Feruloyl Esterase Research Articles

Purification and biochemical characterization of an alkaline feruloyl esterase from Penicillium sumatrense NCH-S2 using rice bran as substrate

ABSTRACT Feruloyl esterases (FAEs) are essential accessory enzymes in the hydrolysis of plant cell wall structure. A novel FAE was obtained from Penicillium sumatrense NCH-S2. Enzyme purification was conducted by ultrafiltration, ammonium sulfate precipitation, anion exchange, and gel filtration chromatography. This FAE has a molecular mass of 36 kDa. Its optimum temperature and pH were 50°C and pH 9.0–10.0, respectively. The FAE demonstrated high pH stability at the pH ranging from 6.0 to 10.0. After enzymatic hydrolysis with FAE, the DPPH free radical scavenging capacity, ferrous ion chelating ability, and total phenolic content (TPC) of defatted rice bran (DRB) hydrolysate significantly increased. Furthermore, the amount of released FA from DRB under a synergistic interaction of FAE and hemicellulose increased by 18–21% in comparison with that of either enzyme acting alone.

Hydrolysis of ferulic acids in corn fiber by a metagenomic feruloyl esterase

A feruloyl esterase (FAE) gene was isolated from rumen microbial metagenome that consisted of 774 bp encoding 258 amino acid residues. The gene was subcloned into pET 32b vector, expressed in Escherichia. coli, and the enzyme was purified in active form. Homology modeling showed that the FAE contained the catalytic triad composed of Ser80-His236-Asp177, and a classical Gly-X-Ser-X-Gly nucleophile motif commonly found in esterases. Under optimum pH and temperature (pH 7.0, 40 °C), 1 nmole FAE catalyzed the release of 19.75 ± 0.24 µg ferulic acid (FA) from 100 mg corn fiber (CF) in 1 h, which represents 3.5% of FA present in saponified CF. Addition of GH10 endoxylanase (XYN) to 0.5 nmole FAE enhanced the yield by 1.1 fold, equivalent to a 5% increase in FA release. Using CF pretreated with hot water, the synergistic effect of adding XYN resulted in 59.0 ± 0.2 mg FA/100 mg CF (5 nmole XYN, 0.5 nmole FAE), equivalent to a 4-fold increase compared to using the untreated CF substrate under the same reaction conditions.

Differential effect of rice husk and xylan on feruloyl esterase activity in Penicillium rubens.

Objective. Analyzed the effect of xylan and rice husk as sole sources of carbon on the FAE activity and expression of faeA and faeB genes of Penicillium rubens (Wisconsin 54-1255), in submerged fermentation. Materials and methods. The fermentations were carried out for 24, 48, and 72 h (28°C/250 rpm), in flasks with a modified Sakamoto medium. FAE activity was determined using the synthetic substrate Ethyl 4-hydroxy-3-methoxycinnamate. The transcription of faeA and faeB was analyzed by RT-PCR. The PCR products were resolved by electrophoresis and analyzed by densitometry. Results. The analysis of gene expression showed that the use of xylan had a positive effect on the expression of both genes. The highest transcriptional activity of both faeA and faeB was detected at 48 h. On the other hand, rice husk showed negative results. The data obtained when the FAE activity was determined, supported the results observed in the gene expression analysis. Conclusion. Different from xylan the rice husk did not demonstrate an inducer effect on the feruloyl esterase activity in P. rubens.

The Role of Endogenous Enzymes during Malting of Barley and Wheat Varieties in the Mitigation of Styrene in Wheat Beer.

