Applied Microbiology and Biotechnology

, Volume 98, Issue 5, pp 2155–2163 | Cite as

A novel thermophilic endo-β-1,4-mannanase from Aspergillus nidulans XZ3: functional roles of carbohydrate-binding module and Thr/Ser-rich linker region

  • Haiqiang Lu
  • Huiying Luo
  • Pengjun Shi
  • Huoqing Huang
  • Kun Meng
  • Peilong Yang
  • Bin YaoEmail author
Biotechnologically relevant enzymes and proteins


The gene man5XZ3 from Aspergillus nidulans XZ3 encodes a multimodular β-mannanase of glycoside hydrolase family 5 that consists of a family 1 carbohydrate-binding module (CBM1), a Thr/Ser-rich linker region, and a catalytic domain. Recombinant Man5XZ3 and its two truncated derivatives, Man5ΔCBM (removing the CBM1) and Man5ΔCL (removing both the CBM1 and linker region), were produced in Pichia pastoris and showed significant variance in the secondary structure. The three enzymes had similar biochemical properties, such as optimal pH and temperature (pH 5.0 and 80 °C) and excellent pH stability at pH 4.0–10.0. Removal of the CBM1 alone could improve the thermostability of Man5XZ3, but further removal of the linker region resulted in worse thermostability. Man5XZ3 retained greater enzyme activity in the presence of an organic solvent (acetone), two detergents (SDS and Triton X-100), and a chaotropic agent (urea) compared with Man5ΔCBM and Man5ΔCL. This study provides an excellent β-mannanase candidate favorable for various industries and primarily demonstrates the relationship between enzyme structure and function.


β-Mannanase Thermophilic CBM Linker region Thermostability 



This research was supported by the National Science Foundation for Distinguished Young Scholars of China (31225026), the National High Technology Research and Development Program of China (863 Program; No. 2012AA022208), and the China Modern Agriculture Research System (CARS-42).

Supplementary material

253_2013_5112_MOESM1_ESM.pdf (151 kb)
ESM 1 (PDF 151 kb)


