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Isolation and characterization of a ferulic acid esterase (Fae1A) from the rumen fungus Anaeromyces mucronatus

Qi, M., Wang, P., Selinger, L.B., Yanke, L.J., Forster, R.J., Mcallister, T.A. (2011). Isolation and characterization of a ferulic acid esterase (Fae1A) from the rumen fungus Anaeromyces mucronatus, 110(5), 1341-1350. http://dx.doi.org/10.1111/j.1365-2672.2011.04990.x

Abstract

Aims: A novel ferulic acid esterase gene from rumen fungus Anaeromyces mucronatus was cloned, heteroexpressed in Escherichia coli and characterized. Methods and Results: A total of 30 clones exhibiting activity on α-naphthyl acetate (α-NA) were isolated from an A. mucronatus YE505 cDNA library. Sequence analysis revealed that these clones represented two esterase-coding sequences. The gene, fae1A, showed highest amino acid sequence identity to CE family 1 esterases from anaerobic micro-organisms such as Orpinomyces sp., Ruminococcus albus and Clostridium thermocellum. The gene comprised 828 nucleotides encoding a polypeptide of 275 amino acids. The coding sequence was cloned into the pET30a expression vector and overexpressed in E. coli BL21 (DE3). Gene product Fae1A was found to exhibit activity against a number of substrates including naphthyl fatty acid esters, p-nitrophenyl fatty acid esters and hydroxylcinnamic acid esters. Conclusions: Fae1A exhibited a lower Km and higher catalytic efficiency (kcat/Km) on ferulic acid esters than on α-NA or p-nitrophenyl acetate, suggesting that it has a higher affinity for ethyl and methyl ferulate than for the acetyl esters. It releases ferulic acid and p-coumaric acid from barley straw. Activity of Fae1A was inhibited by the serine-specific protease inhibitor, phenylmethylsulfonyl fluoride, indicating that a serine residue plays a role in its activity. Significance and Impact of the Study: To our knowledge, this is the first report of characterization of carbohydrate esterase gene from the genus of Anaeromyces. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

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