Abstract
We cloned and sequenced a xylanase gene named xylD from the acidophilic fungus Bispora sp. MEY-1 and expressed the gene in Pichia pastoris. The 1,422-bp full-length complementary DNA fragment encoded a 457-amino acid xylanase with a calculated molecular mass of 49.8 kDa. The mature protein of XYLD showed high sequence similarity to both glycosyl hydrolase (GH) families 5 and 30 but was more homologous to members of GH 30 based on phylogenetic analysis. XYLD shared the highest identity (49.9%) with a putative endo-1,6-β-d-glucanase from Talaromyces stipitatus and exhibited 21.1% identity and 34.3% similarity to the well-characterized GH family 5 xylanase from Erwinia chrysanthemi. Purified recombinant XYLD showed maximal activity at pH 3.0 and 60 °C, maintained more than 60% of maximal activity when assayed at pH 1.5–4.0, and had good thermal stability at 60 °C and remained stable at pH 1.0–6.0. The enzyme activity was enhanced in the presence of Ni2+ and β-mercaptoethanol and inhibited by some metal irons (Hg2+, Cu2+, Pb2+, Mn2+, Li+, and Fe3+) and sodium dodecyl sulfate. The specific activity of XYLD for beechwood xylan, birchwood xylan, 4-O-methyl-d-glucuronoxylan, and oat spelt xylan was 2,463, 2,144, 2,020, and 1,429 U mg−1, respectively. The apparent K m and V max values for beechwood xylan were 5.6 mg ml−1 and 3,622 μmol min−1 mg−1, respectively. The hydrolysis products of different xylans were mainly xylose and xylobiose.
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References
Ahmed S, Riaz S, Jamil A (2009) Molecular cloning of fungal xylanases: an overview. Appl Microbiol Biotechnol 84:19–35
Altschul SF, Lipman DJ (1990) Protein database searches for multiple alignments. Proc Natl Acad Sci 87:5509–5513
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Altschul SF, Madden T, Schaffer A, Zhang J, Zhang Z, Miller W, Lipman D (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Beg QK, Kapoor M, Mahajan L, Hoondal GS (2001) Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol 56:326–338
Beliën T, Joye IJ, Delcour JA, Courtin CM (2009) Computational design-based molecular engineering of the glycosyl hydrolase family 11 B. subtilis XynA endoxylanase improves its acid stability. Protein Eng Des Sel 22:587–596
Biely P (1985) Microbial xylanolytic systems. Trends Biotechnol 3:286–290
Biely P, Vrsanska M, Tenkanen M, Kluepfel D (1997) Endo-beta-1, 4-xylanase families: differences in catalytic properties. J Biotechnol 57:151–166
Chàvez R, Bull P, Eyzaguirre J (2006) The xylanolytic enzyme system from the genus Penicillium. J Biotechnol 23:413–433
Collins T, Gerday C, Feller G (2005) Xylanases, xylanases families and extremophilic xylanases. FEMS Microbiol Rev 29:3–23
Dayhoff MO, Schwartz RM, Orcutt BC (1978) A model of evolutionary change in proteins. In: Dayhoff MO (ed) Atlas of protein sequence and structure, vol. 5, suppl. 3. National Biomedical Research Foundation, Washington, pp 345–352
de Lemos Esteves F, Ruelle V, Lamotte-Brasseur J, Quinting B, Frère JM (2004) Acidophilic adaptation of family 11 endo-β-1, 4-xylanases: modeling and mutational analysis. Protein Sci 13:1209–1218
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
Haegeman A, Vanholme B, Gheysen G (2009) Characterization of a putative endoxylanase in the migratory plant-parasitic nematode Radopholus similes. Mol Plant Pathol 10:389–401
Hurlbert JF, Preston JF III (2001) Functional characterization of a novel xylanase from a corn strain of Erwinia chrysanthemi. J Bacteriol 183:2093–2100
Keen NT, Boyd C, Henrissat B (1996) Cloning and characterization of a xylanase gene from corn strains of Erwinia chrysanthemi. Mol Plant Microb Interact 9:651–657
Khandeparker R, Numan MT (2008) Bifunctional xylanases and their potential use in biotechnology. J Ind Microbiol Biotech 35:635–644
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Li N, Meng K, Wang YR, Shi PJ, Luo HY, Bai YG, Yang PL, Yao B (2008) Cloning, expression, and characterization of a new xylanase with broad temperature adaptability from Streptomyces sp. S9. Appl Microbiol Biotechnol 80:231–240
Liu YG, Whittier RF (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25:674–681
