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Purification and characterisation of two extremely halotolerant xylanases from a novel halophilic bacterium

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Abstract

The present work reports for the first time the purification and characterisation of two extremely halotolerant endo-xylanases from a novel halophilic bacterium, strain CL8. Purification of the two xylanases, Xyl 1 and 2, was achieved by anion exchange and hydrophobic interaction chromatography. The enzymes had relative molecular masses of 43 kDa and 62 kDa and pI of 5.0 and 3.4 respectively. Stimulation of activity by Ca2+, Mn2+, Mg2+, Ba2+, Li2+, NaN3 and isopropanol was observed. The K m and V max values determined for Xyl 1 with 4-O-methyl-d-glucuronoxylan are 5 mg/ml and 125,000 nkat/mg respectively. The corresponding values for Xyl 2 were 1 mg/ml and 143,000 nkat/mg protein. Xylobiose and xylotriose were the major end products for both endoxylanases. The xylanases were stable at pH 4–11 showing pH optima around pH 6. Xyl 1 shows maximal activity at 60°C, Xyl 2 at 65°C (at 4 M NaCl). The xylanases showed high temperature stability with half-lives at 60°C of 97 min and 192 min respectively. Both xylanases showed optimal activity at 1 M NaCl, but substantial activity remained for both enzymes at 5 M NaCl.

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References

  • Bailey MJ, Biely P, Poutanen K (1992) Interlaboratory testing of methods for assay of xylanase activity. J Biotechnol 23:257–270

    CAS  Google Scholar 

  • Bataillon M, Cardinali APN, Castillon N, Duchiron F (2000) Purification and characterization of a moderately thermostable xylanase from Bacillus sp strain SPS-0. Enzyme Microb Technol 26:187–192

    Article  CAS  PubMed  Google Scholar 

  • Bedford MR, Classen HL (1992) The influence of dietary xylanase on intestinal viscosity and molecular weight distribution of carbohydrates in rye-fed broiler chicks. In: Visser J, Someren MAKV, Beldman G, Voragen AGJ (eds) Xylans and xylanases: proceedings of an International Symposium, Wageningen, The Netherlands, December 8–11 1991. Elsevier, Amsterdam. Prog Biotechnol 7:361–370

    CAS  Google Scholar 

  • Beg QK, Kapoor M, Mahajan L, Hoondal GS (2001) Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol 56:326–338

    Article  CAS  PubMed  Google Scholar 

  • Biely P (1985) Microbial xylanolytic systems. Trends Biotechnol 3:286–290

    CAS  Google Scholar 

  • Biely P, Vrsanska M, Tenkanen M, Kluepfel D (1997) Endo-β-1,4-xylanase families: differences in catalytic properties. J Biotechnol 57:151–166

    Article  CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Chivero ET, Mutukumira AN, Zvauya R (2001) Partial purification and characterisation of a xylanase enzyme produced by a micro-organism isolated from selected indigenous fruits of Zimbabwe. Food Chem 72:179–185

    Article  CAS  Google Scholar 

  • Coughlan MP, Hazlewood GP (1993) β-1,4-d-Xylan-degrading enzyme systems: biochemistry, molecular biology and applications. Biotechnol Appl Biochem 17:259–289

    CAS  PubMed  Google Scholar 

  • Elcock AH, McCammon JA (1998) Electrostatic contributions to the stability of halophilic proteins. J Mol Biol 280:731–748

    CAS  PubMed  Google Scholar 

  • Fushinobu S, Ito K, Konno M, Wakagi T, Matsuzawa H (1998) Crystallographic and mutational analyses of an extremely acidophilic and acid-stable xylanase: biased distribution of acidic residues and importance of Asp37 for catalysis at low pH. Protein Eng 11:1121–1128

    Article  CAS  PubMed  Google Scholar 

  • Greasham R, Inamine E (1986). Nutritional Improvement of processes. In: Demain AL, Solomon NA (eds) Manual of microbiology and biotechnology. American Society for Microbiology, Washington, D.C., pp 41–48

  • Gupta S, Bhushan B, Hoondal GS (2000) Isolation, purification and characterization of xylanase from Staphylococcus sp. SG-13 and its application in biobleaching of kraft pulp. J Appl Microbiol 88:325–334