Knowledge of the biochemical processes responsible for the release of phenolic acids (precursors of vinyl aromatics) during malting is important to find mitigation strategies for the toxicologically relevant styrene (formed from cinnamic acid) in wheat beer. Therefore, grain and malts of four barley and three wheat varieties were screened for the activities of various enzymes and the amounts of nonstarch polysaccharides (to which the phenolic acids are bound to a certain extent). During malting, a very strong degradation of β-glucan, synonymous to a depletion of the cell walls, was found, suggesting that a partial degradation of cell walls cannot have an effect on the release of phenolic acids. In barley malts, water-extractable arabinoxylan contents were between 0.59 and 0.79 g/100 g dm and in wheat malts between 0.93 and 1.51 g/100 g dm. Additionally, higher soluble ferulic acid contents in wheat wort compared to barley wort indicated that the degradation of nonstarch polysaccharides has an impact on the release of phenolic acids. For the feruloyl esterase, higher activities were found in malts of the barley varieties. However, this fact was not reflected by the free phenolic acid contents in those malts. Correlation coefficients between the protease activity and the feruloyl esterase, α- and β-amylase, and β-glucanase activities were proven to be insignificant, highlighting that the protease activity had no effect on the activities of these other enzymes.

Comparison of Enzyme Secretion and Ferulic Acid Production by Escherichia coli Expressing Different Lactobacillus Feruloyl Esterases.

Construction of recombinant Escherichia coli strains carrying feruloyl esterase genes for secretory expression offers an attractive way to facilitate enzyme purification and one-step production of ferulic acid from agricultural waste. A total of 10 feruloyl esterases derived from nine Lactobacillus species were expressed in E. coli BL21 (DE3) to investigate their secretion and ferulic acid production. Extracellular activity determination showed all these Lactobacillus feruloyl esterases could be secreted out of E. coli cells. However, protein analysis indicated that they could be classified as three types. The first type presented a low secretion level, including feruloyl esterases derived from Lactobacillus acidophilus and Lactobacillus johnsonii. The second type showed a high secretion level, including feruloyl esterases derived from Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus helveticus. The third type also behaved a high secretion level but easy degradation, including feruloyl esterases derived from Lactobacillus farciminis, Lactobacillus fermentum, and Lactobacillus reuteri. Moreover, these recombinant E. coli strains could directly release ferulic acid from agricultural waste. The highest yield was 140 μg on the basis of 0.1 g de-starched wheat bran by using E. coli expressed L. amylovorus feruloyl esterase. These results provided a solid basis for the production of feruloyl esterase and ferulic acid.

Enzymes produced by solid state fermentation of agro-industrial by-products release ferulic acid in bioprocessed whole-wheat breads

Solid-state fermentation (SSF) presents low cost and the possibility of adding value to waste by generating products rich in enzymes. The production of enzymes by SSF and its application in bakery have been previously reported separately in the literature. However, very few studies combine both approaches to evaluate the feasibility of applying enzymes produced by SSF to bread processing. The objective of this study was to use cocoa bean shell (CBS), wheat bran (WB) and brewer’s spent grain (BSG) for enzyme production by SSF, and to evaluate their addition in breads. Three breads were produced: control bread (CB), bioprocessed bread added with fermented wheat bran (WBB) and bioprocessed bread added with fermented BSG (BSGB). Feruloyl esterase highest activities were 1,730 mU/g for WB fermented for 24 h and 1,128 mU/g for BSG fermented for 72 h. Xylanase highest activities were 547.9 U/g for BSG fermented for 48 h and 868.1 U/g for WB fermented for 72 h. CBS showed the lowest enzymatic activities. Bioprocessing breads with fermented WB and BSG led to an increase in soluble ferulic acid of 159% and 198%, respectively. The combination of SSF enzyme production and bread enzymatic bioprocessing strategies proved to be an effective green option for the valorization of agro-industrial by-products and the production of breads with enhanced ferulic acid content.

Chitosan-based CLEAs from Aspergillus niger type A feruloyl esterase: high-productivity biocatalyst for alkyl ferulate synthesis.

