Sugarcane pith as a lignocellulosic substrate for determination of feruloyl esterase activity

Ferulic acid (FA) limit cell wall digestibility due to its cross linkages between lignin and hemicellulose. Some microorganisms produce esterases that act on the ester linkages, releasing FA from the lignocellulosic substrate. Usually, these enzymes are assayed by using non-cellulosic substrates. The low-recalcitrance pith region of two different low-lignin content sugarcane hybrids (58 and 89) and a reference plant (RB) were used as a new type of substrate for determination of feruloyl esterases activity. These materials were pretreated with commercial cellulases to provide a hemicellulose-enriched substrate. Prepared solids were used to assay Rumen and Clostridium feruloyl esterases. Time-course release of FA pointed out for maximal conversion rates in the range of 1-3%. Therefore, 2% FA conversion was fixed as a target conversion level and enzyme loading was adjusted to reach this value in 5-min reactions. The sugarcane substrates prepared from the hybrids with originally lower lignin content appeared as better substrates owing to their lower recalcitrance. Data enabled to determine the enzymatic activities using this new cellulosic substrate, as well permitted to evaluate pH and temperature dependence of the enzymes. Rumen feruloyl esterase presented an optimum reaction pH in the range of 6.3 to 7.2 and maximal activity at 40 ^oC, agreeing with data obtained with synthetic substrates.