purification of cloned xylanase
6.1 Condition test of induction…………............. 39
6.2 Pretest of purification condition…………....... 40
6.3 Purification and activity assay………………... 41
ⅤResults and discussion
1. Screening and isolation of bacterial strains….…… 43
2. Identification of isolated strain
2.1 Restriction enzyme map and sequences of BL11 16S rDNA………………………........……………………………. 44
2.2 Phylogenetic tree……………………………………… 45
3. Polysaccharide hydrolases assay
3.1 Polysaccharide hydrolases of BL11………….…. 47
3.2 SDS-PAGE and zymography of BL11 xylanase..……… 48
3.3 Growth curve of strain BL11………………………... 48
3.4 Crude xylanase activity of BL11…...…………... 49

4. Molecular cloning of xylanase gene
4.1 Restriction enzyme digestion and recovery of chromosomal DNA…………………………………..…. 51
4.2 Restriction enzyme digestion, dephosphorylation and recovery of vector DNA………………………………… 52
4.3 Ligation, transformation and clone selection.……53
4.4 Clone mapping, sequencing, and blasting…………. 55
4.5 Analyses of pBCX1-ORF3……………………………… 60
5. Overexpression of cloned xylanase (pBCX1-ORF3)
5.1 Primer set design of xylanase gene (xylX) and optimal PCR program................................... 63
5.2 TA cloning, restriction enzyme digestion, and fragment recovery………………………………………………. 64
5.3 Restriction enzyme digestion and fragment recovery of pET25b. ………………………………………………… 66
5.4 Ligation, electroporation, and transformation.. 66
6. Purification of xylanase (pETBX(NX))
6.1 Induced Condition test of pETBX(NX)...……….. 67
6.2 Imidazole elution test………….…………………… 69
6.3 Purification of induced xylanase (pETBX(NX)) and activity assay……………………………………………. 69
ⅥConclusion……………………………………………………. 73
Appendix…………………………………………………….. 81
ⅦReferences………………………………………………….… 88

Aaron, J., T. H. Oakley, A. T. Colin, and C. J. W. Matthew. (2003) Characterization of a family 11 xylanase from Bacillus Subtillis B230 used for paper bleaching. Acta Crystallographica. 59: 627-636.
Bailey, M. J., J. Buchert, and L. Viikari. (1993) Effect of pH on production of xylanase by Trichoderma reesei on xylan and cellulose-based media. Applied Microbiology and Biotechnology. 40: 224-233.
Bajpai, P. (1999) Application of enzymes in the pulp and paper industry. Biotechnology Progress. 15: 147-157.
Balakrishnan, H., M. C. Srinivasan, M. V. Rele, K. Chaundhari, and A. J. Chandwadkar. (2000) Effect of synthetic zeolites on xylanase production from an alkalophilic Bacillus sp. Current Science. 79: 95-99.
Bataillon, M., A. -P. N. Cardinali, N. Castillon, and F. Duchiron. (2000) Purification and characterization of a moderately thermostable xylanase from Bacillus sp. strain SPS-0. Enzyme and Microbial Technology. 26: 187-192.
Beg, O. K., B. Bhushan,M. Kapoor, and G. S. Hoondal. (2000a) Enhanced production of a thermostable xylanase from Streptomyces sp. QG-11-3 and application in biobleaching of eucalyptus kraft pulp. Enzyme and Microbial Technology. 27.
Beg, Q. K., B. Bhushan, M. Kapoor, and G. S. Hoondal. (2000b) Production and characterization of thermostable xylanase and pectinase from Streptomyces sp. QG-11-3. Journal of Industrial Microbiology and Biotechnology. 24: 396-400.
Beg, Q. K., M. Kapoor, L. Mahajan, and G. S. Hoondal. (2001) Microbial xylanases and their industrial applications: a review. Applied Microbiology and Biotechnology. 56: 326-338.
