Abstract
A feruloyl esterase (StFAE-A) produced by Sporotrichum thermophile was purified to homogeneity. The purified homogeneous preparation of native StFAE-A exhibited a molecular mass of 57.0±1.5 kDa, with a mass of 33±1 kDa on SDS-PAGE. The pI of the enzyme was estimated by cation-exchange chromatofocusing to be at pH 3.1. The enzyme activity was optimal at pH 6.0 and 55–60 °C. The purified esterase was stable at the pH range 5.0–7.0. The enzyme retained 70% of activity after 7 h at 50 °C and lost 50% of its activity after 45 min at 55 °C and after 12 min at 60 °C. Determination of k cat/K m revealed that the enzyme hydrolyzed methyl p-coumarate 2.5- and 12-fold more efficiently than methyl caffeate and methyl ferulate, respectively. No activity on methyl sinapinate was detected. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose and it hydrolyzed 4-nitrophenyl 5-O-trans-feruloyl-α-l-arabinofuranoside (NPh-5-Fe-Araf) 2-fold more efficiently than NPh-2-Fe-Araf. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with xylanase from S. thermophile (a maximum of 34% total ferulic acid released after 1 h incubation). StFAE-A by itself could release FA, but at a level almost 47-fold lower than that obtained in the presence of xylanase. The potential of StFAE-A for the synthesis of various phenolic acid esters was tested using a ternary water-organic mixture consisting of n-hexane, 1-butanol and water as a reaction system.
References
Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254
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
Davis BJ (1965) Disc electrophoresis II. Method and application to human serum proteins. Ann NY Acad Sci 121:404–427
Donaghy J, Bronnenmeier K, Soto-Kelly PF, McKay AM (2000) Purification and characterization of an extracellular feruloyl esterase from the thermophilic anaerobe Clostridium stercorarium. J Appl Microbiol 88:458–466
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
Guyot B, Bosquette B, Pina M, Graille J (1997) Esterification of phenolic acids from green coffee with an immobilized lipase from Candida antarctica in solvent-free medium. Biotechnol Lett 19:529–532
Kang X, Frey D.D (2003) High-performance cation-exchange chromatofocusing of proteins. J Chrom 991:117–128
Katapodis P, Vrsanska M, Kekos D, Nerinckx W, Biely P, Claeyssens M, Macris BJ, Christakopoulos P (2003) Biochemical and catalytic properties of an endoxylanase purified from the culture filtrate of Sporotrichum thermophile. Carbohydr Res 338:1881–1890
Khmelnitsky YL, Hilhorst R, Veeger C (1988) Detergentless microemulsions as media for enzymatic reactions. Eur J Biochem 176:265–271
Kroon PA, Faulds CB, Williamson G (1996) Purification and characterization of a novel esterase induced by growth of Aspergillus niger on sugar-beet pulp. Biotechnol Appl Biochem 23:255–262
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
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Mastihubova M, Szemesova J, Biely P (2002) Two efficient ways to 2-O- and 5-O-feruloylated 4-nitrophenyl alpha-l-arabinofuranosides as substrates for differentiation of feruloyl esterases. Tetrahedron Lett 44:1671–1673
Ralet M-C, Faulds CB, Williamson G, Thibault J-F (1994) Degradation of feruloylated oligosaccharides from sugar-beet pulp and wheat bran by ferulic acid esterases from Aspergillus niger. Carbohydr Res 263:257–269
Stamatis H, Sereti V, Kolisis FN (1999) Studies on the enzymatic synthesis of lipophilic derivatives of natural antioxidants. J Am Oil Chem Soc 76:1505–1510
Topakas E, Kalogeris E, Kekos D, Macris BJ, Christakopoulos P (2003a) Production and partial characterization of feruloyl esterase by Sporotrichum thermophile under solid-state fermentation. Proc Biochem 38:1539–1543
Topakas E, Stamatis H, Biely P, Kekos D, Macris BJ, Christakopoulos P (2003b) Purification and characterization of a feruloyl esterase from Fusarium oxysporum catalyzing esterification of phenolic acids in ternary water-organic solvent mixtures. J Biotechnol 102:33–44
Topakas E, Stamatis H, Mastihubova M, Biely P, Kekos D, Macris BJ, Christakopoulos P (2003c) Purification and characterization of a Fusarium oxysporum feruloyl esterase (FoFAE-I) catalysing transesterification of phenolic acid esters. Enzyme Microb Technol 33:729–737
Williamson G, Faulds CB, Kroon PA (1998) Specificity of ferulic acid (feruloyl esterases). Biochem Soc Trans 26:205–209
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Topakas, E., Stamatis, H., Biely, P. et al. Purification and characterization of a type B feruloyl esterase (StFAE-A) from the thermophilic fungus Sporotrichum thermophile . Appl Microbiol Biotechnol 63, 686–690 (2004). https://doi.org/10.1007/s00253-003-1481-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-003-1481-6