Advertisement

Applied Microbiology and Biotechnology

, Volume 102, Issue 5, pp 2191–2201 | Cite as

A novel glucuronoyl esterase from Aspergillus fumigatus—the role of conserved Lys residue in the preference for 4-O-methyl glucuronoyl esters

  • Hung Hiep Huynh
  • Nozomi Ishii
  • Ichiro Matsuo
  • Manabu Arioka
Biotechnologically relevant enzymes and proteins

Abstract

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.

Keywords

Aspergillus fumigatus Catalytic triad Glucuronoyl esterase Methoxy group Preference 

Notes

Acknowledgements

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.

Funding information

This work was supported by a Grant-in-Aid for Scientific Research (No. 16K14879) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) and a research grant from the Institute for Fermentation, Osaka. H. H. H. is a recipient of the MEXT Scholarship.

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 performed by any of the authors.

Supplementary material

253_2018_8739_MOESM1_ESM.pdf (84 kb)
ESM 1 (PDF 84 kb)

References

  1. Adav SS, Ravindran A, Sze SK (2015) Quantitative proteomic study of Aspergillus fumigatus secretome revealed deamidation of secretory enzymes. J Proteome 119:154–168.  https://doi.org/10.1016/j.jprot.2015.02.007 CrossRefGoogle Scholar
  2. Agger JW, Busk PK, Pilgaard B, Meyer AS, Lange L (2017) A new functional classification of glucuronoyl esterases by peptide pattern recognition. Front Microbiol 8:1–8.  https://doi.org/10.3389/fmicb.2017.00309 CrossRefGoogle Scholar
  3. Bååth J, Giummarella N, Klaubauf S, Lawoko M, Olsson L (2016) A glucuronoyl esterase from Acremonium alcalophilum cleaves native lignin-carbohydrate ester bonds. FEBS Lett 590(16):2611–2618.  https://doi.org/10.1002/1873-3468.12290 CrossRefGoogle Scholar
  4. 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
  5. Biely P (2016) Microbial glucuronoyl esterases: ten years after discovery. Appl Environ Microbiol 82(24):7014–7018.  https://doi.org/10.1128/AEM.02396-16 CrossRefPubMedPubMedCentralGoogle Scholar
  6. Biely P, Maloviková A, Uhliariková I, Li XL, Wong DWS (2015) Glucuronoyl esterases are active on the polymeric substrate methyl esterified glucuronoxylan. FEBS Lett 589(18):2334–2339.  https://doi.org/10.1016/j.febslet.2015.07.019 CrossRefPubMedGoogle Scholar
  7. 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(Database):D233–D238.  https://doi.org/10.1093/nar/gkn663 CrossRefPubMedGoogle Scholar
  8. 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
  9. 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
  10. Clark SE, Muslin EH, Henson CA (2004) Effect of adding and removing N-glycosylation recognition sites on the thermostability of barley α-glucosidase. Protein Eng Des Sel 17(3):245–249.  https://doi.org/10.1093/protein/gzh028 CrossRefPubMedGoogle Scholar
  11. 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
  12. d’Errico C, Börjesson J, Ding H, Krogh KBRM, Spodsberg N, Madsen R, Monrad RN (2016) Improved biomass degradation using fungal glucuronoyl esterases-hydrolysis of natural corn fiber substrate. J Biotechnol 219:117–123.  https://doi.org/10.1016/j.jbiotec.2015.12.024 CrossRefPubMedGoogle Scholar
  13. De Santi C, Willassen NP, Williamson A (2016) Biochemical characterization of a family 15 carbohydrate esterase from a bacterial marine arctic metagenome. PLoS One 11(7):1–22.  https://doi.org/10.1371/journal.pone.0159345 CrossRefGoogle Scholar
  14. Ďuranová M, Hirsch J, Kolenová K, Biely P (2009a) Fungal glucuronoyl esterases and substrate uronic acid recognition. Biosci Biotechnol Biochem 73(11):2483–2487.  https://doi.org/10.1271/bbb.90486 CrossRefPubMedGoogle Scholar
  15. Ďuranová M, Špániková S, Wösten HAB, Biely P, De Vries RP (2009b) Two glucuronoyl esterases of Phanerochaete chrysosporium. Arch Microbiol 191(2):133–140.  https://doi.org/10.1007/s00203-008-0434-y CrossRefPubMedGoogle Scholar