Bi, R., X. Sun, and S. Ren. (2000) The study on xylanase fermentation by Aspergillus niger sp. Industrial Microbiology. 30: 53-59.
Biely, P. (1985) Microbial xylanolytic system. Trends in Biotechnology. 3: 286-301.
Bim, M. A. and T. T. Franco. (2000) Extraction in aqueous two-phase systems of alkaline xylanase produced by Bacillus pumilus and its application in kraft pulp bleaching. Jouranl of Chromatography B. 743: 349-356.
Biswas, S. R., S. C. Jana, A. K. Mishra, and G. Nanda. (1989) Production, purification and characterization of xylanase from a hyperxylanolytic mutant of Aspergillus ochracens. Biotechnology and Bioengineering. 35: 244-251.
Blanco, A., P. Diaz, J. Martinez, O. Lopez, C. Soler, and F. I. J. Pastor. (1996) Cloning of a Bacillus sp. BP-23 gene encoding a xylanase with high activity against aryl xylosides. FEMS Microbiology Letters. 137: 285-290.
Blanco, A., P. Diaz, J. Martinez, T. Vidal, A. L. Torres, and F. I. J. Pastor. (1998) Cloning of a new endoglucanase gene from Bacillus sp. BP-23 and characterisation of the enzyme. Performance in paper manufacture from cereal straw. Applied Microbiology and Biotechnology. 50: 48-54.
Blanco, A., P. Diaz, J. Zueco, P. Parascandola, and F. I. J. Pastor. (1999) A multidomain xylanase from a Bacillus sp. with a region homologous to thermostabilizing domains of thermophilic enzymes. Microbiology. 145: 2163-2170.
Blanco, A., T. Vidal, J. F.Colom, and F. I. J. Pastor. (1995) Purification and properties of xylanase A from alkali-Tolerant Bacillus sp. Strain BP-23. Applied Microbiology and Biotechnology. Dec: 4468-4470.
Breccia, J. D., F. Siñeriz, M. D. Baigorí, G. R. Castro, and R. Hatti-Kaul. (1998) Purification and characterization of a thermostable xylanase from Bacillus amyloliquefaciens. Enzyme and Microbial Technology. 22: 42-49.
Christakopoulos, P., W. Nerinekx, D. Kekos, B. Macris, and M. Claeyssens. (1997) The alkaline xylanase III from Fusarium oxysporum F3 belongs to family F/10. Carbohydrate Research. 302: 191-195.
Chuang, S. E., A. L. Chen, and C. C. Chao. (1995) Growth of E. coli at low temperature dramatically increases the transformation frequency by electroporation. Nucleic acid Research. 23: 1641-1648.
Clarke, J. H., K. Davidson, J. E. Rixon, J. R. Halstead, M. P. Fransen, H. J. Gilbert, and G. P. Hazlewood. (2000) A comparsion of enzyme-aided bleaching of softwood paper pulp using combinations of xylanase, mannanase and α-galactosidase. Applied Microbiology and Biotechnology. 53: 661-667.
Cleemput, G., M. Hessing, M. van Oort, M. Deconynck, and J. Delcour. (1997) Purification and characterization of β-D-xylosidase and endo-xylanase from wheat flour. Plant Physiology. 113: 377-386.
Coughlan, M. P. and G. P. Hazlewood. (1993) β-1,4-D-xylan degrading enzyme systems, biochemistry, molecular biology and applications. Biotechnology and Applied Biochemistry. 17: 259-289.
Dahlberg, L., O. Holst, and J. Kristjansson. (1993) Thermostable xylanolytic enzymes from Rhodothermus marinus grown on xylan. Applied Microbiology and Biotechnology. 40: 63-68.
Dalbøge, H. and H. P. Heldt-Hansen. (1994) A novel method for efficient expression cloning of fungal enzyme genes. Molecular Genetics and Genomics. 243: 253-260.