Luo H, Yang J, Yang P, Li J, Huang H, Shi P, Bai Y, Wang Y, Fan Y, Yao B (2009a) Gene cloning and expression of a new acidic family 7 endo-beta-1, 3–1, 4-glucanase from the acidophilic fungus Bispora sp. MEY-1. Appl Microbiol Biotechnol. doi:10.1007/s00253-009-2119-0
Luo H, Wang Y, Wang H, Yang J, Yang Y, Huang H, Yang P, Bai Y, Shi P, Fan Y, Yao B (2009b) A novel highly acidic β-mannanase from the acidophilic fungus Bispora sp. MEY-1: gene cloning and overexpression in Pichia pastoris. Appl Microbiol Biotechnol 82:453–461
Luo H, Wang Y, Li J, Wang H, Yang J, Yang Y, Huang HQ, Fan YL, Yao B (2009c) Cloning, expression and characterization of a novel acidic xylanase, XYL11B, from the acidophilic fungus Bispora sp. MEY-1. Enzyme Microb Technol 45:126–133
Luo H, Li J, Yang J, Wang H, Yang Y, Huang H, Shi P, Yuan T, Fan Y, Yao B (2009d) A thermophilic and acid stable family-10 xylanase from the acidophilic fungus Bispora sp. MEY-1. Extremophiles 13:849–857
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428
Mitreva-Dautova M, Roze E, Overmars H, de Graaff L, Schots A, Helder J, Goverse A, Bakker J, Smant G (2006) A symbiont-independent endo-1, 4-beta-xylanase from the plant-parasitic nematode Meloidogyne incognita. Mol Plant Microb Interact 19:521–529
Nicholas KB, Nicholas Jr HB (1997) GeneDoc: a tool for editing and annotating multiple sequence alignments. (Distributed by the author. http://www.psc.edu/biomed/genedoc)
Polizeli MLTM, Rizzatti ACS, Monti R, Terenzi HF, Jorge JA, Amorim DS (2005) Xylanases from fungi: properties and industrial applications. Appl Microbiol Biotechnol 67:577–591
Sørensen HR, Pedersen S, Jørgensen CT, Meyer AS (2007) Enzymatic hydrolysis of wheat arabinoxylan by a recombinant “minimal” enzyme cocktail containing β-xylosidase and novel endo-1, 4-β-xylanase and α-l-arabinofuranosidase activities. Biotechnol Prog 23:100–107
St John FJ, Rice JD, Preston JF (2006) Characterization of XynC from Bacillus subtilis subsp. subtilis strain 168 and analysis of its role in depolymerization of glucuronoxylan. J Bacteriol 188:8617–8626
Subramaniyan S, Prema P (2002) Biotechnology of microbial xylanases: enzymology, molecular biology, and application. Crit Rev Biotechnol 22:33–64
Suzuki T, Ibata K, Hatsu M, Takamizawa K, Kawai K (1997) Cloning and expression of a 58-kDa xylanase VI gene (xynD) of Aeromonas caviae ME-1 in Escherichia coli which is not categorized as a family F or family G xylanase. J Ferment Bioeng 84:86–89
Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599
Vršanská M, Kolenová K, Puchart V, Biely P (2007) Mode of action of glycoside hydrolase family 5 glucuronoxylan xylanohydrolase from Erwinia chrysanthemi. FEBS J 274:1666–1677
Wang H, Luo H, Bai Y, Wang Y, Yang P, Shi P, Zhang W, Fan Y, Yao B (2009) An acidophilic beta-galactosidase from Bispora sp. MEY-1 with high lactose hydrolytic activity under simulated gastric conditions. J Agric Food Chem 57:5535–5541
Wong KKY, Tan LUL, Saddler JN (1988) Multiplicity of β-1,4-xylanase in microorganisms: functions and applications. Microbiol Rev 52:305–317
Xue Y, Peng J, Wang R, Song X (2009) Construction of the trifunctional enzyme associating the Thermoanaerobacter ethanolicus xylosidase-arabinosidase with the Thermomyces lanuginosus xylanase for degradation of arabinoxylan. Enzyme Microb Technol 45:22–27
Acknowledgements
This work was supported by the National High Technology Research and Development Program of China (863 program, Grant No. 2007AA100601), Key Program of Transgenic Plant Breeding (2008ZX08003-002), and the Earmarked Fund for Modern Agro-industry Technology Research System (nycytx-42-G2-05). We thank Dr. Kenji Fukuda of Obihiro University of Agriculture and Veterinary Medicine, Japan, for his generosity to provide substrate pustulan.
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Huiying Luo and Jun Yang contributed equally to this work.
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Luo, H., Yang, J., Li, J. et al. Molecular cloning and characterization of the novel acidic xylanase XYLD from Bispora sp. MEY-1 that is homologous to family 30 glycosyl hydrolases. Appl Microbiol Biotechnol 86, 1829–1839 (2010). https://doi.org/10.1007/s00253-009-2410-0
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DOI: https://doi.org/10.1007/s00253-009-2410-0