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Characterization of two distinct feruloyl esterases, AoFaeB and AoFaeC, from Aspergillus oryzae


Two hypothetical proteins XP_001818628 and XP_001819091 (designated AoFaeB and AoFaeC, respectively), showing sequence identity with known type-C feruloyl esterases, have been found in the genomic sequence of Aspergillus oryzae. We cloned the putative A. oryzae feruloyl esterase-encoding genes and expressed them in Pichia pastoris. Both purified recombinant AoFaeB (rAoFaeB) and AoFaeC (rAoFaeC) had apparent relative molecular masses of 61,000 and 75,000, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After N-deglycosylation, both proteins had a relative molecular mass of 55,000. The optimum pH for rAoFaeB was 6.0, although it was stable at pH values ranging from 3.0 to 9.0; rAoFaeC had an optimum pH of 6.0 and was stable in the pH range of 7.0–10.0. Thermostability of rAoFaeC was greater than that of rAoFaeB. Whereas rAoFaeC displayed hydrolytic activity toward methyl caffeate, methyl p-coumarate, methyl ferulate, and methyl sinapate, rAoFaeB displayed hydrolytic activity toward methyl caffeate, methyl p-coumarate, and methyl ferulate but not toward methyl sinapate. Substrate specificity profiling of rAoFaeB and rAoFaeC revealed type-B and type-C feruloyl esterases, respectively. Ferulic acid was efficiently released from wheat arabinoxylan when both esterases were applied with xylanase from Thermomyces lanuginosus. Both recombinant proteins also exhibited hydrolytic activity toward chlorogenic acid.

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  1. Ardiansyah, Ohsaki H, Shirakawa H, Koseki T, Komai M (2008) Novel effects of a single administration of ferulic acid on the regulation of blood pressure and the hepatic lipid metabolic profile in stroke-prone spontaneously hypertensive rats. J Agric Food Chem 56:2825–2830

  2. Asther M, Estrada Alvarado MI, Haon M, Navarro D, Asther M, Lesage-Messen L, Record E (2005) Purification and characterization of a chlorogenic acid hydrolase from Aspergillus niger catalyzing the hydrolysis of chlorogenic acid. J Biotechnol 115:47–56

  3. 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 580:5815–5821

  4. Benoit I, Asther A, Bourne Y, Navarro D, Canaan S, Lesage-Meessen L, Herweijer M, Coutinho PM, Asther M, Record E (2007) Gene overexpression and biochemical characterization of the biotechnologically relevant chlorogenic acid hydrolase from Aspergillus niger. Appl Environ Microbiol 73:5624–5632

  5. Benoit I, Danchin EGJ, Bleichrodt RJ, de Vries RP (2008) Biotechnological applications and potential of fungal feruloyl esterases based on prevalence, classification and biochemical diversity. Biotechnol Lett 30:387–396

  6. Brenner S (1998) The molecular evolution of genes and proteins: a tale of two serines. Nature 334:528–530

  7. Conde R, Cueva R, Pablo G, Polaina J, Larriba G (2004) A search for hyperglycosylation signals in yeast glycoproteins. J Biol Chem 279:43789–43798

  8. Crepin VF, Faulds CB, Connerton IF (2003) A non-modular type B feruloyl esterase from Neurospora crassa exhibits concentration-dependent substrate inhibition. Biochem J 370:417–427

  9. Crepin VF, Faulds CB, Connerton IF (2004a) Identification of a type-D feruloyl esterase from Neurospora crassa. Appl Microbiol Biotechnol 63:567–570

  10. Crepin VF, Faulds CB, Connerton IF (2004b) Functional classification of the microbial feruloyl esterases. Appl Microbiol Biotechnol 63:647–652

  11. de Vries RP, van Kuyk PA, Kester HCM, Visser J (2002) The Aspergillus niger faeB gene encodes a second feruloyl esterase involved in pectin and xylan degradation and is specifically induced in the presence of aromatic compounds. Biochem J 363:377–386

  12. Faulds CB, Williamson G (1994) Purification and characterization of a ferulic acid esterase (FAE-III) from Aspergillus niger: Specificity for the phenolic moiety and binding to microcrystalline cellulose. Microbiology 140:779–787

  13. Garcia-Conesa MT, Crepin VF, Goldson AJ, Williamson G, Cummings NJ, Connerton IF, Faulds CB, Kroon PA (2004) The feruloyl esterase system of Talaromyces stipitatus: production of three discrete feruloyl esterases, including a novel enzyme, TsFaeC, with a broad substrate specificity. J Biotechnol 108:227–241

  14. Guglielmetti S, de Noni I, Caracciolo F, Molinari F, Parini C, Mora D (2008) Bacterial cinnamoyl esterase activity screening for the production of a novel functional food product. Appl Environ Microbiol 74:1284–1288

  15. Higgins D, Thompson J, Gibson T (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

  16. Juge N, Williamson G, Puigserver A, Cummings NJ, Connerton IF, Faulds CB (2001) High-level production of recombinant Aspergillus niger cinnamoyl esterase (FAEA) in the methylotrophic yeast Pichia pastoris. FEMS Yeast Res 1:127–132