Dean, J. F. D. and J. D. Anderson. (1991a) Ethylene biosynthesis inducing xylanase Ⅱ: Purification and physical characterization of the enzyme produced by Trichoderma viridae. Plant Physiology. 95: 316-324.
Dean, J. F. D., K. C. Gross, and J. D. Anderson. (1991b) Ethylene biosynthesis inducing xylanase Ⅲ: Product characterization. Plant Physiology. 96: 571-579.
Dervilly, G., C. Leclercq, D. Zimmerman, C. Roue, J.-F. Thibault, and L. Saulnier. (2002) Isolation and characterization of high molecular mass water-soluble arabinoxylans from barley malt. Carbohydrate Polymers. 47: 143-149.
Dervilly, G., J.-F. Thibault, and L. Saulnier. (2001) Experimintal evidence for a semi-flexible conformation for arabinoxylans. Carbohydrate Research. 330: 365-372.
Dey, D., J. Hinge, A. Shendye, and M. Rao. (1992) Purification and properties of extracellular endoxylanases from alkalophilic thermophilic Bacillus sp. Canadian Jouranl of Microbiology. 38: 436-442.
Dhillon, A., J. K. Gupta, and S. Khanna. (2000a) Enhanced production, purification and characterisation of a novel cellulase-poor thermostable, alkalitolerant xylanase from Bacillus circulans AB 16. Process Biochemistry. 35: 849-856.
Dhillon, A., J. K. Gupta, B. M. Jauhari, and S. Khanna. (2000b) 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.
Dobberstein, J. and C. C. Emeis. (1989) β-xylanase produced by Aureobasidium pullulans CBS 58475. Applied Microbiology and Biotechnology. 32: 262-268.

Dower, W. J., J. F. Miller, and C. W. Ragsdale. (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic acid Research. 16: 6123-6145.
Duarte, M. C. T., E. P. Portugal, A. N. Ponezi, M. A. Bim, C. V. Tagliari, and T. T. Franco. (1999) Production and purififation of alkaline xylanases. Bioresource Technology. 68: 49-53.
Elegir, G., G. Szakács, and T. W. Jeffries. (1994) Purification, characterization and substrate specificities of multiple xylanases from Streptomyces sp. strain B-12-2. Applied and Environmental Microbiology. 60: 2609-2615.
Esteban, R., I. R. Villanueva, and T. G. Villa. (1982) β-D-Xylanases of Bacillus circulans WL-12. Canadian Jouranl of Microbiology. 28: 733-741.
Fuchs, Y., A. Saxena, H. R. Gamble, and J. D. Anderson (1989) Ethylene biosynthesis inducing protein from cellulysin in an endoxylanase. Plant Physiology. 89: 138-145.
Gallardo, O., P. Diaz, and F. I. J. Pastor. (2003) Characterization of a Paenibacillus cell-associated xylanase with high activity on aryl-xylosides: a new subclass of family 10 xylanases. Applied Microbiology and Biotechnology. 61: 226-233.
Gallardo, O., P. Diaz, and F. I. J. Pastor. (2004) Cloning and Characterization of Xylanase A from the Strain Bacillus sp. BP-7:Comparison with Alkaline pI-Low Molecular Weight Xylanases of Family 11. Current Microbiology. 48: 276-279.
Gessesse, A. (1998a) Purification ot two thermostable alkaline xylanases from an alkaliphilic Bacillus sp. Applied and Environmental Microbiology. 64: 3533-3535.
Gessesse, A. and G. Mamo. (1998b) Purification and characterization of an alkaline xylanase from alkaliphilic Micrococcus sp. AR-135. Journal of Industrial Microbiology and Biotechnology. 20: 210-214.
Gomes, I., J. Gomes, W. Steiner, and H. Esterbauer. (1992) Production of cellulase and xylanase by a wild strain of Trichoderma viride. Applied Microbiology and Biotechnology. 36: 701-709.
Gomes, J., H. Purkarthofer, M. Hayn, J. Sinner, and W. Steiner. (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.