    Article  CAS  PubMed  Google Scholar 

  • Henrissat B, Bairoch A (1993) New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 293:781–788

    CAS  PubMed  Google Scholar 

  • Honda H, Kudo T, Ikura Y, Horikoshi K (1985) Two types of xylanases of alkalophilic Bacillus sp. No. C-125. Can J Microbiol 31:538–542

    CAS  Google Scholar 

  • Ingvorsen K, Jørgensen BB (1984) Kinetics of sulfate uptake by freshwater and marine species of Desulfovibrio. Arch Microbiol 139:61–66

    CAS  Google Scholar 

  • Johnson KG, Lanthier PH, Gochnauer MB (1986) Studies of two strains of Actinopolyspora halophila, an extremely halophilic actinomycete. Arch Microbiol 143:370–378

    CAS  Google Scholar 

  • Karlsson EN, Dahlberg L, Torto N, Gorton L, Holst O (1998) Enzymatic specificity and hydrolysis pattern of the catalytic domain of the xylanase Xyn1 from Rhodothermus marinus. J Biotechnol 60:23–35

    PubMed  Google Scholar 

  • Kuhad RC, Singh A (1993) Lignocellulose biotechnology—current and future prospects. Crit Rev Biotechnol 13:151–172

    CAS  Google Scholar 

  • Laemmli U (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    PubMed  Google Scholar 

  • Leathers TD (1989) Purification and properties of xylanase from Aureabasidium. J Ind Microbiol 4:341–348

    CAS  Google Scholar 

  • Lopez C, Blanco A, Pastor FIJ (1998) Xylanase production by a new alkali-tolerant isolate of Bacillus. Biotechnol Lett 20:243–246

    Article  CAS  Google Scholar 

  • Maat J, Roza M, Verbakel J, Stam H, Silva MJS, Bosse M, Egmond MR, Hagemans MLD, Gorcom RFM, Hessing JGM, Hondel CAMJJ, Rotterdam C (1992) Xylanases and their application in bakery. In: Visser J, Someren MAKV, Beldman G, Voragen AGJ (eds) Xylans and xylanases: proceedings of an International Symposium, Wageningen, The Netherlands, December 8–11 1991. Elsevier, Amsterdam. Prog Biotechnol 7:349–360

    CAS  Google Scholar 

  • Madern D, Ebel C, Zaccai G (2000) Halophilic adaptation of enzymes. Extremophiles 4:91–98

    CAS  PubMed  Google Scholar 

  • Mevarech M, Frolow F, Gloss LM (2000) Halophilic enzymes: proteins with a grain of salt. Biophys Chem 86:155–164

    CAS  PubMed  Google Scholar 

  • Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428

    CAS  Google Scholar 

  • Minagawa H, Kaneko H, Shimada J (2002) Thermostabilization of enzymes for improvement of computer- aided protein engineering. NEC Res Dev 43:251–255

    CAS  Google Scholar 

  • Polosina YY, Zamyatkin DF, Kostyukova AS, Filimonov VV, Fedorov OV (2002) Stability of Natrialba magadii NDP kinase: comparisons with other halophilic proteins. Extremophiles 6:135–142

    Article  CAS  PubMed  Google Scholar 

  • Puls J, Borchmann A, Gottschalk D, Wiegel J (1988) Xylobiose and xylooligomers. Meth Enzymol 160:528–536

    CAS  Google Scholar 

  • Raj KC, Chandra TS (1996) Purification and characterization of xylanase from alkali- tolerant Aspergillus fischeri Fxn1. FEMS Microbiol Lett 145:457–461

    Article  CAS  PubMed  Google Scholar 

  • Richard SB, Madern D, Garcin E, Zaccai G (2000) Halophilic adaptation: novel solvent protein interactions observed in the 2.9 and 2.6 Å resolution structures of the wild type and a mutant of malate dehydrogenase from Haloarcula marismortui. Biochemistry 39:992–1000

    Article  CAS  PubMed  Google Scholar 

  • Ryu K, Kim J, Dordick JS (1994) Catalytic properties and potential of an extracellular protease from an extreme halophile. Enzyme Microb Technol 16:266–275

