Heterologous production of a feruloyl esterase from Pleurotus sapidus synthesizing feruloyl‐saccharide esters

Abstract

The feruloyl esterase (FAE) gene EST1 from the basidiomycete Pleurotus sapidus was heterologously expressed in Escherichia coli and Pichia pastoris. Catalytically active recombinant Est1 was secreted using P. pastoris as a host. For expression in P. pastoris, the expression vector pPIC9K was applied. The EST1 gene was cloned with an N-terminal α-mating factor pre-pro sequence and expressed under the control of a methanol inducible alcohol oxidase 1 promotor. Est1 was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. The recombinant Est1 showed optima at pH 5.0 and 50 °C, and released ferulic acid from saccharide esters and from the natural substrate destarched wheat bran. Substrate specificity profile and descriptor-based analysis demonstrated unique properties, showing that Est1 did not fit into the current FAE classification model. Transferuloylation synthesis of feruloyl-saccharide esters was proven for mono- and disaccharides.

Multimodular fused acetyl–feruloyl esterases from soil and gut Bacteroidetes improve xylanase depolymerization of recalcitrant biomass

“…FjCE1 was active on all model FAE substrates and, similar to previous reports of other FAEs [38,39], the highest activity amongst all FAE model substrates was observed on MFA, which out of the substrates most closely resembles the ferulic acid ester links found in xylan. While BoCE1 only exhibited trace activity on two of the FAE model substrates (MFA and MSA), it is possible that the enzyme might perform better on a model substrate which is more similar to the structures found in xylan, such as 5-O-trans-feruloyl-l-arabinofuranose, as previously reported for an FAE from the fungus Pleurotus sapidus [38,40]. The FAE family is very diverse with the CE1 grouping currently representing only a small sub-section (sub-families 5 and 6 based on the proposed classification by Dilokpimol et al [26]).…”

The Synthetic Potential of Fungal Feruloyl Esterases: A Correlation with Current Classification Systems and Predicted Structural Properties

“…The most lipophilic product (PFA) was synthesized in highest yields unanimously by FAEs belonging to SF6, as well as by Fae125 from T. wortmannii belonging to SF5 During analysis of transesterification products, two peaks or three peaks (18.3-19.1 min) were detected for pentose and hexose substituted esters, respectively. We propose that these peaks reflect feruloylation of sugar anomers with slightly differing lipophilicity as it is generally accepted that sugar acylation occurs only at the primary O-5 hydroxyl group [9,12,25]. In the case of sugar alcohols, two major peaks (18.8-19.1 min) with similar lipophilicity, suggesting that feruloylation could have occurred as well in secondary hydroxyl groups of the polyol, and one additional distinctive peak with higher lipophilicity (23.9 min) were detected suggesting that the polyol could have undergone diferuloylation ( Figure S2).…”

“…StFaeC from S. thermophile (syn M. thermophila) catalyzed the synthesis of feruloylated arabino-oligosaccharides (with a different degree of polymerization) but no yields were quantified [23]. Est1 (SF12) from Pleurotus sapidus successfully synthesized feruloylated derivatives of glucose, fructose, galactose, sucrose, and maltose, but not lactose, via transesterification of MFA in sugar-saturated aqueous medium [25]. Yields were low, which is in accordance with our findings regarding AFA synthesis among tannase-related (SF1-3, 9-11) and choline esterase-related (SF12-13) phylogenetic subfamilies.…”

Diversity of fungal feruloyl esterases: updated phylogenetic classification, properties, and industrial applications

“…FAEs catalyze the hydrolysis of the substrate following the mechanism utilized by serine proteases [63] with a conserved Ser-His-Asp/Glu catalytic triad [64]. Glu as a part of catalytic triad, instead of Asp, was recently reported in several Basidiomycetes, which is uncommon among FAEs, but found in some members of the α/β-hydrolase-fold superfamily [57, 65, 66]. Differences in amino acid residues within loops and domains that situate in close proximity to the catalytic and substrate binding sites enable different FAEs to target different substrates [59, 62, 64].…”

“…This includes antioxidant activity, probiotic effects, and inhibition against glycation which are of interest by a wide range of applications in food, pharmaceutical, and cosmetic industries [135]. The advantage of using transesterification over hydrolases or transferases is the flexibility of their acceptor molecules, which can vary from different carbohydrates [66, 136, 137], aliphatic and aromatic alcohols [138, 139], and glycerol [140, 141] to propolis [142]. In the latter case, FAEs can also be used for impoverishing the allergenicity of propolis by specifically removing esters of caffeic acid under hydrolytic conditions [143].…”