Abstract
Xylanase A of Thermotoga neapolitana contains binding domains both at the N- and C-terminal ends of the catalytic domain. In the N-terminal position it contains two carbohydrate-binding modules (CBM) which belong to family 22. These CBMs bind xylan but not to cellulose. The gene encoding the mature peptide of these CBMs was fused with an alkaline active GH10 xylanase from Bacillus halodurans S7 and expressed in Escherichia coli. The (His)6 tagged hybrid protein was purified by immobilized metal affinity chromatography and characterized. Xylan binding by the chimeric protein was influenced by NaCl concentration and pH of the binding medium. Binding increased with increasing salt concentration up to 200 mM. Higher extent of binding was observed under acidic conditions. The fusion of the CBM structures enhanced the hydrolytic efficiency of the xylanase against insoluble xylan, but decreased the stability of the enzyme. The optimum temperature and pH for the activity of the xylanase did not change.
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Altschhul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Bolam DN, Ciruela A, McQueen-Mason S, Simpson P, Williamson MP, Rixon JE, Hazlewood GP, Gilbert HJ (1998) Pseudomonas cellulose-binding domains mediate their effects by increasing enzyme substrate proximity. Biochem J 331:775–781
Boraston AB, Warren ARJ, Kilburn DG (2001) β-1,3-Glucan binding by a thermostable carbohydrate-binding module from Thermotoga maritima. Biochemistry 40:14679–14685
Collins T, Gerday C, Feller G (2005) Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol Rev 29:3–23
Coutinho PM, Henrissat B (1999) Carbohydrate-active enzymes: an integrated database approach. In: Gilbert HJ, Davies GJ, Henrissat B, Svensson B (eds) Recent advances in carbohydrate bioengineering. The Royal Society of Chemistry, Cambridge, pp 3–12
Creagh AL, Ong E, Jervis E, Kilburn DG, Haynes CA (1996) Binding of the cellulose-binding domain of exoglucanase Cex from Cellulomonas fimi to insoluble microcrystalline cellulose is entropically driven. Proc Natl Acad Sci USA 93:12229–12234
Dias FMV, Goyal A, Gilbert HJ, Prates JAM, Ferreira LMA, Fontes CMGA (2004) The N-terminal family 22 carbohydrate-binding module of xylanase 10B of Clostridium themocellum is not a thermostabilizing domain. FEMS Microbiol Lett 238:71–78
Din N, Gilkes NR, Tekant B, Miller J, Robert C, Warren RAJ, Kilburn DG (1991) Non-hydrolytic disruption of cellulose fibres by the binding domain of a bacterial cellulase. Bio/Technol 9:1096–1099
Feng JX, Karita S, Fujino E, Fujino T, Kimura T, Sakka K, Ohmiya K (2000) Cloning, sequencing, and expression of the gene encoding a cell-bound multi-domain xylanase from Clostridium josui, and characterization of the translated product. Biosci Biotechnol Biochem 64:2614–2624
Fernandes AC, Fontes CMGA, Gilbert HJ, Hazlewood GP, Fernandes TH, Ferreira LMA (1999) Homologus xylanases from Clostridium thermocellum: evidence for bi-functional activity, synergism between xylanase catalytic modules and the presence of xylan-binding domains in enzyme complexes. Biochem J 342:105–110
Gibbs MD, Reeves RA, Farrington GK, Anderson P, Williams DP, Bergquist PL (2000) Multidomain and multifunctional glycosyl hydrolases from the extreme thermophile Caldicellulosiruptor isolate Tok7B.1. Curr Microbiol 40:333–340
Grant WD, Mwatha E, Jones BE (1990) Alkaliphiles: ecology, diversity and applications. FEMS Microbiol Rev 75:255–270
Hachem MA, Karlsson EN, Bartonek-Roxå E, Raghothama S, Simpson PJ, Gilbert HJ, Williamson MP, Holst O (2000) Carbohydrate-binding modules from a thermostable Rhodothermus marinus xylanase: cloning, expression and binding studies. Biochem J 345:53–60
Harhangi HR, Freelove ACJ, Ubhayasekera W, van Dinther M, Steenbakkers PJM, Akhmanova A, van der Drift C, Jetten MSM, Mowbray SL, Gilbert HJ, den Camp H (2003) Cel6A, a major exoglucanase from the cellulosome of the anaerobic fungi Piromyces sp E2 and Piromyces equi. Biochim Biophys Acta-Gene Struct Expr 1628:30–39
Henrissat B, Davies GJ (2000) Glycoside hydrolases and glycosyltransferases. Families, modules, and implications for genomics. Plant Physiol 124:1515–1519
Hilden L, Johansson G (2004) Recent developments on cellulases and carbohydrate-binding modules with cellulose affinity. Biotechnol Lett 26:1683–1693
Irwin D, Jung ED, Wilson DB (1994) Characterization and sequence of a Thermomonospora fusca xylanase. Appl Environ Microbiol 60:763–770
Ito Y, Tomita T, Roy N, Nakano A, Sugawara-Tomita N, Watanabe S, Okai N, Abe N, Kamio Y (2003) Cloning, expression, and cell surface localization of Paenibacillus sp. strain W-61 xylanase 5, a multidomain xylanase. Appl Environ Microbiol 69:6969–6978
Karita S, Sakka K, Ohmiya K (1996) Cellulose-binding domains confer an enhanced activity against insoluble cellulose to Ruminococcus albus endoglucanase IV. J Ferment Bioeng 81:555–556
Kataeva IA, Blum DL, Li X-L, Ljungdahl LG (2001) Do domain interactions of glycosyl hydrolases from Clostridium thermocellum contribute to protein thermostability? Protein Eng 14:167–172