The enzymatic synthesis of alkyl ferulates is an important reaction in cosmetic and pharmaceutical chemistries, since it may allow to expand the biorefinery concept valorizing biomass wastes enriched in ferulic acid. However, robust biocatalysts for that purpose are scarce. Herein, we have immobilized the type A feruloyl esterase from Aspergillus niger (AnFaeA) as cross-linked enzyme aggregates, employing chitosan as co-feeder (ChCLEAs). High immobilization yields and relative activity recovery were attained in all assessed conditions (> 93%). Furthermore, we enhanced the thermal stability of the soluble enzyme 32-fold. AnFaeA-ChCLEAs were capable to quantitatively perform the solvent-free direct esterification of short- to medium-chain alkyl ferulates (C4-C12) in less than 24 h. By raising the operational temperature to 50 °C, AnFaeA-ChCLEAs transformed 350 mM ferulic acid into isopentyl ferulate with a space-time yield of 46.1 g of product × L-1 × day-1, 73-fold higher than previously reported. The overall sustainability of this alkyl ferulate production bioprocess is supported by the high total turnover number (TTN 7 × 105) and the calculated green metrics (E factor = 30). Therefore, we herein present a robust, efficient, and versatile heterogeneous biocatalyst useful for the synthesis of a wide diversity of alkyl ferulates. KEY POINTS: • CLEAs of feruloyl esterase A from A. niger using chitosan as co-feeder were obtained. • Microenvironment of the biocatalysts allowed to obtain C1 to C18 alkyl ferulates. • Biocatalyst at boundary conditions showed a high productivity of 46 g/L day. Graphical Abstract.

Advance diversity of enzymatically modified arabinoxylan from wheat chaff

Hydrolysates of arabinoxylan extracted from wheat chaff were prepared using different enzymatic treatments with an emphasis on improvements in their anti-diabetic, antioxidant and functional characteristics. The extracted arabinoxylan was subjected to enzymatic hydrolysis using individual xylanase, arabinofuranosidase, and feruloyl esterase, and their combinations. In all obtained hydrolysates, peaks corresponding to molecular weight lower than 38 kDa were noticed, while non-hydrolysed arabinoxylan had only peaks corresponding to 580 and 38 kDa. Results indicated that applied enzymes could hydrolyse polymeric arabinoxylan while their synergistic actions successfully modified its structure reflecting in lowered viscosity. Besides, it has been observed that the synergistic actions of enzymes improved the biological activities of arabinoxylan more than twice. Chemometric classification analysis showed that synergistic enzymes' actions were predominantly responsible for the improvement of biological activities. It indicated that they might be a useful tool for diversification and enhancement of biological activities of arabinoxylan from wheat chaff.

Enzymatic Production of Xylooligosaccharides from Alkali-Solubilized Arabinoxylan from Sugarcane Straw and Coffee Husk

Enzymatic hydrolysis of lignocellulosic biomass has opened new perspectives for the production of biofuels and functional food ingredients, such as prebiotic xylooligosaccharides (XOS). This study aimed to improve the arabinoxylan extraction process from sugarcane straw (SS) and coffee husk (CH) to produce short-chain XOS by enzymatic hydrolysis using ideal enzyme mixture formulations. The improvement of arabinoxylan extraction process provided the highest extraction yield of about 40.35% and arabinoxylan extraction efficiency around 82.32% with insignificant lignin content values. In a second step, a central composite rotatable design was used to develop an enzyme mixture for XOS production from SS and CH arabinoxylan. Three commercial enzymes were tested, endoxylanase (GH11), α-l-arabinofuranosidase (GH51), and feruloyl esterase (CE1). An efficient enzyme mixture was developed, affording high XOS concentrations from SS arabinoxylan around 10.23 g L−1 and CH arabinoxylan 8.45 g L−1.

Feruloyl esterase activity and its role in regulating the feruloylation of maize cell walls

Cell walls of grasses have ferulic acid (FA) ester-linked to the arabinosyl substitutions of arabinoxylan (AX). Feruloyl esterases (FAE) are carboxylic acid esterases that release FA from cell walls and synthetic substrates. Despite the importance of FA for cell wall recalcitrance and in response to biotic and abiotic stresses, the physiological function of plant FAEs remains unclear. Here, we developed a simple method for the determination of FAE activity (ZmFAE) in maize using the total protein extract and investigated its role in regulating the feruloylation of cell wall. The method includes a single protein extraction and enzymatic reaction with protein concentration as low as 65 μg at 35 °C for 30 min, using methyl ferulate as the substrate. The methodology allowed the determination of the apparent Km (392.82 μM) and Vmax (79.15 pkat mg−1 protein). We also found that ZmFAE activity was correlated (r = 0.829) with the levels of FA in seedling roots, plant roots and leaves of maize. Furthermore, the exposure to osmotic stress resulted in a 50% increase in ZmFAE activity in seedling roots. These data suggest that FAE-catalyzed reaction is important for cell wall feruloylation during plant development and in response to abiotic stress. We conclude proposing a model for the feruloylation and deferuloylation of AX, which explains the role of FAE in regulating the levels of ester-linked FA. Our model might orient further studies investigating the role of plant FAEs and assist strategies for genetic engineering of grasses to obtain plants with reduced biomass recalcitrance.