Gomes, J., I. Gomes, K. Terler, N. Gubala, G. Ditzelmuller, and W. Steiner. (2000) Optimisation of culture medium and condition for α-L-arabinofuranosidase production by the extreme thermophilic eubacterium Rhodothermus marinus. Enzyme and Microbial Technology. 27: 414-422.
Grabski, A. C., and T. W. Jeffries. (1991) Production, purification and characterization of β-(1-4)-Endoxylanase of Streptomyces roseiscleroticus. Applied and Environmental Microbiology. 57: 987-991.
Gupta, N., V. S. Reddy, S. Maiti, and A. Ghosh. (2000) Cloning, expression, and sequence analysis of the gene encoding the alkali-stable, thermostable endoxylanase from alkalophilic, mesophilic Bacillus sp. strain NG-27. Applied and Environmental Microbiology. June: 2631-2635.
Henrissat, B. (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochemistry Journal. 280: 309-316.
Henrissat, B., and A. Bairoch. (1993) New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochemistry Journal. 293: 781-788.
Honda, H., T. Kudo, Y. Ikura, and K. Horikoshi. (1985) Two types of xylanases of alkalophilic Bacillus sp NC-125. Canadian Jouranl of Microbiology. 31: 538-542.
Hreggvidsson, G. O., E. Kaiste, O. Holst, G. Eggertsson, A. Palsdottir, and J. K. Kristjansson. (1996) An extremely thermostable cellulase from the thermophilic eubacterium Rhodothermus marinus. Applied and Environmental Microbiology. 62: 3047-3049.
Iverson, T. and S. Wannstrom. (1986) Lignin-carbohydrate bonds in a residual lignin isolated from pine kraft pulp. Holzforschung. 40: 19-28.
Jeffries, T. W. (1996) Biochemistry and genetics of microbial xylanases. Current Oppinion in Biotechnology. 7: 337-342.
Jeong, K. J., P. C. Lee, I. Y. Park, M. S. Kim, and S. C. Kim. (1998) Molecular cloning and charaterization of an endoxylanase gene of Bacillus sp. in Escherichia coli. Enzyme and Microbial Technology. 22: 599-605.
Jimenez, L., C. Martinze, I. Perez, and F. Lopez. (1997) Biobleaching procedures for pulp and agri-cultural residues using Phanerochaete chrysosporium and enzymes. Process Biochemistry. 4: 297-304.
Kandeler, E. and K. E. Böhm. (1996) Temporal dynamics of microbial biomass, xylanase activity, N-mineralization and potential nitrification in different tillage system. Applied Soil Ecology. 8: 181-191.
Kandeler, E., J. Luxhaei, D. Tscherko, and J. Magid. (1999a) Xylanase, invertase and protease activities at the soil litter interface of a sandy loam. Soil Biology and Biochemistry. 31: 1171-1179.
Kandeler, E., M. Stemmer, and E. M. Klimanek. (1999b) Response of soil microbial biomass, urease and xylanase within particle size fractions to long term soil management. Soil Biology and Biochemistry. 31: 2161-2173.
Karlsson, O., B. Pettersson, and U. Westermark. (2001) Linkages between residual lignin and carbohydrates in bisulphite (manefite) pulps. Journal of Pulp and Paper Science. 27: 310-315.
Khasin, A., I. Alchanati, and Y. Shoham. (1993) Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6. Applied and Environmental Microbiology. 59: 1725-1730.
Kulkarni, N., A. Shendue, and M. Rao. (1999) Molecular and biotechnological aspects of xylanases. FEMS Microbiology Reviews. 23: 411-456.
Kulkarni, N. J. C., and M. Rao. (1995) Characterization of the recombinant xylanases in Escherichia coli from an alkaliphilic thermophilic Bacillus sp. NCIM 59. Enzyme and Microbial Technology. 17: 972-976.
