Development of a Novel Biocatalytic Approach for the Synthesis of Feruloylated Glycosides by Feruloyl Esterase Expressed in Selected Multi-Enzymatic Extracts

Methyl ferulate and isolated feruloylated non-digestible oligosaccharides (NDOs) from sugar-beet pulp and wheat bran were used as substrates to evaluate the levels of feruloyl esterase (FAE) activity and substrate specificity of twenty multi-enzymatic preparations. The synthesis of feruloylated monosaccharides by FAEs expressed in the six best multi-enzymatic preparations from Bacillus spp. (Ceremix), Humicola spp. (Depol 740L), Aspergillus oryzae (Flavourzyme), Bacillus amyloliquefaciens (Multifect P 3000), Bacillus subtilis (RP-1), and Trichoderma reesei (Depol 670L) was investigated using a surfactant-less organic microemulsion system as reaction medium. The highest bioconversion yield of feruloylated arabinose (37%) and galactose (61%) was obtained using Multifect P 3000 and Depol 670L as biocatalysts, respectively, in n-hexane, 1-butanol and 3-(N-morpholino)ethanesulfonic acid (MES)--NaOH buffer reaction mixture (51:46:3, v/v/v). Using n-hexane, 2-butanone and MES--NaOH (51:46:3, v/v/v), Depol 670L resulted in the highest bioconversion yield of feruloylated xylose (37%). Feruloylated arabinose, galactose and xylose demonstrated potential antioxidant properties and their chemical structures were confirmed by APCI-MS. Depol 740L from Humicola spp. was enriched on FAE activity (2.5-fold) by ammonium sulfate precipitation. The enriched FAE extract exhibited the highest esterifying activity for the feruloylation of sucrose (13%) in n-hexane, 2-butanone and MES-NaOH (51:46:3, v/v/v). As compared to their corresponding ferulic acid, the feruloylated di- and NDOs demonstrated similar or higher potential antioxidant properties. By varying the media composition, the highest bioconversion yields were obtained in n-hexane, 2-butanone and MES-NaOH (log P of 1.8) using arabinobiose (8%), xylobiose (9%) and raffinose (11%) as glycoside substrates. However, using galactobiose as substrate, the highest bioconversion yield (27%) was obtained in n-hexane, 1,4-dioxane and MES-NaOH (log P of 1.6). RSM, based on a five level, four-factor central composite rotatable design revealed that enzyme amount and substrate molar ratio were the most important variables for the bioconversion yield of feruloylated raffinose. The optimum synthesis conditions were as follows: temperature of 35°C; ferulic acid to raffinose molar ratio of 3:1; water content of 3% (v/v); and enzyme amount of 345 enzymatic FAE units. The predicted value was 12.0% (119.7 muM) and the actual experimental value was 11.9% (119.4 muM).