Functional characterization of a thermostable endoglucanase belonging to glycoside hydrolase family 45 from Fomitopsis palustris
- 66 Downloads
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.
KeywordsGlycoside hydrolase Cellulase Endoglucanase Fomitopsis palustris
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.
This article does not contain any studies with human participants or animals by any of the authors.
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- Nelson N (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J Biol Chem 153(2):375–380Google Scholar
- 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
- 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
- Somogyi M (1952) Notes on sugar determination. J Biol Chem 195(1):19–23Google Scholar
- 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