La-Grange, D. C., M. Claeyssens, I. S. Pretorius, and W. H. van-Zyl. (2000) Coexpression of the Bacillus pumilus beta-xylosidase (xynB) gene with the Trichoderma reesei beta-xylanase-2 (xyn2) gene in the yeast Saccharomyces cerevisiae. Applied Microbiology and Biotechnology. 54: 195-200.
Lappalainen, A. (1986) Purification and characterization of xylanolytic enzymes from Trichoderma reesei. Biotechnology and Applied Biochemistry. 8: 437-448.
Lebeda, A., L. Luhova, M. Sedla, and D. Jan (2001) The role of enzymes in plant-fungal pathogens interactions. Zeitschrift fur Pflanzenkrankheiten und Pflanzenschutz. 108: 89-95.
Lin, J., L. M. Ndlovu, S. Singh, and B. Pillay. (1999) Purification and biochemical characteristics of β-D-xylanase from a thermophilic fungus, Thermomyces languginosus-SSBP. Biotechnology and Applied Biochemistry. 30: 73-79.
Lopéz-Fernández, C. L., J. Rodríguez, A. S. Ball, J. L. C. Patiño, M. I. P. Leblic, and M. E. Arias. (1998) Application of the affinity binding of xylanases to oat spelt xylan in the purification of endoxylanase CM 2 from Streptomyces chattanoogensis CECT 3336. Applied Microbiology and Biotechnology. 50: 284-287.
Lotan, T. and R. Fluhr. (1990) Xylanase, a novel elictior of pathogensis releated proteins in tobacco, uses a non-ethylene pathway for induction. Plant Physiology. 93: 811-821.
Lundgren, K. R., L. Bergkvist, S. Hogman, H. Joves, G. Eriksson, T. Bartfai, J. V. D. Laan, E. Rosenberg, and Y. Shoham. (1994) TCF Mill Trail on softwood pulp with Korsnas thermostable and alkaline stable xylanases T6. FEMS Microbiology Reviews. 13: 365-371.
Luthi, E., N. B. Jasmat, and P. L. Bergquist. (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.
Lymar, E. S., B. Li, and V. Renganathan. (1995) Purification and characterization of cellulose-binding β-glucosidase from cellulose-degrading cultures of Phanerochaete chrysosporium. Applied and Environmental Microbiology. 61: 2976-2980.
Madlala, A. M., S. Bissoon, S. Singh, and L. Christove (2001) Xylanase induced reduction of chlorine dioxide consumption during elemental choline-free bleaching of different pulp types. Biotechnology Letters. 23: 345-353.
Maheswari, M. U., and T. S. Chandra. (2000) Production and potential applications of a xylanase from a new strain of Streptomyces roseiscleroticus. World Journal of Microbiology and Biotechnology. 16: 257-263.
Mathrani, I. M. and B. K. Ahring. (1992) Thermophilic and alkalophilic xylanases from several Diclyoglomus isolates. FEMS Microbiology Letters. 38: 23-31.
Mishra, C., I. T. Forrester, B. D. Kelley, R. R. Burgess, and G. F. Leatham. (1990) Characterization of a major xylanase purified from Lentinula edodes cultures grown on a commerical solid lignocellulosic substrate. Applied Microbiology and Biotechnology. 36: 226-232.


Morales, P., A. Madarro, and J. A. Perez-Gonzalez. (1993) Purification and characterization of alkaline xylanase from Bacillus polymyxa. Applied and Environmental Microbiology. 59: 1376-1382.
Morales, P. J., M. Sendra, and J. A. Perez-Gonzalez. (1995) Purification and characterization of an arabinofuranosidase from Bacillus polymyxa expressed in Bacillus subtilis. Applied Microbiology and Biotechnology. 44: 112-117.
Nakamura, S., K. Wakabayashi, R. Nakai, R. Aono, and K. Horikoshi. (1993) Purification and some prpperties of an alkaline xylanase from alkaliphilic Bacillus sp. strain 41M-1. Applied and Environmental Microbiology. 59: 2311-2316.
