A novel glucuronoyl esterase from Aspergillus fumigatus—the role of conserved Lys residue in the preference for 4-O-methyl glucuronoyl esters
Cellulose in plant cell walls is mainly covered by hemicellulose and lignin, and thus efficient removal of these components is thought to be a key step in the optimal utilization of lignocellulose. The recently discovered carbohydrate esterase (CE) 15 family of glucuronoyl esterases (GEs) which cleave the linkages between the free carboxyl group of d-glucuronic acid in hemicellulose and the benzyl groups in lignin residues could contribute to this process. Herein, we report the identification, functional expression, and enzymatic characterization of a GE, AfGE, from the filamentous fungus Aspergillus fumigatus. AfGE was heterologously expressed in Aspergillus oryzae, and the purified enzyme displayed the ability to degrade the synthetic substrates mimicking the ester linkage between hemicellulose and lignin. AfGE is a potentially industrially applicable enzyme due to its characteristic as a thermophilic enzyme with the favorable temperature of 40–50 °C at pH 5. Molecular modeling and site-directed mutagenesis studies of AfGE demonstrated that Lys209 plays an important role in the preference for the substrates containing 4-O-methyl group in the glucopyranose ring.
KeywordsAspergillus fumigatus Catalytic triad Glucuronoyl esterase Methoxy group Preference
We are grateful to Dr. Daisuke Hagiwara (Medical Mycology Research Center, Chiba University, Japan) for providing the genomic DNA of the A. fumigatus Af293 strain.
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 performed by any of the authors.
- Benoit I, Asther M, Sulzenbacher G, Record E, Marmuse L, Parsiegla G, Gimbert I, Asther M, Bignon C (2006) Respective importance of protein folding and glycosylation in the thermal stability of recombinant feruloyl esterase A. FEBS Lett 580(25):5815–5821. https://doi.org/10.1016/j.febslet.2006.09.039 CrossRefPubMedGoogle Scholar
- Cerqueira GC, Arnaud MB, Inglis DO, Skrzypek MS, Binkley G, Simison M, Miyasato SR, Binkley J, Orvis J, Shah P, Wymore F, Sherlock G, Wortman JR (2014) The Aspergillus Genome Database : multispecies curation and incorporation of RNA-Seq data to improve structural gene annotations. Nucleic Acids Res 42:705–710. https://doi.org/10.1093/nar/gkt1029 CrossRefGoogle Scholar
- Charavgi MD, Dimarogona M, Topakas E, Christakopoulos P, Chrysina ED (2013) The structure of a novel glucuronoyl esterase from Myceliophthora thermophila gives new insights into its role as a potential biocatalyst. Acta Crystallogr Sect D Biol Crystallogr 69(1):63–73. https://doi.org/10.1107/S0907444912042400 CrossRefGoogle Scholar
- d’Errico C, Jørgensen JO, Krogh KBRM, Spodsberg N, Madsen R, Monrad RN (2015) Enzymatic degradation of lignin-carbohydrate complexes (LCCs): model studies using a fungal glucuronoyl esterase from Cerrena unicolor. Biotechnol Bioeng 112(5):914–922. https://doi.org/10.1002/bit.25508 CrossRefPubMedGoogle Scholar
- Katsimpouras C, Bénarouche A, Navarro D, Karpusas M, Dimarogona M, Berrin JG, Christakopoulos P, Topakas E (2014) Enzymatic synthesis of model substrates recognized by glucuronoyl esterases from Podospora anserina and Myceliophthora thermophila. Appl Microbiol Biotechnol 98(12):5507–5516. https://doi.org/10.1007/s00253-014-5542-9 CrossRefPubMedGoogle Scholar
- Sasagawa T, Matsui M, Kobayashi Y, Otagiri M, Moriya S, Sakamoto Y, Ito Y, Lee CC, Kitamoto K, Arioka M (2011) High-throughput recombinant gene expression systems in Pichia pastoris using newly developed plasmid vectors. Plasmid 65(1):65–69. https://doi.org/10.1016/j.plasmid.2010.08.004 CrossRefPubMedGoogle Scholar
- Topakas E, Moukouli M, Dimarogona M, Vafiadi C, Christakopoulos P (2010) Functional expression of a thermophilic glucuronyl esterase from Sporotrichum thermophile: identification of the nucleophilic serine. Appl Microbiol Biotechnol 87(5):1765–1772. https://doi.org/10.1007/s00253-010-2655-7 CrossRefPubMedGoogle Scholar
- Uchima CA, Tokuda G, Watanabe H, Kitamoto K, Arioka M (2011) Heterologous expression and characterization of a glucose-stimulated β-glucosidase from the termite Neotermes koshunensis in Aspergillus oryzae. Appl Microbiol Biotechnol 89(6):1761–1771. https://doi.org/10.1007/s00253-010-2963-y CrossRefPubMedGoogle Scholar
- Vafiadi C, Topakas E, Biely P, Christakopoulos P (2009) Purification, characterization and mass spectrometric sequencing of a thermophilic glucuronoyl esterase from Sporotrichum thermophile. FEMS Microbiol Lett 296(2):178–184. https://doi.org/10.1111/j.1574-6968.2009.01631.x CrossRefPubMedGoogle Scholar
- Wong DWS, Chan VJ, McCormack AA, Hirsch J, Biely P (2012) Functional cloning and expression of the Schizophyllum commune glucuronoyl esterase gene and characterization of the recombinant enzyme. Biotechnol Res Int 2012:951267. https://doi.org/10.1155/2012/951267 CrossRefPubMedPubMedCentralGoogle Scholar