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. A maximum of
205±5.7 µg FA and 0.84±0.1 µg diFA were released (37°C, pH 6.5, 2 hr) when a saturating amount of
RuFae4 (23 nmole for 100 mg WIA) was used. These yields represent 48.3% of FA, and 6.6% of diFAs present in the
WIA substrate. 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 it was primarily the GH10 enzyme contributing synergism to FA/diFA release in mixed reactions.
Keywords: Arabinoxylan, ferulic acid, ferulic acid esterase, feruloyl esterase, metagenomics.
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