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Expression, characterization and structural modelling of a feruloyl esterase from the thermophilic fungus Myceliophthora thermophila

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Abstract

A ferulic acid esterase (FAE) from the thermophilic fungus Myceliophthora thermophila (synonym Sporotrichum thermophile), belonging to the carbohydrate esterase family 1 (CE-1), was functionally expressed in methylotrophic yeast Pichia pastoris. The putative FAE from the genomic DNA was successfully cloned in P. pastoris X-33 to confirm that the enzyme exhibits FAE activity. The recombinant FAE was purified to its homogeneity (39 kDa) and subsequently characterized using a series of model substrates including methyl esters of hydroxycinnamates, alkyl ferulates and monoferuloylated 4-nitrophenyl glycosides. The substrate specificity profiling reveals that the enzyme shows a preference for the hydrolysis of methyl caffeate and p-coumarate and a strong preference for the hydrolysis of n-butyl and iso-butyl ferulate. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose, whilst it was found capable of de-esterifying acetylated glucuronoxylans. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with an M3 xylanase from Trichoderma longibrachiatum (a maximum of 41% total FA released after 1 h incubation). Prediction of the secondary structure of MtFae1a was performed in the PSIPRED server whilst modelling the 3D structure was accomplished by the use of the HH 3D structure prediction server.

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Acknowledgements

The authors thank Dr. Peter Biely from Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia, for supplying the acetylated glucuronoxylans. They would also like to thank Dr. Dimitrios J. Stravopodis, Assistant Professor of the Department of Cell Biology and Biophysics, Faculty of Biology, National and Kapodistrian University of Athens, for his scientific contribution on the OEPCR technique. Maria Moukouli thanks the Hellenic State Scholarships Foundation for a grant.

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  1. BIOtechMASS Unit, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str, Zografou Campus, 15700, Athens, Greece

    Evangelos Topakas, Maria Moukouli, Maria Dimarogona & Paul Christakopoulos

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  1. Evangelos Topakas
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  2. Maria Moukouli
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  3. Maria Dimarogona
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  4. Paul Christakopoulos
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Correspondence to Paul Christakopoulos.

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Evangelos Topakas and Maria Moukouli made equal contribution to this work.

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Topakas, E., Moukouli, M., Dimarogona, M. et al. Expression, characterization and structural modelling of a feruloyl esterase from the thermophilic fungus Myceliophthora thermophila . Appl Microbiol Biotechnol 94, 399–411 (2012). https://doi.org/10.1007/s00253-011-3612-9

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