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

Abstract

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.

Full Article

Hydrolysis of Ferulic Acids in Corn Fiber by a Metagenomic Feruloyl Esterase

Dominic W. S. Wong,^a,* Victor J. Chan,^a and Hans Liao ^b

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-His_236-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.

Keywords: Feruloyl esterase; Ferulic acid esterase; Ferulic acid; Metagenomics; Corn fiber

Contact information: a: Western Regional Research Center, United States Department of Agriculture, Agricultural Research Service, Albany, CA, USA; b: Cargill Biotechnology Development Center, Minneapolis, MN, USA; *Corresponding author: Dominic.Wong@ars.usda.gov

INTRODUCTION

In the wet-milling process, the corn is steeped, coarsely ground, and processed with hydrocyclones to remove the germ (for corn oil), finely ground again to release the starch and gluten (zein and glutelin) from the fiber, and filtered over screens to catch the fiber, which is then pressed and dried for use in animal feed. Corn fiber (CF) is a mixture of coarse fiber from kernel pericarp or hull and fine fiber from endosperm cellular materials in the corn kernel. The major components consist of 50% hemicellulose, 20% cellulose, 11% to 23% starch, 15% protein, 13% lignin and ash, and 2% to 3% oil. In 2017 (NASS), 6.7 x 10⁵ tons of corn was wet-milled. The corn fiber is produced in yields of 80 to 110 g/kg total corn kernel. The corn-ethanol industry produces approximately 6.4 x 10⁴ tons of corn fiber yearly. The corn fiber is almost completely insoluble because of the hemicellulose fraction, which has a complex structure of heteroxylan, consisting of a -(1,4)-xylopyranosyl (Xylp) backbone attached with a variety of side groups (Biely et al. 2016). Over 70% of the Xylp residues have arabinofuranosyl, acetyl, and glucuronyl side units. Oligosaccharide side chains containing galactose, xylose, and arabinose are also found. Some of the arabinofuranosyl side units are ester-linked with ferulic acid moieties. Due to the complexity of this structural architecture, CF is highly recalcitrant to enzymatic degradation. There have been efforts to characterize the remaining (non-degraded) oligosaccharides that are resistant to mild acid pretreatment and simultaneous saccharification and fermentation (as used in corn ethanol production). It has been shown that resistant oligosaccharides represented 50% of the starting material, which survives pretreatment, enzymatic hydrolysis, and fermentation. All of these complex xylan oligomers were found to contain ferulic acid, diferulates, acetic acid, galactose, arabinose, and uronic acid groups (Appeldoorn et al. 2010). A common feature was (part of) an -L-galactopyranosyl-(1,2)--D-xylopyranosyl-(1,2)-5-O–trans-feruloyl-L-arabinofuranosyl side chain attached to the O-3 position of the -1,4 linked xylose residue (Agger et al. 2010; Appeldoorn et al. 2013). The recalcitrance of CF hemicellulose can be ascribed to feruloylation of the side units and/or backbone of the xylan structure, which have not received adequate attention in enzymatic hydrolysis in biomass conversion.

A variety of bacteria and fungi are known to produce feruloyl esterases (FAE, EC 3.1.1.73) that catalyze the cleavage of feruloyl-arabinose (Araf-FA) ester bonds linking ferulic acids to arabino-furanosyl side groups of the xylan main chain. It has been proposed that accessory enzymes, including FAE, be used to augment the hydrolytic efficiency of cellulases and hemicellulases in biomass conversion, thus requiring less severe pretreatment conditions (Mosier et al. 2005; Akin 2007). Furthermore, FAE are known to act synergistically with endo-xylanases, because short-chain feruloylated xylo-oligosaccharides are better substrates with a higher rate of Araf-FA cleavage (Wong et al. 2013). Other xylanolytic accessory enzymes that act on various side chains may also enhance the hydrolysis of xylan main chain and in turn the ferulic acid linkage (Vardakou et al. 2004; Wong 2006). Direct cloning of metagenomes provides a powerful tool to explore the sequence space of unculturable microbial communities for novel gene discovery and biocatalyst development. The objective of this paper is to report the application of a metagenomic feruloyl esterase (FAE-C3) for the hydrolysis of CF, and characterize its synergistic action with endo-xylanases.