Xylooligosaccharides production by commercial enzyme mixture from agricultural wastes and their prebiotic and antioxidant potential

Advancement in industrial biotechnology offers potential opportunities for economic utilization of agro industrial biomass for the production of value-added products. Xylooligosaccharides (XOS) are non-digestible food ingredients with prebiotic properties for selectively promoting the growth of probiotics providing many health benefits and several applications on food and pharmaceutical industry. The present study deal with enzymatic production of XOS from xylan extracted from different agroindustrial wastes, namely sugarcane straw (SS) and coffee husk (CH) using an optimized enzymatic mixture with endo-xylanase (GH11), α-l-arabinofuranosidase (GH51) and Feruloyl Esterase (CE1). The XOS profile concentration was quantified by HPAEC-PAD using standard (X2-X6) from Megazyme®. The efficient enzymatic mixture achieved a high total XOS concentration using SS xylan (10.23 ± 0.56 g/L) and CH xylan (8.45 ± 0.65 g/L). Three of four probiotic cultures of Lactobacillus and Bifidobacterium tested were able to utilize XOS produced from agricultural wastes and showed remarkable growth in the media containing XOS, consuming preferentially the X2 and X3 fractions as the sole source of carbon. The XOS produced also exhibited a considerable resistance to hydrolysis of digestive enzymes, as well as an concentration dependent anti-oxidant activity achieving until 78% in a XOS concentration of 2 g. L−1. Thus, the results showed that XOS produced from these agricultural residues have great prebiotic potential and good antioxidant activity; therefore, it can be used in food-related applications as functional ingredients.

Optimizing Immobilization and Stabilization of Feruloyl Esterase from Humicola Insolens and its Application for the Feruloylation of Oligosaccharides

Feruloyl esterase (FAE)-catalyzed esterification reaction is as a potential route for the biosynthesis of feruloylated oligosaccharides as functional ingredients. Immobilization of FAE from Humicola insolens on metal chelate-epoxy supports was investigated. The study of effects of immobilization parameters using response surface methodology revealed the significance of enzyme/support ratio (3.25-29.25 mg/g support), immobilization time (14-38 h), buffer molarity (0.27-1.25 M) and pH (4.0-8.0). The interactions between enzyme-to-support ratio/buffer molarity and enzyme-to-support ratio/pH were found to be critical for the modulation of the immobilization activity yield and the retention of specific activity, respectively. Optimum conditions for FAE-immobilization on metal chelate Sepabeads® EC-EP R were identified to be 22.75 mg FAE/g support, pH of 5.0, 27.7 h and buffer molarity of 0.86 M. At these conditions, an activity yield of 82.4%, a specific activity retention of 143.4%, and an enzyme activity of 395.4 μmol/min. g support were achieved. Further incubation of the immobilized FAE at pH 10.0 improved its thermostability. Increasing the pore size of the epoxy support improved the retention of FAE hydrolytic activity and the esterifying efficiency of the immobilized biocatalyst. Optimally immobilized and stabilized FAE on metal chelate-epoxy support retained up to 92.9% of the free enzyme feruloylation efficiency to xylooligosaccharides..

Functional Validation of Two Fungal Subfamilies in Carbohydrate Esterase Family 1 by Biochemical Characterization of Esterases From Uncharacterized Branches.