  16. Grosdidier A, Zoete V, Michielin O (2011) SwissDock, a protein-small molecule docking web service based on EADock DSS. Nucleic Acids Res 39(suppl):270–277.  https://doi.org/10.1093/nar/gkr366 CrossRefGoogle Scholar
  17. Han Y, Lei XG (1999) Role of glycosylation in the functional expression of an Aspergillus niger phytase (phyA) in Pichia pastoris. Arch Biochem Biophys 364(1):83–90.  https://doi.org/10.1006/abbi.1999.1115 CrossRefPubMedGoogle Scholar
  18. Hüttner S, Klaubauf S, de Vries RP, Olsson L (2017) Characterisation of three fungal glucuronoyl esterases on glucuronic acid ester model compounds. Appl Microbiol Biotechnol 101(13):5301–5311.  https://doi.org/10.1007/s00253-017-8266-9 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Huynh HH, Arioka M (2016) Functional expression and characterization of a glucuronoyl esterase from the fungus Neurospora crassa: identification of novel consensus sequences. J Gen Appl Microbiol 62(5):1–8.  https://doi.org/10.2323/jgam.2016.03.004 CrossRefGoogle Scholar
  20. Jeffries TW (1994) Biodegradation of lignin and hemicelluloses. In: Ratledge C (ed) Biochemistry of microbial degradation. Kluwer academic Publisher, Madison, pp 233–277CrossRefGoogle Scholar
  21. 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
  22. Latgé J-P (1999) Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev 12(2):310–350PubMedPubMedCentralGoogle Scholar
  23. Li X-L, Špániková S, de Vries RP, Biely P (2007) Identification of genes encoding microbial glucuronoyl esterases. FEBS Lett 581(21):4029–4035.  https://doi.org/10.1016/j.febslet.2007.07.041 CrossRefPubMedGoogle Scholar
  24. Mabashi Y, Kikuma T, Maruyama J, Arioka M (2006) Development of a versatile expression plasmid construction system for Aspergillus oryzae and its application to visualization of mitochondria. Biosci Biotechnol Biochem 70(8):1882–1889.  https://doi.org/10.1271/bbb.60052 CrossRefPubMedGoogle Scholar
  25. Motohashi K (2015) Seamless ligation cloning extract (SLiCE) method using cell lysates from laboratory Escherichia coli strains and its application to slip site-directed mutagenesis. Methods Mol Biol 1498:349–357.  https://doi.org/10.1007/978-1-4939-6472-7_23 CrossRefGoogle Scholar
  26. Pokkuluri PR, Duke NEC, Wood SJ, Cotta MA, Li XL, Biely P, Schiffer M (2011) Structure of the catalytic domain of glucuronoyl esterase Cip2 from Hypocrea jecorina. Proteins Struct Funct Bioinforma 79(8):2588–2592.  https://doi.org/10.1002/prot.23088 CrossRefGoogle Scholar
  27. 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
  28. Špániková S, Biely P (2006) Glucuronoyl esterase—novel carbohydrate esterase produced by Schizophyllum commune. FEBS Lett 580(19):4597–4601.  https://doi.org/10.1016/j.febslet.2006.07.033 CrossRefPubMedGoogle Scholar
  29. Špániková S, Poláková M, Joniak D, Hirsch J, Biely P (2007) Synthetic esters recognized by glucuronoyl esterase from Schizophyllum commune. Arch Microbiol 188(2):185–189.  https://doi.org/10.1007/s00203-007-0241-x CrossRefPubMedGoogle Scholar
  30. Sunner H, Charavgi M-D, Olsson L, Topakas E, Christakopoulos P (2015) Glucuronoyl esterase screening and characterization assays utilizing commercially available benzyl glucuronic acid ester. Molecules 20(10):17807–17817.  https://doi.org/10.3390/molecules201017807 CrossRefPubMedGoogle Scholar
  31. 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
  32. 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
  33. 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
  34. 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

Copyright information

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

Authors and Affiliations

  • Hung Hiep Huynh
    • 1
  • Nozomi Ishii
    • 2
  • Ichiro Matsuo
    • 2
  • Manabu Arioka
    • 1
  1. 1.Department of BiotechnologyThe University of TokyoTokyoJapan
  2. 2.Department of Chemistry and Chemical BiologyGunma UniversityMaebashiJapan

Personalised recommendations