  17. Kainz E, Gallmetzer A, Hatzl C, Nett JH, Li H, Schinko T, Pachlinger R, Berger H, Reyes-Domingues Y, Bernreiter A, Gerngross T, Wildt S, Strauss J (2008) N-Glycan modification in Aspergillus species. Appl Environ Microbiol 74:1076–1086

  18. Koseki T, Ito Y, Furuse S, Ito K, Iwano K (1996) Conversion of ferulic acid into 4-vinylguaiacol, vanillin and vanillic acid in model solutions of shochu. J Ferment Bioeng 82:46–50

  19. Koseki T, Takahashi K, Fushinobu S, Iefuji H, Iwano K, Hashizume K, Matsuzawa H (2005) Mutational analysis of a feruloyl esterase from Aspergillus awamori involved in substrate discrimination and pH dependence. Biochim Biophys Acta 1722:200–208

  20. Koseki T, Takahashi K, Handa T, Yamane Y, Fushinobu S, Hashizume K (2006) N-linked oligosaccharides of Aspergillus awamori feruloyl esterase are important for thermostability and catalysis. Biosci Biotechnol Biochem 70:2476–2480

  21. Koseki T, Mese Y, Fushinobu S, Masaki K, Fujii T, Ito K, Shiono Y, Murayama T, Iefuji H (2008) Biochemical characterization of a glycoside hydrolase family 61 endoglucanase from Aspergillus kawachii. Appl Microbiol Biotechnol 77:1279–1285

  22. Kroon PA, Garcia-Conesa MT, Fillingham IJ, Hazlewood GP, Williamson G (1999) Release of ferulic acid dehydrogimers from plant cell walls by feruloyl esterases. J Sci Food Agric 79:428–434

  23. Kroon PA, Williamson G, Fish NM, Archer DB, Belshaw NJ (2000) A modular esterase from Penicillium funiculosum which releases ferulic acid from plant cell walls and binds crystalline cellulose contains a carbohydrate binding module. Eur J Biochem 267:6740–6752

  24. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

  25. Lesage-Meessen L, Delattre M, Haon M, Thibault JF, Ceccaldi BC, Brunerie P, Asther M (1996) A two-step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus. J Biotechnol 50:107–113

  26. Levasseur A, Benoit I, Asther M, Asther M, Record E (2004) Homologous expression of the feruloyl esterase B gene from Aspergillus niger and characterization of the recombinant enzyme. Protein Expr Purif 37:126–133

  27. Mathew S, Abraham TE (2004) Ferulic acid: an antioxidant found naturally in plant cell walls and feruloyl esterases involved in its release and their applications. Crit Rev Biotechnol 24:59–83

  28. Moukouli M, Topakas E, Christakopoulos P (2008) Cloning, characterization and functional expression of an alkalitorelant type C feruloyl esterase from Fusarium oxysporum. Appl Microbiol Biotechnol 79:245–254

  29. Oddou J, Stentelaire C, Lesage-Meesen L, Asther M, Ceccaldi BC (1999) Improvement of ferulic acid bioconversion into vanillin by use of high-density cultures of Pycnoporus cinnabarinus. Appl Microbiol Biotechnol 53:1–6

  30. Puchart V, Vršanská M, Mastihubová M, Topakas E, Vafiadi C, Faulds CB, Tenkanen M, Christakopoulos P, Biely P (2007) Substrate and positional specificity of feruloyl esterases for monoferuloylated and monoacetylated 4-nitrophenyl glycoside. J. Biotechnol 127:235–243

  31. Reddy A, Gibbs BS, Liu YL, Coward JK, Changchien LM, Maley F (1999) Glycosylation of the overlapping sequins in yeast external invertase: effect of amino acid variation on site selectively in vivo and in vitro. Glycobiology 9:547–555

  32. Shin HD, Chen RR (2007) A type B feruloyl esterase from Aspergillus nidulans with broad pH applicability. Appl Microbiol Biotechnol 73:1323–1330

  33. Sundberg M, Poutanen K, Makkanen P, Linko M (1990) An extracellular esterase of Aspergillus awamori. Biotechnol Appl Biochem 12:670–680

  34. Tenkanen M, Schuseil J, Puls J, Poutanen K (1991) Production, purification and characterization of an esterase liberating phenolic acids from lignocellulosics. J Biotechnol 18:69–84

  35. Topakas E, Vafiadi C, Christakopoulos P (2007) Microbial production, characterization and applications of feruloyl esterases. Process Biochem 42:497–509

  36. Williamson G, Kroon PA, Faulds CB (1998) Hairy plant polysaccharides: a close shave with microbial esterases. Microbiology 144:2011–2023

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This work was supported by the Japan Society for the Promotion of Science, with Grant-in-Aid for Scientific Research no. 20580071 (to T. K.).

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Correspondence to Takuya Koseki.

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Koseki, T., Hori, A., Seki, S. et al. Characterization of two distinct feruloyl esterases, AoFaeB and AoFaeC, from Aspergillus oryzae . Appl Microbiol Biotechnol 83, 689–696 (2009).

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  • Feruloyl esterase
  • Plant cell wall
  • Ferulic acid
  • Aspergillus oryzae