The fungal members of Carbohydrate Esterase family 1 (CE1) from the CAZy database include both acetyl xylan esterases (AXEs) and feruloyl esterases (FAEs). AXEs and FAEs are essential auxiliary enzymes to unlock the full potential of feedstock. They are being used in many biotechnology applications including food and feed, pulp and paper, and biomass valorization. AXEs catalyze the hydrolysis of acetyl group from xylan, while FAEs release ferulic and other hydroxycinnamic acids from xylan and pectin. Previously, we reported a phylogenetic analysis for the fungal members of CE1, establishing five subfamilies (CE1_SF1–SF5). Currently, the characterized AXEs are in the subfamily CE1_SF1, whereas CE1_SF2 contains mainly characterized FAEs. These two subfamilies are more related to each other than to the other subfamilies and are predicted to have evolved from a common ancestor, but target substrates with a different molecular structure. In this study, four ascomycete enzymes from CE1_SF1 and SF2 were heterologously produced in Pichia pastoris and characterized with respect to their biochemical properties and substrate preference toward different model and plant biomass substrates. The selected enzymes from CE1_SF1 only exhibited AXE activity, whereas the one from CE1_SF2 possessed dual FAE/AXE activity. This dual activity enzyme also showed broad substrate specificity toward model substrates for FAE activity and efficiently released both acetic acid and ferulic acid (∼50%) from wheat arabinoxylan and wheat bran which was pre-treated with a commercial xylanase. These fungal AXEs and FAEs also showed promising biochemical properties, e.g., high stability over a wide pH range and retaining more than 80% of their residual activity at pH 6.0–9.0. These newly characterized fungal AXEs and FAEs from CE1 have high potential for biotechnological applications. In particular as an additional ingredient for enzyme cocktails to remove the ester-linked decorations which enables access for the backbone degrading enzymes. Among these novel enzymes, the dual FAE/AXE activity enzyme also supports the evolutionary relationship of CE1_SF1 and SF2.

A novel feruloyl esterase with high rosmarinic acid hydrolysis activity from Bacillus pumilus W3

A novel feruloyl esterase (BpFae12) with rosmarinic acid (RA) hydrolysis activity was isolated from Bacillus pumilus W3 and expressed in Escherichia coli BL21 (DE3). With RA as a substrate, the optimal pH and temperature of BpFae12 were pH 8.0 and 50 °C, respectively. The specific enzyme activity was 12.8 U·mg−1. BpFae12 showed the highest activity and substrate affinity toward RA (Vmax of 13.13 U·mg−1, Km of 0.41 mM). Moreover, it also presented strong hydrolysis performance against chlorogenic acid (190.17 U·mg−1). RA was effectively Hydrolyzed into more bioactive caffeic acid and 3,4-dihydroxyphenyllactic acid by BpFae12, which have potential applications in the food industry.

Evaluating Feruloyl Esterase—Xylanase Synergism for Hydroxycinnamic Acid and Xylo-Oligosaccharide Production from Untreated, Hydrothermally Pre-Treated and Dilute-Acid Pre-Treated Corn Cobs

Agricultural residues are considered the most promising option as a renewable feedstock for biofuel and high valued-added chemical production due to their availability and low cost. The efficient enzymatic hydrolysis of agricultural residues into value-added products such as sugars and hydroxycinnamic acids is a challenge because of the recalcitrant properties of the native biomass. Development of synergistic enzyme cocktails is required to overcome biomass residue recalcitrance, and achieve high yields of potential value-added products. In this study, the synergistic action of two termite metagenome-derived feruloyl esterases (FAE5 and FAE6), and an endo-xylanase (Xyn11) from Thermomyces lanuginosus, was optimized using 0.5% (w/v) insoluble wheat arabinoxylan (a model substrate) and then applied to 1% (w/v) corn cobs for the efficient production of xylo-oligosaccharides (XOS) and hydroxycinnamic acids. The enzyme combination of 66% Xyn11 and 33% FAE5 or FAE6 (protein loading) produced the highest amounts of XOS, ferulic acid, and p-coumaric acid from untreated, hydrothermal, and acid pre-treated corn cobs. The combination of 66% Xyn11 and 33% FAE6 displayed an improvement in reducing sugars of approximately 1.9-fold and 3.4-fold for hydrothermal and acid pre-treated corn cobs (compared to Xyn11 alone), respectively. The hydrolysis product profiles revealed that xylobiose was the dominant XOS produced from untreated and pre-treated corn cobs. These results demonstrated that the efficient production of hydroxycinnamic acids and XOS from agricultural residues for industrial applications can be achieved through the synergistic action of FAE5 or FAE6 and Xyn11.

