Abstract
A genomic library of the extremely thermophilic eubacterial strain Rt8B.4 was constructed in λZapII and screened for the expression of xylanase activity. One recombinant bacteriophage showed xylanase, xylosidase and arabinosidase activity. Sequence analysis and homology comparisons showed that this plasmid derivative, pNZ2011, was composed of 6.7 kb thermophilic DNA and contained what appeared to be an operon-like structure involving genes associated with xylose metabolism. The xylanase gene, xynA was shown to code for a multi-domain protein. Xylanase activity was shown to be associated with the carboxy-terminal domain (domain 2) by deletion analysis and also by selezctive polymerase chain reaction (PCR) amplification and expression of the individual domains. Denaturing polyacrylamide gel analysis of the protein encoded by the PCR product showed three main overexpressed proteins to be present in cell extracts, presumably caused by proteolytic degradation in the Escherichia coli host. The xylanase activity from domain 2 is associated with a 36-kDa protein, which is stable at 70°C for at least 12 h at pH 7. The small size of this active enzymatic domain and its temperature stability suggest that it may be of value in the enzyme-enhanced bleaching of kraft pulp.
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Aduse Opoku J, Tao L, Ferretti JJ, Russell RR (1991) Biochemical and genetic analysis of Streptococcus mutans α-galactosidase. J Gen Microbiol 137: 757–764
Appleyard RK (1958) Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from Escherichia coli K12. Genetics 39: 440–452
Bahl H, Burchhardt G, Wienecke A (1991) Nucleotide sequence of two Clostridium thermosulfurogenes EM1 genes homologous to Escherichia coli genes encoding integral membrane components of binding protein-dependent transport systems. FEMS Microbiol Lett 81: 83–88
Bajpai P, Bajpai PK (1992) Biobleaching of kraft pulp. Process Biochem 27: 319–325
Bergquist PL, Gibbs MD, Saul DJ, Te’o VSJ, Dwivedi PP, Morris DM, Donald A. Karle A (1993a) Cloned enzymes from thermophilic bacteria that degrade xylan and mannan. In: Proceedings of the Seventh International Conference on Wood and Paper Chemistry, Beijing. Vol 2: pp 596–604
Bergquist PL, Gibbs MD, Saul DJ, Te’o VSJ, Dwivedi PP, Morris D (1993b) Molecular genetics of thermophilic bacterial genes coding for enzymes involved in cellulose and hemicellulose degradation. In: Shimada K, Hoshino S, Ohimiya K, Sakka K, Kobayashi Y, Karita S (eds) Genetics, biodiversity and ecology in biodegradation of lignocellulose. pp 276–285. Uni Publishers, Co, Tokyo
Biely P (1985) Microbial xylanolytic systems. Trends Biotechnol 3: 286–290
Biely P, Kluepfel D, Morosoli R, Shareck F (1993) Mode of action of three endo-β-l,4-xylanases of Streptomyces lividans. Biochim Biophys Acta 1162: 246–254
Bullock WO, Fernandez JM, Short JM (1987) XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with β-galactosidase selection. Biotechniques 5: 376–379
Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12: 387–395
Donald KAG, Karle A, Gibbs MD, Bergquist PL (1994) Production of a bacterial thermophilic xylanase in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 42: 309–312
Gilkes NR, Henrissat B, Kilburn DG, Miller RC, Narren RAJ (1991) Domains in Microbial β-l,4-glycaneses: sequence conservation, function and enzyme families. Microbiol Revs 55: 303–315
Grépinet O, Chebrou M, Béguin P (1988) Purification of Clostridium thermocellum xylanase Z expressed in Escherichia coli and identification of the corresponding product in the culture medium of C. thermocellum. J Bacteriol 170: 4576–4581