Nakamura, S., Y. Ishiguro, R. Nakai, K. Wakabayashi, R. Aono, and K. Horikoshi. (1995) Purification and characterization of a thermophilic alkaline xylanase from thermoalkaliphilic Bacillus sp. strain TAR-1. Jouranl of Chromatography B. 1: 7-15.
Onysko, K. A. (1993) Biological bleaching of chemical pulps: a review. Biotechnology Advances. 11: 179-198.
Pastor, F. I. J., X. Pujol, A. Blanco, T. Vidal, A. L. Torres, and P. Diaz. (2001) Molecular cloning and characterization of a multidomain endoglucanase from Paenibacillus sp BP-23:evaluation of its performance in pulp refining. Applied Microbiology and Biotechnology. 55: 61-68.
Pereira, P. S., H. Paveia, M. C. Ferreira, and M. R. A. Barros. (2003) A New Look at Xylanases. Molecular Biotechnology. 24: 257-281.
Pereira, P. S., J. Duarte, and M. C. Ferreira. (2000) Electroelution as a simple and fast protein purification method:isolation of an extracellular xylanase from Bacillus sp. CCMI 966. Enzyme and Microbial Technology. 27: 95-99.
Prade, R. A. (1995) Xylanases: from biology to biotechnology. Biotechnology Genetic Engineering Reviews. 13: 101-131.
Puls, J. (1997) Chemistry and biochemistry of hemicelluloses: relationship between hemicellulose structure and enzymes required for hydrolysis. Macromolecular Symposia. 120: 183-196.
Ratanakhanokchai, K., K. L. Kyu, and M. Tanticharoen. (1999) Purification and properties of a xylan-binding endoxylanase from alkaliphilic Bacillus sp. strain K-1. Applied and Environmental Microbiology. 65: 694-697.
Ratto, M., K. Poutanen, and L. Viikari. (1992) Production of xylanolytic enzymes by an alkalitolerant Bacillus circulans strain. Applied Microbiology and Biotechnology. 37: 470-475.
Rincón, M. T., S. I. McCrae, J. Kirby, K. P. Scott, and H. J. Flint. (2001) EndB, a multidomain family 44 cellulase from Ruminococcus flavefaciens 17, binds to cellulose via a novel cellulose-binding module and to another protein via a dockerin domain. Applied and Environmental Biotechnology. 67: 4426-4431.
Ritschkoff, A.-C., J. Buchert, and L. Viikari. (1994) Purification and characterization of a thermophylic xylanase from the brown-rot fungus Gloeophyllum trabeum. Jouranl of Biotechnology. 32: 67-74.
Sá-Pereira, P., J. Duarte, and M. Costa-Ferreira. (2000) Electroelution as a simple and fast protein purification method, isolation of an extracellular xylanase from Bacillus sp. CCMI 966. Enzyme and Microbial Technology. 27: 95-99.
Saha, B. C. (2001) Xylanase from a newly isolated Fusarium verticillioides capable of utilizing corn fiber xylan. Applied Microbiology and Biotechnology. 56: 762-766.
Sales, B. C., R. B. Cunha, W. Fontes, M. V. Sousa, and E. X. F. Filho. (2000) Purification and characterization of a new xylanase from Acrophialophora nainiana. Jouranl of Biotechnology. 81: 199-204.
Sardar, M., I. Roy, and M. N. Gupta. (2000) Simultaneous purification and immobilization of Aspergillus niger xylanase on the reversible soluble polymer Eudragit TM L-100. Enzyme and Microbial Technology. 27: 672-679.
Shallom, D. and Y. Shoham. (2003) Microbial hemicellulases. Current Oppinion in Microbiology. 6: 219-228.
Shoham, Y., Z. Schwartz, A. Khasin, O. Gat, Z. Zosim, and E. Rosenberg (1992) Delignification of wood pulp by a thermostable xylanase from Bacillus stearothermophilus strain T-6. Biodegradation. 3: 207-218.