  1. Abdel-Fattah AF, Hashem AM, Ismail AMS, El-Refai MA (2009) Purification and some properties of β-mannanase from Aspergillus oryzae NRRL 3448. J Appl Sci Res 5:2067–2073Google Scholar
  2. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCentralPubMedCrossRefGoogle Scholar
  3. Anbarasan S, Jänis J, Paloheimo M, Laitaoja M, Vuolanto M, Karimäki J, Vainiotalo P, Leisola M, Turunen O (2010) Effect of glycosylation and additional domains on the thermostability of a family 10 xylanase produced by Thermopolyspora flexuosa. Appl Environ Microbiol 76:356–360PubMedCentralPubMedCrossRefGoogle Scholar
  4. Bai Y, Wang J, Zhang Z, Shi P, Luo H, Huang H, Luo C, Yao B (2010) Expression of an extremely acidic β-1,4-glucanase from thermoacidophilic Alicyclobacillus sp. A4 in Pichia pastoris is improved by truncating the gene sequence. Microb Cell Factories 9:33CrossRefGoogle Scholar
  5. Berka RM, Grigoriev IV, Otillar R, Salamov A, Grimwood J, Reid I, Ishmael N, John T, Darmond C, Moisan MC, Henrissat B, Coutinho PM, Lombard V, Natvig DO, Lindquist E, Schmutz J, Lucas S, Harris P, Powlowski J, Bellemare A, Taylor D, Butler G, de Vries RP, Allijn IE, van den Brink J, Ushinsky S, Storms R, Powell AJ, Paulsen IT, Elbourne LD, Baker SE, Magnuson J, Laboissiere S, Clutterbuck AJ, Martinez D, Wogulis M, de Leon AL, Rey MW, Tsang A (2011) Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris. Nat Biotechnol 29:922–927PubMedCrossRefGoogle Scholar
  6. Boraston AB, Bolam DN, Gilbert HJ, Davies GJ (2004) Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Biochem J 382:769–781PubMedCentralPubMedCrossRefGoogle Scholar
  7. 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 72:248–254PubMedCrossRefGoogle Scholar
  8. Bruins ME, Janssen AE, Boom RM (2001) Thermozymes and their applications: a review of recent literature and patents. Appl Biochem Biotechnol 90:155–186PubMedCrossRefGoogle Scholar
  9. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 37:D233–D238PubMedCentralPubMedCrossRefGoogle Scholar
  10. Chauhan PS, Puri N, Sharma P, Gupta N (2012) Mannanases: microbial sources, production, properties and potential biotechnological applications. Appl Microbiol Biotechnol 93:1817–1830PubMedCrossRefGoogle Scholar
  11. Chitte RR, Deshmukh SV, Kanekar PP (2011) Production, purification, and biochemical characterization of a fibrinolytic enzyme from thermophilic Streptomyces sp. MCMB-379. Appl Biochem Biotechnol 165:1406–1413PubMedCrossRefGoogle Scholar
  12. Couturier M, Feliu J, Haon M, Navarro D, Lesage-Meessen L, Coutinho PM, Berrin JG (2011) A thermostable GH45 endoglucanase from yeast: impact of its atypical multimodularity on activity. Microb Cell Factories 10:103CrossRefGoogle Scholar
  13. Dhawan S, Kaur J (2007) Microbial mannanases: an overview of production and applications. Crit Rev Biotechnol 27:197–216PubMedCrossRefGoogle Scholar
  14. Ding M, Teng Y, Yin Q, Zhao J, Zhao F (2008) The N-terminal cellulose-binding domain of EGXA increases thermal stability of xylanase and changes its specific activities on different substrates. Acta Biochim Biophys Sin 40:949–954PubMedCrossRefGoogle Scholar
  15. Do BC, Dang TT, Berrin JG, Haltrich D, To KA, Sigoillot JC, Yamabhai M (2009) Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-β-mannosidase from Aspergillus niger BK01. Microb Cell Factories 8:59CrossRefGoogle Scholar
  16. Gentzsch M, Tanner W (1997) Protein-O-glycosylation in yeast: protein-specific mannosyltransferases. Glycobiology 7:481–486PubMedCrossRefGoogle Scholar
  17. Goto M, Tsukamoto M, Kwon I, Ekino K, Furukawa K (1999) Functional analysis of O-linked oligosaccharides in threonine/serine-rich region of Aspergillus glucoamylase by expression in mannosyltransferase-disruptants of yeast. Eur J Biochem 260:596–602PubMedCrossRefGoogle Scholar
  18. Guillén D, Sánchez S, Rodríguez-Sanoja R (2006) Carbohydrate-binding domains: multiplicity of biological roles. Appl Microbiol Biotechnol 85:1241–1249CrossRefGoogle Scholar
  19. Hägglund P, Eriksson T, Collén A, Nerinckx W, Claeyssens M, Stålbrand H (2003) A cellulose-binding module of the Trichoderma reesei β-mannanase Man5A increases the mannan-hydrolysis of complex substrates. J Biotechnol 101:37–48PubMedCrossRefGoogle Scholar
  20. Henrissat B, Davies G (1997) Structural and sequence-based classification of glycoside hydrolases. Curr Opin Struct Biol 7:637–644PubMedCrossRefGoogle Scholar
  21. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  22. Li N, Shi P, Yang P, Wang Y, Luo H, Bai Y, Zhou Z, Yao B (2009) A xylanase with high pH stability from Streptomyces sp. S27 and its carbohydrate-binding module with/without linker-region-truncated versions. Appl Microbiol Biotechnol 83:99–107PubMedCrossRefGoogle Scholar
