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Applied Microbiology and Biotechnology

, Volume 102, Issue 15, pp 6515–6523 | Cite as

Functional characterization of a thermostable endoglucanase belonging to glycoside hydrolase family 45 from Fomitopsis palustris

Biotechnologically relevant enzymes and proteins

Abstract

A gene encoding an endoglucanase belonging to subfamily C of glycoside hydrolase family 45 (GH45) was identified in the brown rot fungus Fomitopsis palustris and functionally expressed in Pichia pastoris. The recombinant protein displayed hydrolytic activities toward various substrates such as carboxymethyl cellulose, phosphoric acid swollen cellulose, glucomannan, lichenan, and β-glucan. In particular, the enzyme had a unique catalytic efficiency on β-1,4-glucans rather than mixed β-1,3/1,4-glucans as compared to other GH45 endoglucanases. The fungal enzyme was relatively thermostable, retaining more than 91.4% activity at 80 °C for 1 h. Site-directed mutagenesis studies revealed that the mutants N95D and D117N had significantly reduced enzymatic activities, indicating that both residues are essential for the catalytic reaction. Our study expands knowledge and understanding of the catalytic mechanism of GH45 subfamily C enzymes and also suggests that this thermostable endoglucanase from F. palustris has great potential in industrial applications.

Keywords

Glycoside hydrolase Cellulase Endoglucanase Fomitopsis palustris 

Notes

Acknowledgements

This research was supported by the Chung-Ang University Excellent Student Scholarship in 2012.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals by any of the authors.

Supplementary material

253_2018_9075_MOESM1_ESM.pdf (1.1 mb)
ESM 1 (PDF 1113 kb)

