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Applied and Environmental Microbiology, September 2003, p. 5622-5626, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5622-5626.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Purification and Properties of a Feruloyl Esterase Involved in Lignocellulose Degradation by Aureobasidium pullulans

Karl Rumbold,1 Peter Biely,2 Maria Mastihubová,2 Marinka Gudelj,3 Georg Gübitz,3 Karl-Heinz Robra,3 and Bernard A. Prior1*

Department of Microbiology, University of Stellenbosch, 7602 Matieland, South Africa,1 Institute of Chemistry, Slovak Academy of Sciences, 84238 Bratislava, Slovakia,2 Institute for Environmental Biotechnology, Graz University of Technology, 8010 Graz, Austria3

Received 12 February 2003/ Accepted 29 June 2003

The lignocellulolytic fungus Aureobasidium pullulans NRRL Y 2311-1 produces feruloyl esterase activity when grown on birchwood xylan. Feruloyl esterase was purified from culture supernatant by ultrafiltration and anion-exchange, hydrophobic interaction, and gel filtration chromatography. The pure enzyme is a monomer with an estimated molecular mass of 210 kDa in both native and denatured forms and has an apparent degree of glycosylation of 48%. The enzyme has a pI of 6.5, and maximum activity is observed at pH 6.7 and 60°C. Specific activities for methyl ferulate, methyl p-coumarate, methyl sinapate, and methyl caffeate are 21.6, 35.3, 12.9, and 30.4 µmol/min/mg, respectively. The pure feruloyl esterase transforms both 2-O and 5-O arabinofuranosidase-linked ferulate equally well and also shows high activity on the substrates 4-O-trans-feruloyl-xylopyranoside, O-{5-O-[(E)-feruloyl]-{alpha}-L-arabinofuranosyl}-(1,3)-O-ß-D-xylopyranosyl-(1,4)-D-xylopyranose, and p-nitrophenyl-acetate but reveals only low activity on p-nitrophenyl-butyrate. The catalytic efficiency (kcat/Km) of the enzyme was highest on methyl p-coumarate of all the substrates tested. Sequencing revealed the following eight N-terminal amino acids: AVYTLDGD.

* Corresponding author. Mailing address: Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa. Phone: 27-21-808-5856. Fax: 27-21-808-5846. E-mail: bap{at}sun.ac.za.

Applied and Environmental Microbiology, September 2003, p. 5622-5626, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5622-5626.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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