Cloning of a Novel Feruloyl Esterase from Rumen Microbial Metagenome for Substantial Yield of Mono- and Diferulic Acids from Natural Substrates

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.