Optimization of fermentation conditions for the production of recombinant feruloyl esterase from Burkholderia pyrrocinia B1213.

Statistical experimental designs were used to optimize conditions for recombinant Burkholderia pyrrocinia feruloyl esterase (BpFae) production in bacteria under lactose induction. After optimization by single factor design, Plackett-Burman design, steepest ascent design and the response surface method, the optimal conditions for BpFae production were: 6g/L lactose, pH 5.5, pre-induced period 5h, 23°C, shaker rotational speed of 240rpm, medium volume of 50mL/250mL, inoculum size 0.2% (v/v), and a post-induced period of 32h in a Luria-Bertani culture. The produced BpFae activity was 7.43U/mL, which is 2.92 times higher than that obtained under optimal conditions using IPTG as the inducer. BpFae activity was 4.82U/mL in a 5L fermenter under the abovementioned optimal conditions. BpFae produced a small amount of ethyl acetate but had no effect on the synthesis of other important esters in Baijiu. The results underpin further investigations into BpFae characterization and potential applications.

Feruloyl Esterases for Biorefineries: Subfamily Classified Specificity for Natural Substrates.

Feruloyl esterases (FAEs) have an important role in the enzymatic conversion of lignocellulosic biomass by decoupling plant cell wall polysaccharides and lignin. Moreover, FAEs release anti-oxidative hydroxycinnamic acids (HCAs) from biomass. As a plethora of FAE candidates were found in fungal genomes, FAE classification related to substrate specificity is an indispensability for selection of most suitable candidates. Hence, linking distinct substrate specificities to a FAE classification, such as the recently classified FAE subfamilies (SF), is a promising approach to improve the application of these enzymes for a variety of industrial applications. In total, 14 FAEs that are classified members of SF1, 5, 6, 7, 9, and 13 were tested in this research. All FAEs were investigated for their activity toward a variety of substrates: synthetic model substrates, plant cell wall-derived substrates, including lignin, and natural substrates. Released HCAs were determined using reverse phase-ultra high performance liquid chromatography coupled to UV detection and mass spectrometry. Based on this study, FAEs of SF5 and SF7 showed the highest release of FA, pCA, and diFAs over the range of substrates, while FAEs of SF6 were comparable but less pronounced for diFAs release. These results suggest that SF5 and SF7 FAEs are promising enzymes for biorefinery applications, like the production of biofuels, where a complete degradation of the plant cell wall is desired. In contrast, SF6 FAEs might be of interest for industrial applications that require a high release of only FA and pCA, which are needed as precursors for the production of biochemicals. In contrast, FAEs of SF1, 9 and 13 showed an overall low release of HCAs from plant cell wall-derived and natural substrates. The obtained results substantiate the previous SF classification as a useful tool to predict the substrate specificity of FAEs, which eases the selection of FAE candidates for industrial applications.

The crystal structure of a Fusariumoxysporum feruloyl esterase that belongs to the tannase family.

Feruloyl esterases are enzymes of industrial interest that catalyse the hydrolysis of the ester bond between hydroxycinnamic acids such as ferulic acid and sugars present in the plant cell wall. Although there are several structures of biochemically characterized feruloyl esterases available, the structural determinants of their substrate specificity are not yet fully understood. Here, we present the crystal structure of a feruloyl esterase from Fusariumoxysporum (FoFaeC) at 2.3Å resolution. Similar to the two other tannase-like feruloyl esterases, FoFaeC features a large lid domain covering the active site with potential regulatory role and a disulphide bond that brings together the serine and histidine of the catalytic triad. Differences are mainly observed in the metal coordination site and the substrate binding pocket. ENZYMES: E.C.3.1.1.73. DATABASES: The sequence of FoFaeC has been deposited with UniProt with accession code A0A1D3S5H0_FUSOX and the atomic coordinates of the three-dimensional structure with Protein Data Bank, with PDB code: 6FAT.

