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Applied and Environmental Microbiology, December 2008, p. 7482-7489, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.00807-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Novel Family of Carbohydrate Esterases, Based on Identification of the Hypocrea jecorina Acetyl Esterase Gene{triangledown} ,{dagger}

Xin-Liang Li,1* Christopher D. Skory,2 Michael A. Cotta,1 Vladimir Puchart,3 and Peter Biely3

Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, United States Department of Agriculture-Agricultural Research Service, 1815 N. University Street, Peoria, Illinois 61604,1 Bioproducts and Biocatalysis Research Unit, National Center for Agricultural Utilization Research, United States Department of Agriculture-Agricultural Research Service, 1815 N. University Street, Peoria, Illinois 61604,2 Institute of Chemistry, Slovak Academy of Sciences, 845 38 Bratislava, Slovakia3

Received 8 April 2008/ Accepted 6 October 2008

Plant cell walls have been shown to contain acetyl groups in hemicelluloses and pectin. The gene aes1, encoding the acetyl esterase (Aes1) of Hypocrea jecorina, was identified by amino-terminal sequencing, peptide mass spectrometry, and genomic sequence analyses. The coded polypeptide had 348 amino acid residues with the first 19 serving as a secretion signal peptide. The calculated molecular mass and isoelectric point of the secreted enzyme were 37,088 Da and pH 5.89, respectively. No significant homology was found between the predicated Aes1 and carbohydrate esterases of known families, but putative aes1 orthologs were found in genomes of many fungi and bacteria that produce cell wall-degrading enzymes. The aes1 transcript levels were high when the fungal cells were induced with sophorose, cellulose, oat spelt xylan, lactose, and arabinose. The recombinant Aes1 produced by H. jecorina transformed with aes1 under the cellobiohydrolase I promoter displayed properties similar to those reported for the native enzyme. The enzyme hydrolyzed acetate ester bond specifically. Using 4-nitrophenyl acetate as substrate, the activity of the recombinant enzyme was enhanced by D-xylose, D-glucose, cellobiose, D-galactose, and xylooligosaccharides but not by arabinose, mannose, or lactose. With the use of 4-nitrophenyl-β-D-xylopyranoside monoacetate as substrate in a β-xylosidase-coupled assay, Aes1 hydrolyzed positions 3 and 4 with the same efficiency while the H. jecorina acetylxylan esterase 1 exclusively deacetylated the position 2 acetyl group. Aes1 was capable of transacetylating methylxyloside in aqueous solution. The data presented demonstrate that Aes1 and other homologous microbial proteins may represent a new family of esterases for lignocellulose biodegradation.

* Corresponding author. Present address: Youtell Biotechnology, Inc., Suite 100, North Creek Corporate Center, 19310 North Creek Parkway, Bothell, WA 98011. Phone: (425) 485-8218. Fax: (425) 485-8830. E-mail: shinli88{at}hotmail.com

{triangledown} Published ahead of print on 31 October 2008.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, December 2008, p. 7482-7489, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.00807-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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