Kittur FS, Mangala SL, Rusd AA, Kitaoka M, Tsujibo H, Hayashi K (2003) Fusion of family 2b carbohydrate-binding module increases the catalytic activity of a xylanase from Thermotoga maritima to soluble xylan. FEBS Lett 549:147–151
Kulkarni N, Shendye A, Rao M (1999) Molecular and biotechnological aspects of xylanases. FEMS Microbiol Rev 23:411–456
Mamo G, Hatti-Kaul R, Mattiasson B (2006a) A thermostable alkaline active endo-β-1-4-xylanase from Bacillus halodurans S7: purification and characterization. Enzyme Microb Technol (in press)
Mamo G, Delgado O, Martinez A, Mattiasson B, Hatti-Kaul R (2006b) Cloning, sequence analysis and expression of a gene encoding an endoxylanase from Bacillus halodurans S7. Mol Biotechnol 33:149–160
Mangala SL, Kittur FS, 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. J Mol Catal B: Enzym 21:221–230
Meissner K, Wassenberg D, Liebl W (2000) The thermostabilizing domain of the modular xylanase XynA of Thermotoga maritima represents a novel type of binding domain with affinity for soluble xylan and mixed-linkage β-1,3/β-1,4-glucan. Mol Microbiol 36:898–912
Melack JM, Kilham P (1974) Photosynthetic rate of phytoplankton in East African alkaline saline lakes. Limnol Oceanogr 19:743–755
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428
Notenboom V, Boraston AB, Kilburn DG, Rose DR (2001) Crystal structures of the family 9 carbohydrate-binding module from Thermotoga maritima Xylanase 10A in native and ligand-bound forms. Biochemistry 40:6248–6256
Pei-Ji G, Guan-Jun C, Tian-Hong W, Ying-Shu Z, Jie L (2001) Non-hydrolytic disruption of crystalline structure of cellulose by cellulose binding domain and linker sequence of cellobiohydrolase I from Penicillium janthinellum. Acta Biochim Biophys Sin 33:13–18
Sakaguchi K, Kiyohara M, Watanabe N, Yamaguchi K, Ito M, Kawamura T, Tanaka I (2004) Preparation and preliminary X-ray analysis of the catalytic module of beta-1,3-xylanase from the marine bacterium Vibrio sp. AX-4. Acta Crystallogr D Biol Crystallogr 60:1470–1472
Sakka K, Takada G, Karita S, Ohmiya K (1996) Identification and characterization of cellulose-binding domains in xylanase A of Clostridium stercorarium. Ann NY Acad Sci 782:241–251
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor
Shin ES, Yang MJ, Hwa Jung K, Kwon EJ, Jung JS, Park SK, Kim J, Yun HD, Kim H (2002) Influence of the transposition of the thermostabilizing domain of Clostridium thermocellum xylanase (XynX) on xylan binding and thermostabilization. Appl Environ Microbiol 68:3496–3501
Simpson PJ, Jamieson SJ, Hachem MA, Karlsson EN, Gilbert HJ, Holst O, Williamson MP (2002) The solution structure of the CBM4-2 carbohydrate binding module from a thermostable Rhodothermus marinus xylanase. Biochemistry 41:5712–5719
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85
Sun JL, Sakka K, Karita S, Kimura T, Ohmiya K (1998) Adsorption of Clostridium stercorarium xylanase A to insoluble xylan and the importance of the CBDs to xylan hydrolysis. J Ferment Bioeng 85:63–68
Sunna A, Antranikian G (1997) Xylanolytic enzymes from fungi and bacteria. Crit Rev Biotechnol 17:39–67
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence weighing, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 76:4350–4354
Tomme P, Warren RAJ, Gilkes NR (1995) Cellulose hydrolysis by bacteria and fungi. Adv Microb Physiol 37:1–81
van Solingen P, Meijer D, van der Kleij WAH, Barnett C, Bolle R, Power SD, Jones BE (2001) Cloning and expression of an endocellulase gene from a novel streptomycete isolated from an East African soda lake. Extremophiles 5:333–341
Wassenberg D, Schurig H, Liebl W, Jaenicke R (1997) Xylanase XynA from the hyperthermophilic bacterium Thermotoga maritima: structure and stability of the recombinant enzyme and its isolated cellulose-binding domain. Protein Sci 6:1718–1726
Winterhalter C, Heinrich P, Candussio A, Wich G, Liebl W (1995) Identification of a novel cellulose-binding domain within the multidomain 120 kDa xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima. Mol Microbiol 15:431–444
Zverlov V, Piotukh K, Dakhova O, Velikodvorskaya G, Borriss R (1996) The multidomain xylanase A of the hyperthermophilic bacterium Thermotoga neapolitana is extremely thermoresistant. Appl Microbiol Biotechnol 45:245–247
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This study was supported by The Swedish Agency for International Development Co-operation (Sida/SAREC).
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Communicated by G. Antranikian.
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Mamo, G., Hatti-Kaul, R. & Mattiasson, B. Fusion of carbohydrate binding modules from Thermotoga neapolitana with a family 10 xylanase from Bacillus halodurans S7. Extremophiles 11, 169–177 (2007). https://doi.org/10.1007/s00792-006-0023-4
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DOI: https://doi.org/10.1007/s00792-006-0023-4