Carrier-bound and carrier-free immobilization of type A feruloyl esterase from Aspergillus niger: Searching for an operationally stable heterogeneous biocatalyst for the synthesis of butyl hydroxycinnamates

Feruloyl esterases synthesize butyl hydroxycinnamates, molecules possessing interesting biological properties, nonetheless, they exhibit a low stability under synthesis conditions in organic solvents, restricting its use. To enhance its operational stability in synthesis, we immobilized type A feruloyl esterase from Aspergillus niger (AnFAEA) using several carrier-bound and carrier-free strategies. The most active biocatalysts were: 1) AnFAEA immobilized on epoxy-activated carriers (protein load of 0.6 mgenzyme x mg−1carrier) that recovered 91 % of the initial hydrolytic activity, and 2) AnFAEA aggregated and cross-linked in the presence of 5 mg of BSA and 15 mM of glutaraldehyde (AnFAEA-amino-CLEAs), which exhibited 385 % of its initial hydrolytic activity; both using 4-nitrophenyl butyrate as substrate. The AnFAEA-amino-CLEAs were 12.7 times more thermostable at 60 °C than the AnFAEA immobilized on epoxy-activated carrier, thus AnFAEA-amino-CLEAs were selected for further characterization. Interestingly, during methyl sinapate hydrolysis (pH 7.2 and 30 °C), AnFAEA-amino-CLEAs KM was 15 % higher, while during butyl sinapate synthesis the KM was reduced in 63 %, both compared with the soluble enzyme. The direct esterification of butyl sinapate at solvent free conditions using sinapic acid 50 mM, reached 95 % conversion after 24 h employing AnFAEA-amino-CLEAs, which could be used for 10 cycles without significant activity losses, demonstrating their outstanding operational stability.

Multimodular fused acetyl\u2013feruloyl esterases from soil and gut Bacteroidetes improve xylanase depolymerization of recalcitrant biomass

BackgroundPlant biomass is an abundant and renewable carbon source that is recalcitrant towards both chemical and biochemical degradation. Xylan is the second most abundant polysaccharide in biomass after cellulose, and it possesses a variety of carbohydrate substitutions and non-carbohydrate decorations which can impede enzymatic degradation by glycoside hydrolases. Carbohydrate esterases are able to cleave the ester-linked decorations and thereby improve the accessibility of the xylan backbone to glycoside hydrolases, thus improving the degradation process. Enzymes comprising multiple catalytic glycoside hydrolase domains on the same polypeptide have previously been shown to exhibit intramolecular synergism during degradation of biomass. Similarly, natively fused carbohydrate esterase domains are encoded by certain bacteria, but whether these enzymes can result in similar synergistic boosts in biomass degradation has not previously been evaluated.ResultsTwo carbohydrate esterases with similar architectures, each comprising two distinct physically linked catalytic domains from families 1 (CE1) and 6 (CE6), were selected from xylan-targeting polysaccharide utilization loci (PULs) encoded by the Bacteroidetes species Bacteroides ovatus and Flavobacterium johnsoniae. The full-length enzymes as well as the individual catalytic domains showed activity on a range of synthetic model substrates, corn cob biomass, and Japanese beechwood biomass, with predominant acetyl esterase activity for the N-terminal CE6 domains and feruloyl esterase activity for the C-terminal CE1 domains. Moreover, several of the enzyme constructs were able to substantially boost the performance of a commercially available xylanase on corn cob biomass (close to twofold) and Japanese beechwood biomass (up to 20-fold). Interestingly, a significant improvement in xylanase biomass degradation was observed following addition of the full-length multidomain enzyme from B. ovatus versus the addition of its two separated single domains, indicating an intramolecular synergy between the esterase domains. Despite high sequence similarities between the esterase domains from B. ovatus and F. johnsoniae, their addition to the xylanolytic reaction led to different degradation patterns.ConclusionWe demonstrated that multidomain carbohydrate esterases, targeting the non-carbohydrate decorations on different xylan polysaccharides, can considerably facilitate glycoside hydrolase-mediated hydrolysis of xylan and xylan-rich biomass. Moreover, we demonstrated for the first time a synergistic effect between the two fused catalytic domains of a multidomain carbohydrate esterase.