    CAS  PubMed  Google Scholar 

  • Saake B, Kruse T, Puls J (2001) Investigation on molar mass, solubility and enzymatic fragmentation of xylans by multi-detected SEC chromatography. Bioresour Technol 80:195–204

    Article  CAS  PubMed  Google Scholar 

  • Shavers CL, Parson ML, Deming SN (1979) Simplex optimization of chemical systems. J Chem Educ 56:307–309

    CAS  Google Scholar 

  • Silva C, da Fonseca AS, Neto SDL, Ximenes ED, Puls J, Ferreira EX (2000) Evaluation of hydrolysis products of xylans by xylan-degrading enzymes from Humicola grisea var. thermoidea and Aspergillus fumigatus Fresenius. World J Microbiol Biotechnol 16:81–83

    Article  Google Scholar 

  • Tisi LC, White PJ, Squirrell DJ, Murphy MJ, Lowe CR, Murray JAH (2002) Development of a thermostable firefly luciferase. Anal Chim Acta 457:115–123

    Article  CAS  Google Scholar 

  • Tull D, Withers SG (1994) Mechanisms of cellulases and xylanases—a detailed kinetic study of the exo-beta-1,4-glycanase from Cellulomonas fimi. Biochemistry 33:6363–6370

    CAS  PubMed  Google Scholar 

  • Wainø M, Ingvorsen K (1999) Production of halostable β-mannanase and β-mannosidase by strain NN, a new extremely halotolerant bacterium. Appl Microbiol Biotechnol 52:675–680

    Article  Google Scholar 

  • Wainø M, Ingvorsen K (2003) Production of halostable β-mannanase and β-mannosidase by the extremely halophilic archaeon Halorhabdus utahensis. Extremophiles (in press)

  • Wainø M, Tindall BJ, Schumann P, Ingvorsen K (1999) Gracilibacillus gen. nov., with description of Gracilibacillus halotolerans gen. nov., sp. nov.; transfer of Bacillus dipsosauri to Gracilibacillus dipsosauri comb. nov., and Bacillus salexigens to the genus Salibacillus gen. nov., as Salibacillus salexigens comb. nov. Int J Syst Bacteriol 49:1–11

    Google Scholar 

  • Winterhalter C, Liebl W (1995) Two extremely thermostable xylanases of the hyperthermophilic bacterium Thermotoga maritima MSB8. Appl Environ Microbiol 61:1810–1815

    CAS  Google Scholar 

  • Wong KKY, Saddler JN (1992) Trichoderma xylanases, their properties and application. Crit Rev Biotechnol 12:413–435

    CAS  Google Scholar 

  • Wong KKY, Tan LUL, Saddler JN (1988) Multiplicity of β-1,4-xylanase in microorganisms: functions and applications. Microbiol Rev 52:305–317

    PubMed  Google Scholar 

  • Wright DB, Banks DD, Lohman JR, Hilsenbeck JL, Gloss LM (2002) The effect of salts on the activity and stability of Escherichia coli and Haloferax volcanii dihydrofolate reductases. J Mol Biol 323:327–344

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We thank Dr. Jürgen Puls for generous help with product analysis and Jens Frisbæk Sørensen at Danisco A/S for performing the isoelectric focusing analysis. Rene Trinderup is also acknowledged of his information on the characteristics of strain CL8.

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Authors and Affiliations

  1. Department of Microbial Ecology, Institute of Biological Sciences, University of Aarhus, Ny Munkegade, Building 540, 8000, Aarhus, Denmark

    Peter Langborg Wejse & Kjeld Ingvorsen

  2. Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, 8000, Aarhus, Denmark

    Kim Kusk Mortensen

Authors
  1. Peter Langborg Wejse
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  2. Kjeld Ingvorsen
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  3. Kim Kusk Mortensen
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Corresponding author

Correspondence to Kjeld Ingvorsen.

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Communicated by W.D. Grant

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Wejse, P.L., Ingvorsen, K. & Mortensen, K.K. Purification and characterisation of two extremely halotolerant xylanases from a novel halophilic bacterium. Extremophiles 7, 423–431 (2003). https://doi.org/10.1007/s00792-003-0342-7

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