EXPERIMENTAL

Cloning of Feruloyl Esterase

Metagenomic DNA was isolated from microflora of a cow’s rumen, and it was used to construct a ZAP library and screened for FAE enzyme activity. The identified gene was subcloned in pET32b and transformed in BL21. The protein was purified to homo-geneity, using previously reported procedures (Wong et al. 2013, 2015; Shang et al. 2018).

Bioinformatics

Vector NTI (Informax, Bethesda, MD, USA) and Geneious (Biomatters Ltd., Auckland, New Zealand) were used for sequence analysis. The gene sequence was submitted to GenBank with the following accession number: MH785296.

Electrophoresis

The purified enzyme was run on a 4% to 12% Bis-Tris NuPAGE gel using a 50 mM buffer solution of 3-morpholinopropane-1-sulfonic acid (MOPS) at constant 100 V for 2 h and stained with SimplyBlue Safe stain (Invitrogen, Carlsbad, CA, USA). The protein and marker bands were analyzed by image analysis software (Alpha Inotech, AlphaImager, San Jose, CA, USA). For pI determination, the enzyme protein was run on an electrofocusing gel (pH 3 to 10, Invitrogen, Carlsbad, CA, USA) using Serva IEF markers 3−10 mix (Biophoretics, Reno, NV, USA, Heidelberg, Germany).

Enzyme Activity Measurement

A typical enzyme reaction mixture contained 100 mg CF, various nmole concentrations of FAE-C3 in 2 mL 50 mM K₂HPO₄, pH 7.0 buffer, and was incubated for 2 h in a 40 °C shaker bath. For all reactions, the CF was milled for three times for 10 s each (Micro-Mill, Technilab Instruments, Vineland, NJ, USA), washed 5 times with water, and oven-dried at 50 °C. Before using in enzyme reaction, the CF was exhaustively washed 4 times with water. Activity was expressed as g FA released from 100 mg substrate per hour measured by high performance liquid chromatography analysis. The HPLC system (Gilson 307 HPLC, Middleton, WI, USA) consisted of a Gilson 307 pump equipped with a Brownlee analytical C18 m ODS column (260 × 4.5 mm), using a mobile phase of water/formic acid/acetonitrile (7/1/2 v/v) at a flow rate of 0.2 mL/min at ambient temperature. The UV-absorbance detector was set at 315 nm.

Optimum pH and Temperature

For pH optimum, the reaction mixture of 100 mg CF and 0.4 nmole FAE-C3 in 1 mL universal buffer of varying pH was incubated for 2 h in a 37 °C shaker bath. To determine temperature optimum, the enzyme reaction mixture was incubated in 100 mM K₂HPO₄ buffer, at pH 7.0 at temperatures from 20 °C to 70 °C for 2 h.

Hot Water Pretreatment of Corn Fiber

The pretreatment reactor tube with a working volume of 30 mL was made out of stainless steel with dimensions of 1″ OD × 4.5″ L × 0.065″ thickness, capped with 1″ swagelock end fittings. The CF (1.5 g) was soaked overnight in 28.5 mL H₂O, then autoclaved for 20 min at 121°C at 20 to 21 psi. The pretreated CF was washed 4 times with H₂O before using for enzyme reaction.

Alkali Hydrolysis of Corn Fiber

To 200 mg CF, 6 mL 1 N NaOH was added, and the mixture was incubated for 16 h in a 37 °C shaker bath (Bartolome et al. 1997). Following centrifugation, the supernatant was recovered, and adjusted to a pH ≤ 2 with HCl. Ferulic acid products were then extracted with ethyl acetate (3 × 3 mL), dried under N₂, and dissolved in CH₃OH:H₂O (1:1 v/v) for HPLC analysis.

