A novel feruloyl esterase from rumen microbial metagenome: Gene cloning and enzyme characterization in the release of mono- and diferulic acids

Authors: Wong, Dominic; Takeoka, Gary; Chan, Victor; LIAO, HANS - Opx Biotechnologies, Inc; MARAKAMI, MARIO - Brazilian Biosciences National Laboratory (LNBIO)

Submitted to: Protein and Peptide Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/10/2015
Publication Date: 6/29/2015
Citation: Wong, D., Takeoka, G.R., Chan, V.J., Liao, H., Marakami, M. 2015. A novel feruloyl esterase from rumen microbial metagenome: Gene cloning and enzyme characterization in the release of mono- and diferulic acids. Protein and Peptide Letters. 22(2):681-688.

Interpretive Summary: Plant cell walls are rich in ferulic acids present as side groups of various polysaccharides. These phenolic acids are oxidatively coupled through its aromatic ring or aliphatic group to form diferulates, resulting in polysaccharide-polysaccharide, polysaccharide-lignin, and polysaccharide-protein polymeric dimers. Ferulic acid-mediated crosslinking is a major cause of the recalcitrance of biomass against enzymatic digestion. Our laboratory has isolated and characterized several genes for feruloyl esterases, one of which encoding an enzyme that could release both mono- and diferulates from xylan polymers. The yield increased significantly with cooperative action of a family 10 endoxylanase. This report presents extensive characterization of the gene and the enzyme as potential use in increasing the digestibility of plant cell wall polymers.

Technical Abstract: A feruloyl esterase (FAE) gene was isolated from a rumen microbial metagenome, cloned into E. coli, and expressed in active form. The enzyme (RuFae4) was classified as a Type D feruloyl esterase based on its action on synthetic substrates and ability to release diferulates. The RuFae4 alone released ferulic acid (FA) and diferulic acid (diFA) from wheat insoluble arabinoxylan (WIA) and other natural substrates. The diFA released was confirmed by mass spectrometry. Using a saturating amount of RuFae4 (23 nmole for 100 mg WIA), a maximum of 205±5.7 'g FA and 0.84±0.1 'g diFA were released (37oC, pH 6.5, 2 hr). These yields represent 48.3% of FA, and 6.6% of diFAs compared to those present in saponified WIA. Addition of GH10 endoxylanase (EX) to RuFae4 both at 1 nmole concentrations increased the release of FA and diFAs by 17 and 10 fold, respectively. Addition of GH11 EX resulted in smaller increase in the amount of both FA and diFAs. Applying additive amount of the two enzymes did not lead to additive increase in the product yields, suggesting that the GH11 enzyme did not contribute synergistically to FA/diFA release in mixed reactions.