References

  1. Beguin P, Aubert JP (1994) The biological degradation of cellulose. FEMS Microbiol Rev 13(1):25–58.  https://doi.org/10.1016/0168-6445(94)90099-X CrossRefPubMedGoogle Scholar
  2. Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, Kiefer F, Gallo Cassarino T, Bertoni M, Bordoli L, Schwede T (2014) SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 42(W1):W252–W258.  https://doi.org/10.1093/nar/gku340 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 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 10:103.  https://doi.org/10.1186/1475-2859-10-103 CrossRefGoogle Scholar
  4. Davies GJ, Tolley SP, Henrissat B, Hjort C, Schulein M (1995) Structures of oligosaccharide-bound forms of the endoglucanase V from Humicola insolens at 1.9 Å resolution. Biochemistry-US 34(49):16210–16220.  https://doi.org/10.1021/bi00049a037 CrossRefGoogle Scholar
  5. Eberhardt RY, Gilbert HJ, Hazlewood GP (2000) Primary sequence and enzymic properties of two modular endoglucanases, Cel5A and Cel45A, from the anaerobic fungus Piromyces equi. Microbiology 146(Pt 8):1999–2008.  https://doi.org/10.1099/00221287-146-8-1999 CrossRefPubMedGoogle Scholar
  6. Edman P (1949) A method for the determination of the amino acid sequence in peptides. Arch Biochem 22(3):475PubMedGoogle Scholar
  7. Emalfrab MA, Burlingame RP, Olson PT, Sinitsyn AP, Parriche M, Bousson JC, Pynnonen CM, Punt PJ, Van Zeijl CMJ (2003) Transformation system in the field of filamentous fungal hosts. U.S. Patent 6,573,086 B1Google Scholar
  8. Heckman KL, Pease LR (2007) Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc 2(4):924–932.  https://doi.org/10.1038/nprot.2007.132 CrossRefPubMedGoogle Scholar
  9. Henrissat B, Davies G (1997) Structural and sequence-based classification of glycoside hydrolases. Curr Opin Struc Biol 7(5):637–644.  https://doi.org/10.1016/S0959-440x(97)80072-3 CrossRefGoogle Scholar
  10. Hirvonen M, Papageorgiou AC (2003) Crystal structure of a family 45 endoglucanase from Melanocarpus albomyces: mechanistic implications based on the free and cellobiose-bound forms. J Mol Biol 329(3):403–410.  https://doi.org/10.1016/S0022-2836(03)00467-4 CrossRefPubMedGoogle Scholar
  11. Hong CY, Lee SY, Ryu SH, Kim M (2017) Whole-genome de novo sequencing of wood rot fungus Fomitopsis palustris (ATCC62978) with both a cellulolytic and ligninolytic enzyme system. J Biotechnol 251:156–159.  https://doi.org/10.1016/j.jbiotec.2017.04.009 CrossRefPubMedGoogle Scholar
  12. Igarashi K, Ishida T, Hori C, Samejima M (2008) Characterization of an endoglucanase belonging to a new subfamily of glycoside hydrolase family 45 of the basidiomycete Phanerochaete chrysosporium. Appl Environ Microbiol 74(18):5628–5634.  https://doi.org/10.1128/Aem.00812-08 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Isikgor FH, Becer CR (2015) Lignocellulosic biomass: a sustainable platform for the production of bio-based chemicals and polymers. Polym Chem-UK 6(25):4497–4559.  https://doi.org/10.1039/c5py00263j CrossRefGoogle Scholar
  14. Ji HW, Cha CJ (2010) Identification and functional analysis of a gene encoding β-glucosidase from the brown-rot basidiomycete Fomitopsis palustris. J Microbiol 48(6):808–813.  https://doi.org/10.1007/s12275-010-0482-2 CrossRefPubMedGoogle Scholar
  15. Kadowaki MA, Camilo CM, Muniz AB, Polikarpov I (2015) Functional characterization and low-resolution structure of an endoglucanase Cel45A from the filamentous fungus Neurospora crassa OR74A: thermostable enzyme with high activity toward lichenan and β-glucan. Mol Biotechnol 57(6):574–588.  https://doi.org/10.1007/s12033-015-9851-8 CrossRefPubMedGoogle Scholar
  16. Karim N, Shibuya H, Kikuchi T (2011) Analysis of expressed sequence tags from the wood-decaying fungus Fomitopsis palustris and identification of potential genes involved in the decay process. J Microbiol Biotechnol 21(4):347–358.  https://doi.org/10.4014/jmb.1010.10048 PubMedGoogle Scholar
  17. Karlsson J, Siika-aho M, Tenkanen M, Tjerneld F (2002) Enzymatic properties of the low molecular mass endoglucanases Cel12A (EG III) and Cel45A (EG V) of Trichoderma reesei. J Biotechnol 99(1):63–78.  https://doi.org/10.1016/S0168-1656(02)00156-6 CrossRefPubMedGoogle Scholar
  18. Karnaouri AC, Topakas E, Christakopoulos P (2014) Cloning, expression, and characterization of a thermostable GH7 endoglucanase from Myceliophthora thermophila capable of high-consistency enzymatic liquefaction. Appl Microbiol Biotechnol 98(1):231–242.  https://doi.org/10.1007/s00253-013-4895-9 CrossRefPubMedGoogle Scholar
  19. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33(7):1870–1874.  https://doi.org/10.1093/molbev/msw054 CrossRefPubMedGoogle Scholar
  20. Letunic I, Bork P (2016) Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res 44(W1):W242–W245.  https://doi.org/10.1093/nar/gkw290 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Li DC, Li AN, Papageorgiou AC (2011) Cellulases from thermophilic fungi: recent insights and biotechnological potential. Enzyme Res 2011:308730.  https://doi.org/10.4061/2011/308730 PubMedPubMedCentralGoogle Scholar
