Feruloyl esterase (rumen microorganism)

Uncharacterized side activities of enzymes can have significant negative effects on reaction products and yields. Hence, their identification and characterization is crucial for the development of successful reaction systems. Here, we report the presence of feruloyl esterase activity in CtXyn5A from Acetivibrio thermocellus besides its well-known arabinoxylanase activity for the first time. Both reaction types appear to be catalysed in the same active site in two subsequential steps. The ferulic acid substituent is cleaved off first, followed by the hydrolysis of the xylan backbone. The esterase activity on complex carbohydrates was found to be higher than the one of a designated ferulic acid esterase (E-FAERU). Therefore, we conclude that the enzyme exhibits a dual function rather than an esterase side activity.

A series of polymers, including chitosan (CS), carboxymethylcellulose (CMC) and a chitosan–gelatin (CS–GEL) hybrid polymer, were functionalized with ferulic acid (FA) derived from the enzymatic treatment of arabinoxylan through the synergistic action of two enzymes, namely, xylanase and feruloyl esterase. Subsequently, the ferulic acid served as the substrate for laccase from Agaricus bisporus (AbL) in order to enzymatically functionalize the above-mentioned polymers. The successful grafting of the oxidized ferulic acid products onto the different polymers was confirmed through ultraviolet–visible (UV–Vis) spectroscopy, attenuated total reflectance (ATR) spectroscopy, scanning electron microscopy (SEM) and nuclear magnetic resonance (NMR) spectroscopy. Additionally, an enhancement of the antioxidant properties of the functionalized polymers was observed according to the DDPH and ABTS protocols. Finally, the modified polymers exhibited strong antimicrobial activity against bacterial populations of Escherichia coli BL21DE3 strain, suggesting their potential application in pharmaceutical, cosmeceutical and food industries.

The success of establishing bioeconomies replacing current economies based on fossil resources largely depends on our ability to degrade recalcitrant lignocellulosic biomass. This study explores the potential of employing various enzymes acting synergistically on previously pretreated agricultural side streams (corn bran, oat hull, soluble and insoluble oat bran). Degrees of synergy (oligosaccharide yield obtained with the enzyme combination divided by the sum of yields obtained with individual enzymes) of up to 88 were obtained. Combinations of a ferulic acid esterase and xylanases resulted in synergy on all substrates, while a laccase and xylanases only acted synergistically on the more recalcitrant substrates. Synergy between different xylanases (glycoside hydrolase (GH) families 5 and 11) was observed particularly on oat hulls, producing a yield of 57%. The synergistic ability of the enzymes was found to be partly due to the increased enzyme stability when in combination with the substrates.

The Gibbs energy of reaction (Δ^Rg) is the key quantity in the thermodynamic characterization of biological reactions. Its calculation requires precise standard Gibbs energy of reaction (Δ^Rg⁺) values. The value of Δ^Rg⁺ is usually determined by measuring the apparent (concentration-dependent) equilibrium constants K, e.g., the molality-based K_m. However, the thermodynamically consistent determination of Δ^Rg⁺ requires the thermodynamic (activity-based) equilibrium constant K_a. These values (K_m and K_a) are equal only if the ratio of the activity coefficients of the reactants to the activity coefficients of the products (K_γ) is equal to unity. In this work, the impact of K_γ on the estimation of K_a for biological reactions was investigated using methyl ferulate (MF) hydrolysis as a model reaction. The value of K_γ was experimentally determined from K_m values that were measured at different reactant concentrations. Moreover, K_γ was independently predicted using the thermodynamic model ePC-SAFT. Both the experimentally determined and the predicted K_γ values indicate that this value cannot be assumed to be unity in the considered reaction. In fact, in the reaction conditions considered in this work, K_γ was shown to be in the range of 3 < K_γ < 6 for different reactant molalities (2 < mmol MF kg^-1 < 10). The inclusion of K_γ and thus the use of the thermodynamically correct K_a value instead of K_m lead to remarkable differences (almost 40%) in the determination of Δ^Rg⁺. Moreover, the new value for Δ^Rg⁺ increases the concentration window at which the reaction can thermodynamically occur. The influence of additives was also investigated both experimentally and theoretically. Both procedures consistently indicated that the addition of NaCl (0 to 1 mol kg^-1 water) moderately decreased the value of K_γ, which means that the values of K_m increase and that a higher amount of products is obtained as a result of the addition of salt. Additionally, K_m was found to strongly depend on pH. A ten-fold increase in the K_m values was observed in the pH range of 6 to 7; this increase corresponds to a change of more than 100% in the value of Δ^Rg⁺.