Silveria, F. Q. P., M. V. Sousa, C. A. O. Ricart, A. M. F. Milagres, C. L. Medeiros, and E. X. F. Filho. (1999) A new xylanase from Trichoderma harzianum strain. Journal of Industrial Microbiology and Biotechnology. 23: 682-685.
Singh, S., B. Pillay, V. Dilsook, and B. A. Prior. (2000) Production and properties of hemicellulases by a Thermomyces lanuginosus strain. Jouranl of Applied Micribiology. 88: 975-982.
Steiner, W., R. M. Lafferty, I. Gomes, and H. Esterbauer. (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.
Stemmer, S., M. Gerzabek, and E. Kandeler. (1999) Invertase and xylanase activity of bulk soil and particle size fraction during maize straw decomposition. Soil Biology and Biochemistry. 31: 9-18.
Subramaniyan, S. and P. Prema. (2000) Cellulase-free xylanases from Bacillus and other microorganisms. FEMS Microbiology Letters. 183: 1-7.
Subramaniyan, S. and P. Prema. (2001) Control of xylanase production with out protease activity in Bacillus sp. by the selected nitrogen source. Biotechnology Letters. 23: 369-371.
Subramaniyan, S. and P. Prema. (2002) Biotechnology of microbial xylanases: enzymology, molecular biology, and application. Critical Reviews in Biotechnology. 22: 33-64.
Sung, W. L., C. K. Luk, D. M. Zahab, and W. Wakarchuk. (1993) Overexpression of the Bacillus subtilis and circulans xylanases in Escherichia coli. Protein Expression and Purification. 4: 200-206.
Sunna, A., J. Puls, and G. Antranikian. (1996) Purification and characterization of two thermostable endo-β-xylanases from Thermotoga thermarum. Biotechnology and Applied Biochemistry. 24: 177-185.
Teleman, A., M. Tenkanen, A. Jacobs, and O. Dahlman. (2002) Characterization of ο-acetyl-(4-ο-methylglucurono) xylan isolated from birch and beech. Carbohydrate Researchs. 337: 373-377.
Tseng, M. J., M. N. Yap, K. Ratanakhanokchai, K. L. Kyu, and S. T. Chen. (2002) Purification and characterization of two cellulase free xylanases from an alkaliphilic Bacillus firmus. Enzyme and Microbial Technology. 30: 590-595.
Viikari, L., A. Kantelinen, J. Sundquist, and M. Linko. (1994) Xylanase in bleaching. From an idea to industry. FEMS Microbiology Reviews. 13: 335-350.
Wong, K. K. Y., L. U. L. Tan, and J. N. Saddler. (1988) Multiplicity of B-1, 4-xylanase in microorganisms: Functions and applications. Microbiology Reviews. 52: 305-327.
Wood, P. J., J. D. Erfle, and R. M. Teather. (1988a) Use of complex formation between congo red and polysaccharides in detection and assay of polysaccharide hydrolases. Methods in Enzymology. 160: 59-74.
Wood, T. M. and K. M. Bhat. (1988b) Methods for measuring cellulase activities. Methods in Enzymology. 160: 87-112.
Yamura, I., T. Koga, T. Matsumoto, and T. Kato. (1997) Purification and some properties of Endo-1,4-β-D-xylanase from a fresh water molusc, Pomacea insularus. Bioscience Biotechnology and Biochemistry. 61: 615-620.
Yang, V. W., Z. Zhuang, G. Elegir, and T. W. Jeffries. (1995) Alkaline-active xylanase produced by an alkaliphilic Bacillus sp isolated from kraft pulp. Journal of Industrial Microbiology. 15: 434-441.
Zilliox, C. and P. Debeire. (1998) Hydrolysis of wheat straw by a thermostable endoxylanase. Adsorption and kinetic studies. Enzyme and Microbial Technology. 22: 58-63.