  23. Lu H, Zhang H, Shi P, Luo H, Wang Y, Yang P, Yao B (2013) A family 5 β-mannanase from the thermophilic fungus Thielavia arenaria XZ7 with typical thermophilic enzyme features. Appl Microbiol Biotechnol. doi: 10.1007/s00253-012-4656-1 Google Scholar
  24. Luo H, Wang K, Huang H, Shi P, Yang P, Yao B (2012) Gene cloning, expression, and biochemical characterization of an alkali-tolerant β-mannanase from Humicola insolens Y1. J Ind Microbiol Biotechnol 39:547–555PubMedCrossRefGoogle Scholar
  25. Maijala P, Kango N, Szijarto N, Viikari L (2012) Characterization of hemicellulases from thermophilic fungi. Antonie van Leeuwenhoek 101:905–917PubMedCrossRefGoogle Scholar
  26. Mamo G, Thunnissen M, Hatti-Kaul R, Mattiasson B (2009) An alkaline active xylanase: insights into mechanisms of high pH catalytic adaptation. Biochimie 91:1187–1196PubMedCrossRefGoogle Scholar
  27. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  28. Moreira LRS, Filho EXF (2008) An overview of mannan structure and mannan-degrading enzyme systems. Appl Microbiol Biotechnol 79:165–178PubMedCrossRefGoogle Scholar
  29. Naganagouda K, Salimath PV, Mulimani VH (2009) Purification and characterization of endo-β-1,4-mannanase from Aspergillus niger gr for application in food processing industry. J Microbiol Biotechnol 19:1184–1190PubMedGoogle Scholar
  30. Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786PubMedCrossRefGoogle Scholar
  31. Pham TA, Berrin JG, Record E, To KA, Sigoillot JC (2010) Hydrolysis of softwood by Aspergillus mannanase: role of a carbohydrate-binding module. J Biotechnol 148:163–170PubMedCrossRefGoogle Scholar
  32. Puchart V, Vrsanská M, Svoboda P, Pohl J, Ogel ZB, Biely P (2004) Purification and characterization of two forms of endo-β-1,4-mannanase from a thermotolerant fungus, Aspergillus fumigatus IMI 385708 (formerly Thermomyces lanuginosus IMI 158749). Biochim Biophys Acta 1674:239–250PubMedCrossRefGoogle Scholar
  33. Ravalason H, Herpoël-Gimbert I, Record E, Bertaud F, Grisel S, deWeert S, van den Hondel CAMJJ, Asther M, Petit-Conil M, Sigoillot JC (2009) Fusion of a family 1 carbohydrate binding module of Aspergillus niger to the Pycnoporus cinnabarinus laccase for efficient softwood kraft pulp biobleaching. J Biotechnol 142:220–226PubMedCrossRefGoogle Scholar
  34. Semimaru T, Goto M, Furukawa K, Hayashida S (1995) Functional analysis of the threonine- and serine-rich Gp-I domain of glucoamylase I from Aspergillus awamori var. kawachi. Appl Environ Microbiol 61:2885–2890PubMedCentralPubMedGoogle Scholar
  35. Shallom D, Shoham Y (2003) Microbial hemicellulases. Curr Opin Microbiol 6:219–228PubMedCrossRefGoogle Scholar
  36. Singh S, Madlala AM, Prior BA (2003) Thermomyces lanuginosus: properties of strains and their hemicellulases. FEMS Microbiol Rev 27:3–16PubMedCrossRefGoogle Scholar
  37. Sunna A (2010) Modular organization and functional analysis of dissected modular β-mannanase CsMan26 from Caldicellulosiruptor Rt8B.4. Appl Microbiol Biotechnol 86:189–200PubMedCrossRefGoogle Scholar
  38. Turner P, Mamo G, Karlsson EN (2007) Potential and utilization of thermophiles and thermostable enzymes in biorefining. Microb Cell Factories 6:9CrossRefGoogle Scholar
  39. van Zyl WH, Rose SH, Trollope K, Görgens JF (2010) Fungal β-mannanases: mannan hydrolysis, heterologous production and biotechnological applications. Process Biochem 45:1203–1213CrossRefGoogle Scholar
  40. Whitmore L, Wallace BA (2008) Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases. Biopolymers 89:392–400PubMedCrossRefGoogle Scholar
  41. Yan XX, An XM, Gui LL, Liang DC (2008) From structure to function: insights into the catalytic substrate specificity and thermostability displayed by Bacillus subtilis mannanase BCman. J Mol Biol 379:535–544PubMedCrossRefGoogle Scholar
  42. Yeoman CJ, Han Y, Dodd D, Schroeder CM, Mackie RI, Cann IK (2010) Thermostable enzymes as biocatalysts in the biofuel industry. Adv Appl Microbiol 70:1–55PubMedCrossRefGoogle Scholar
  43. Zhao J, Shi P, Huang H, Li Z, Yuan T, Yang P, Luo H, Bai Y, Yao B (2012) A novel thermoacidophilic and thermostable endo-β-1,4-glucanase from Phialophora sp. G5: its thermostability influenced by a distinct β-sheet and the carbohydrate-binding module. Appl Microbiol Biotechnol 95:947–955PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Haiqiang Lu
    • 1
  • Huiying Luo
    • 1
  • Pengjun Shi
    • 1
  • Huoqing Huang
    • 1
  • Kun Meng
    • 1
  • Peilong Yang
    • 1
  • Bin Yao
    • 1
    Email author
  1. 1.Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research InstituteChinese Academy of Agricultural SciencesBeijingPeople’s Republic of China

Personalised recommendations