Abstract
A complete gene, xyl10C, encoding a thermophilic endo-1,4-β-xylanase (XYL10C), was cloned from the acidophilic fungus Bispora sp. MEY-1 and expressed in Pichia pastoris. XYL10C shares highest nucleotide and amino acid sequence identities of 57.3 and 49.7%, respectively, with a putative xylanase from Aspergillus fumigatus Af293 of glycoside hydrolase family 10. A high expression level in P. pastoris (73,400 U ml−1) was achieved in a 3.7–l fermenter. The purified recombinant XYL10C was thermophilic, exhibiting maximum activity at 85°C, which is higher than that reported from any fungal xylanase. The enzyme was also highly thermostable, exhibiting ~100% of the initial activity after incubation at 80°C for 60 min and >87% of activity at 90°C for 10 min. The half lives of XYL10C at 80 and 85°C were approximately 45 and 3 h, respectively. It had two activity peaks at pH 3.0 and 4.5–5.0 (maximum), respectively, and was very acid stable, retaining more than 80% activity after incubation at pH 1.5−6.0 for 1 h. The enzyme was resistant to Co2+, Mn2+, Cr3+ and Ag+. The specific activity of XYL10C for oat spelt xylan was 18,831 U mg−1. It also had wide substrate specificity and produced simple products (65.1% xylose, 25.0% xylobiose and 9.9% xylan polymer) from oat spelt xylan.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modeling. Bioinformatics 22:195–201
Beg QK, Kapoor M, Mahajan L, Hoondal GS, Terenzi HF, Jorge JA, Amorim DS (2005) Xylanases from fungi: properties and industrial applications. Appl Microbiol Biotechnol 67:577–591
Biely P (1985) Microbial xylanolytic systems. Trends Biotechnol 3:286–290
Biely P, Vrsanska M, Tenkanen M, Kluepfel D (1997) Endo-β-1,4-xylanase families: differences in catalytic properties. J Biotechnol 57:151–166
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
Cao Y, Yang P, Shi P, Wang Y, Luo H, Meng K, Zhang Z, Wu N, Yao B, Fan Y (2007) Purification and characterization of a novel protease-resistant α-galactosidase from Rhizopus sp. F78 ACCC 30795. Enzyme Microb Tech 41:835–841
Collins T, Gerday C, Feller G (2005) Xylanases, xylanases families and extremophilic xylanases. FEMS Microbiol Rev 29:3–23
Damaso MCT, Almeida MS, Kurtenbach E, Martins OB, Andrade CMMC, Pereira N Jr, Albano RM (2003) Optimized expression of a thermostable xylanase from Thermomyces lanuginosus in Pichia pastoris. Appl Environ Microbiol 69:6064–6072
Decelle B, Tsang A, Storms RK (2004) Cloning, functional expression and characterization of three Phanerochaete chrysosporium endo-1, 4-β-xylanases. Curr Genet 46:166–175
Han Y, Wilson DB, Lei XG (1999) Expression of an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae. Appl Environ Microbiol 65:1915–1918
Henrissat B, Bairoch A (1993) New families in the classification of glycosyl hydrolases on amino acid sequence similarities. Biochem J 293:781–788
Kimura T, Ito J, Kawano A, Makino T, Kondo H, Karita S, Sakka K, Ohmiya K (2000) Purification, characterization, and molecular cloning of acidophilic xylanase from Penicillium sp. 40. Biosci Biotechnol Biochem 64:1230–1237
Kulkarni N, Shendye A, Rao M (1999) Molecular and biotechnological aspects of xylanases. FEMS Microbiol Rev 23:411–456
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 HY, Wang YR, Wang H, Yang J, Yang YH, Huang HQ, Yang PL, Bai YG, Shi PJ, Fan YL, Yao B (2009) 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
Maalej I, Belhaj I, Masmoudi FN, Belghith H (2008) Highly thermostable xylanase of the thermophilic fungus Talaromyces thermophilus: purification and characterization. Appl Biochem Biotechnol 158:200–212
McCarthy AA, Morris DD, Bergquist PL, Baker EN (2000) Structure of XynB, a highly thermostable beta-1, 4-xylanase from Dictyoglomus thermophilum Rt46B.1, at 1.8 Å resolution. Acta Crystallogr D Biol Crystallogr 56:1367–1375
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428
Nakamura S, Wakabayashi K, Nakai R, Aono R, Horikoshi K (1993) Purification and some properties of an alkaline xylanase from alkaliphilic Bacillus sp. strain 41M–1. Appl Environ Microbiol 59:2311–2316
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)
Ritschkoff AC, Buchert J, Viikari L (1994) Purification and characterization of a thermophilic xylanase from the brown-rot fungus Gloeophyllum trabeum. J Biotechnol 32:67–74
Simpson HD, Haufler UR, Daniel RM (1991) An extremely thermostable xylanase from the thermophilic eubacterium Thermotoga. Biochem J 277:413–417
Subramaniyan S, Prema P (2002) Biotechnology of microbial xylanases: enzymology, molecular biology, and application. Crit Rev Biotechnol 22:33–64
Sunna A, Antranikian G (1996) Growth and production of xylanolytic enzymes by the extreme thermophilic anaerobic bacterium Thermotoga thermarum. Appl Microbiol Biotechnol 45:671–676
Tuohy MG, Coughlan MP (1992) Production of thermostable xylan-degrading enzymes by Talaromyces emersonii. Biores Technol 39:131–137
Acknowledgments
This work was supported by the National High Technology Research and Development Program of China (863 program, Grant No. 2007AA100601), the Chinese Program on Research for Public Good (Grant No. 2005DIB4J038), and the 948 program of the Ministry of Agriculture (Grant No. 2007-Z3).
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by A. Driessen.
Rights and permissions
About this article
Cite this article
Luo, H., Li, J., Yang, J. et al. A thermophilic and acid stable family-10 xylanase from the acidophilic fungus Bispora sp. MEY-1. Extremophiles 13, 849–857 (2009). https://doi.org/10.1007/s00792-009-0272-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00792-009-0272-0