Synergistic Activity with Added Accessory Enzymes

To 100 mg of CF, 0.5 nmole FAE-C3 was added, supplemented with endo-xylanase (XYN GH10 from Cellvibrio mixtus), -L-arabinofuranosidase (ABF from Bifidobacterium sp.), and acetylxylan esterase (AXE from Orpinomyces sp.) (Megazyme, Bray, Ireland) in various nmole concentrations. The XYN, ABF, and AXE were added individually and in combinations to the reaction. The mixture was incubated for 2 h in a 40 °C shaker bath.

RESULTS AND DISCUSSION

Isolation, Cloning, and Bioinformatics of the FAE Gene

The genomic insert isolated from the metagenomic library contained (1) a TlpA-like sequence of 1,323 bp (136 amino acid residues) at the 5′ end, and (2) an esterase domain (FAE-C3) of 774 bp (258 residues). The FAE-C3 gene was subcloned into pET 32b vector as a fusion protein containing a thioredoxin (Trx) tag, a Met start codon and a 6xHis tag. A BLASTP search reveals the amino acid sequence related to the primary structures of Prevotella oris C735 feruloyl esterase (EF148533), Lactobacillus johnsonii cinnamoyl esterase (ADD11991), and Bacteroides vulgatus / fold hydrolase (WP_117885235) with identity percentages of 65.2%, 42.1%, and 59.2%, respectively (Fig. 1) (Chenna et al. 2003). Homology modeling showed an / hydrolase fold commonly observed in various esterases (Fig. 2) (Guex et al. 2009). The FAE-C3 is closely related to the Lactobacillus johnsonii feruloyl esterase (3S2Z) structure. The L. johnsonii enzyme has a 3D structure consisting of a central  sheet of eight parallel strands flanked by -helices (Lei et al. 2011). Based on sequence and structural comparison, the FAE-C3 contains the catalytic triad composed of Ser₈₀ His₂₀₅Asp₁₇₇, and a classical Gly₃₉-X-Ser₄₁-X-Gly₄₃ nucleophile motif commonly found in esterases (Brenner 1988).

Fig. 1. Multiple sequence alignment of FAE-C3 with Prevotella oris C735 feruloyl esterase (EF148533), Lactobacillus johnsonii cinnamoyl esterase (ADD11991), and Bacteroides vulgatus  fold hydrolase (WP_117885235), using the Geneious software. The Gly₃₉-X-Ser₄₁-X-Gly₄₃ motif sequence is highlighted with a striated bar above the residues. The catalytic triad Ser₁₀₆ His₂₀₅Asp₁₇₇ is indicated with stars above the residues.

Fig. 2. SWISS-MODEL homology modeling of FAE-C3 based on comparison with the crystal structure of Lactobacillus johnsonii enzyme (3s2z.1), showing the /-fold architecture with the essential Ser₈₀ labeled (at the 3 helix). Elements of secondary structure are colored from the N- to the C-terminal ends in the order of violet, blue, green, yellow, orange, and red

Enzyme Activity on Corn Fiber

The enzyme FAE-C3 was characterized using CF as the substrate. An enzyme unit was defined as the amount of enzyme that catalyzes the formation of 1 g of ferulic acid product per 100 mg of substrate per hour reaction. The FAE showed a pH optimum at pH ≥ 7, and a temperature optimum of 40 °C (Figs. 3A and 3B). Under the described reaction conditions (100 mg CF, in 1 mL pH 7.0 buffer, incubated for 2 h at 40 °C), 1 nmole FAE catalyzed the release of 19.75 ± 0.24 g FA from 100 mg CF/h equivalent to 3.5% of total FA content in the 100 mg CF (as determined by alkaline hydrolysis) used in the reaction mixture. Using optimum temperature and pH, the effect of enzyme concentration was determined. The sharpest increase in FA release occurred in the first nmole FAE range (Fig. 3C). Thereafter, the slope started to level progressively with increasing enzyme concentration. This may suggest that some FA residues were relatively less accessible and more resistant, requiring higher enzyme concentration for hydrolytic cleavage. Two possible factors may be involved: (1) The FA residues could be crosslinked, resulting in resistance to enzymatic hydrolysis; and (2) The FA may have been located at interior positions of the polymer, rendering it inaccessible to the FAE enzyme. Recent studies show that GH67 -glucuronidases remove only the uronic acid linked to the non-reducing end of the glucuronyl xylooligosaccharides (Bielty et al. 1997; Pell et al. 2004). An analogy may be drawn that the FAE-C3 hydrolyzes only or cleaves at a higher rate the Araf-FA substitution linked at the non-reducing end of the feruloyl oligosaccharide.