  22. Liu G, Wei X, Qin Y, Qu Y (2010) Characterization of the endoglucanase and glucomannanase activities of a glycoside hydrolase family 45 protein from Penicillium decumbens 114-2. J Gen Appl Microbiol 56(3):223–229.  https://doi.org/10.2323/jgam.56.223 CrossRefPubMedGoogle Scholar
  23. Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42(D1):D490–D495.  https://doi.org/10.1093/nar/gkt1178 CrossRefPubMedGoogle Scholar
  24. Luo H, Wang Y, Wang H, Yang J, Yang Y, Huang H, Yang P, Bai Y, Shi P, Fan Y, 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 Biotchnol 82(3):453–461.  https://doi.org/10.1007/s00253-008-1766-x CrossRefGoogle Scholar
  25. Maki M, Leung KT, Qin W (2009) The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass. Int J Biol Sci 5(5):500–516.  https://doi.org/10.7150/ijbs.5.500 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Nakamura A, Ishida T, Kusaka K, Yamada T, Fushinobu S, Tanaka I, Kaneko S, Ohta K, Tanaka H, Inaka K, Higuchi Y, Niimura N, Samejima M, Igarashi K (2015) “Newton’s cradle” proton relay with amide–imidic acid tautomerization in inverting cellulase visualized by neutron crystallography. Sci Adv 1(7):e1500263.  https://doi.org/10.1126/sciadv.1500263 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Nelson N (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J Biol Chem 153(2):375–380Google Scholar
  28. Okamoto K, Sugita Y, Nishikori N, Nitta Y, Yanase H (2011) Characterization of two acidic β-glucosidases and ethanol fermentation in the brown rot fungus Fomitopsis palustris. Enzym Microb Technol 48(4–5):359–364.  https://doi.org/10.1016/j.enzmictec.2010.12.012 CrossRefGoogle Scholar
  29. Payne CM, Knott BC, Mayes HB, Hansson H, Himmel ME, Sandgren M, Ståhlberg J, Beckham GT (2015) Fungal cellulases. Chem Rev 115(3):1308–1448.  https://doi.org/10.1042/bst0200046 CrossRefPubMedGoogle Scholar
  30. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25(13):1605–1612.  https://doi.org/10.1002/jcc.20084 CrossRefPubMedGoogle Scholar
  31. Rubin EM (2008) Genomics of cellulosic biofuels. Nature 454(7206):841–845.  https://doi.org/10.1038/nature07190 CrossRefPubMedGoogle Scholar
  32. Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1(6):2856–2860.  https://doi.org/10.1038/nprot.2006.468 CrossRefPubMedGoogle Scholar
  33. Shimokawa T, Shibuya H, Nojiri M, Yoshida S, Ishihara M (2008) Purification, molecular cloning, and enzymatic properties of a family 12 endoglucanase (EG-II) from Fomitopsis palustris: role of EG-II in larch holocellulose hydrolysis. Appl Environ Microbiol 74(18):5857–5861.  https://doi.org/10.1128/Aem.00435-08 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7(1):539.  https://doi.org/10.1038/msb.2011.75 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Somogyi M (1952) Notes on sugar determination. J Biol Chem 195(1):19–23Google Scholar
  36. Song BC, Kim KY, Yoon JJ, Sim SH, Lee K, Kim YS, Kim YK, Cha CJ (2008) Functional analysis of a gene encoding endoglucanase that belongs to glycosyl hydrolase family 12 from the brown-rot basidiomycete Fomitopsis palustris. J Microbiol Biotechnol 18(3):404–409PubMedGoogle Scholar
  37. Valadares F, Gonçalves TA, Gonçalves DS, Segato F, Romanel E, Milagres AM, Squina FM, Ferraz A (2016) Exploring glycoside hydrolases and accessory proteins from wood decay fungi to enhance sugarcane bagasse saccharification. Biotechnol Biofuels 9(1):110.  https://doi.org/10.1186/s13068-016-0525-y CrossRefPubMedPubMedCentralGoogle Scholar
  38. Vlasenko E, Schülein M, Cherry J, Xu F (2010) Substrate specificity of family 5, 6, 7, 9, 12, and 45 endoglucanases. Bioresour Technol 101(7):2405–2411.  https://doi.org/10.1016/j.biortech.2009.11.057 CrossRefPubMedGoogle Scholar
  39. Wood TM (1988) Preparation of crystalline, amorphous, and dyed cellulase substrates. Method Enzymol 160:19–25.  https://doi.org/10.1016/0076-6879(88)60103-0 CrossRefGoogle Scholar
  40. Yoon JJ, Kim YK (2005) Degradation of crystalline cellulose by the brown-rot basidiomycete Fomitopsis palustris. J Microbiol 43(6):487–492PubMedGoogle Scholar
  41. Yoon JJ, Cha CJ, Kim YS, Son DW, Kim YK (2007) The brown-rot basidiomycete Fomitopsis palustris has the endo-glucanases capable of degrading microcrystalline cellulose. J Microbiol Biotechnol 17(5):800–805PubMedGoogle Scholar
  42. Yoon JJ, Kim KY, Cha CJ (2008) Purification and characterization of thermostable β-glucosidase from the brown-rot basidiomycete Fomitopsis palustris grown on microcrystalline cellulose. J Microbiol 46(1):51–55.  https://doi.org/10.1007/s12275-007-0230-4 CrossRefPubMedGoogle Scholar
  43. Zhao J, Shi P, Li Z, Yang P, Luo H, Bai Y, Wang Y, Yao B (2012) Two neutral thermostable cellulases from Phialophora sp. G5 act synergistically in the hydrolysis of filter paper. Bioresour Technol 121:404–410.  https://doi.org/10.1016/j.biortech.2012.07.027 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Systems BiotechnologyChung-Ang UniversityAnseongRepublic of Korea
  2. 2.IT Convergence Materials R&BD Group, Chungcheong Regional DivisionKorea Institute of Industrial Technology (KITECH)CheonanRepublic of Korea

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