Huynh TV, Young RA, Davis RA (1985) Constructing and screening cDNA libraries in λgt10 and λgt11. In: Glover D (ed) DNA cloning. pp 56–110. Oxford University Press, Oxford
Kreuzer P, Gaertner D, Allmansberger R, Hillen W (1989) Identification and sequence analysis of the Bacillus subtilis W23 xylR gene and xyl operator. J Bacteriol 171: 3840–3845
Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680–685
Lever M (1973) Colorimetric and fluorimetric carbohydrate determination with p-hydroxybenzoic acid hydrazide. Biochem Med 7: 274–281
Lüthi E, Bhana Jasmat N, Bergquist PL (1990a) Xylanase from the extremely thermophilic bacterium “Caldocellum saccharolyticum”: Overexpression of the gene in Escherichia coli and characterisation of the gene product. Appl Environ Microbiol 56: 2677–2683
Lüthi E, Love DR, McAnulty J, Wallace C, Caughey PA, Saul DJ, Bergquist PL (1990b) Cloning, sequence analysis, and expression of genes encoding xylan-degrading enzymes from the thermophile “Caldocellum saccharolyticum”. Appl Environ Microbiol 56: 1017–1024
Perlman D, Halvorson HO (1983) A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides. J Mol Biol 167: 391–409
Rainey FA, Donnison AM, Janssen PH, Saul DJ, Rodrigo AG, Bergquist PL, Daniel RM, Stackebrandt E, Morgan HW (1994) Description of Caldicellulosiruptor saccharolyticus gen. nov. sp. nov: an obligately anaerobic, extremely thermophilic, cellulolytic bacterium. FEMS Microbiol Lett 120: 263–266
Saake B, Clarke T, Puls J (1993) Fundamental investigations on the reaction of xylanases and mannanases on Sprucewood chemical pulps. In: Proceedings of the Seventh International Symposium on Wood Pulping Chemistry. Beijing, vol 2 pp 605–613
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY
Saul DJ, Williams LC, Grayling RA, Chamley LW, Love DR, Bergquist PL (1990) celB, a gene coding for a bifunctional cellulase from the extreme thermophile “Caldocellum saccharolyticuni”. Appl Environ Microbiol 56: 3117–3124
Saul DJ, Rodrigo AG, Reeves RA, Williams LC, Borges KM, Bergquist PL (1993) Phylogeny of twenty Thermus isolates constructed from 16s rRNA gene sequence data. Int J System Bacteriol 43: 754–760
Schauder B, Blöcher H, Frank R, McCarthy JEG (1987) Inducible expression vectors incorporating the Escherichia coli atp E translational initiation region. Gene 52: 279–283
Short JM, Fernandez JM, Sarge JA, Huse WD (1988) λZap: a bacteriophage λ expression vector with in vivo excision properties. Nucleic Acids Res 16: 7583–7600
Sissons CH, Sharrock KR, Daniel RM, Morgan HW (1987) Isolation of cellulolytic anaerobic extreme thermophiles from New Zealand thermal sites. Appl Environ Microbiol 53: 832–838
Swofford DL (1993) PAUP: Phylogenetic Analysis Using Parsimony, Version 3.1.1, Smithsonian Institute, Washington, D.C.
Teather RM, Wood PJ (1982) Use of Congo red polysaccharide interaction in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol 143: 777–780
Viikari L, Kantelinen A, Buchert J, Puls J (1994) Enzymatic accessibility of xylans in lignocellulosic materials. Appl Microbiol Biotechnol 41: 124–129
Von Heijne G (1983) Pattern of amino acids near signal sequence cleavage sites. Eur J Biochem 133: 17–21
Watson MEE (1984) Compilation of published signal sequences. Nucleic Acids Res 12: 5145–5164
Yannish-Perron C, Viera J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33: 103–199
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Dwivedi, P.P., Gibbs, M.D., Saul, D.J. et al. Cloning, sequencing and overexpression in Escherichia coli of a xylanase gene, xynA from the thermophilic bacterium Rt8B.4 genus Caldicellulosiruptor . Appl Microbiol Biotechnol 45, 86–93 (1996). https://doi.org/10.1007/s002530050653
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DOI: https://doi.org/10.1007/s002530050653