Fig. 3. FAE-catalyzed release of ferulic acid from CF. (A) Effect of pH (reaction conditions: 100 mg CF, 1 mL universal buffer of varying pH, 0.4 nmole FAE-C3, 37 °C for 2 h); (B) Effect of incubation temperature (reaction conditions: 100 mg CF, 1 mL 0.1 M K₂HPO₄ pH 7.0 buffer, 0.4 nmole FAE, varying incubation temperature for 2 h); and (C) Effect of enzyme concentration (reaction conditions: 100 mg CF, 1 mL K₂HPO₄ pH 7.0 buffer, 0.5 to 14 nmole FA-C3E, 40 °C for 2 h). Enzyme unit is defined as the amount of enzyme that catalyzes the formation of 1 µg of ferulic acid product per 100 mg CF/h at 40 °C

Effect of Endo-xylanase on FAE Reaction

The synergistic action of FAE-C3 with endo-xylanase was investigated by adding increasing concentrations of XYN (1.0 nmole to 20 nmole) at a constant concentration (0.5 nmole) of FAE. An average increase of 1.11 ± 0.14 g of FA was observed for each doubling of the concentration of XYN (Figs. 4A and 4B).

Fig. 4. Effect of endo-xylanase on FAE-catalyzed release of FA analyzed by HPLC: (A) CF and (B) pre-treated CF (reaction conditions: 100 mg CF, 1 mL 0.1 M K₂HPO₄ pH 7.0 buffer, 0.5 nmole FAE, varying concentrations (0.5 to 40 nmoles) XYN, 40 °C for 2 h). The “B” label on the x-axis stands for “Blank”, which contained only 100 mg CF (without enzymes).

The synergistic effect of adding XYN was equivalent to a 5% or 1.23 fold increase in FA release over the use of FAE-C3 alone. The release of FA from CF and corn bran by FAE in synergism has been reported to be in a similar range, 1.21 and 1.19 fold, respectively (Wong et al. 2013). These amounts are less than using wheat bran or wheat insoluble arabinoxylan as substrates (6.72 and 2.72 fold increase) (Wong et al. 2013). Various investigations have shown that FAE activity may be enhanced by the synergistic action of endo-xylanase because shorter chain xylooligosaccharides are more susceptible (hydrolyzed at faster rates by FAE) than long chain substrates (Faulds and Williamson 1993, 1995; Vardakou et al. 2004; Wong 2006; Wong et al. 2013). The other two accessory enzymes, ABF and AXE, did not show synergistic effect on the CF substrate under similar experimental conditions.

Fig. 5. Mixed endo-xylanase and FAE-C3 on hydrolysis of the xylan main chain assayed by DNSA (3,5-dinitrosalicyclic acid) (reaction conditions: 100 mg CF compared to pre-treated CF, 1 mL 0.1 M K₂HPO₄ pH 7.0 buffer, 0.5 nmole FAE-C3, varying concentrations (0.5 to 40 nmoles) XYN, 40 °C for 2 h)

Effect of Hot Water Pretreatment of Corn Fiber on FA Hydrolysis

Pretreatment has been a key step of cellulosic ethanol production for improving hydrolysis efficiency. The pretreatment loosened the substrate structure, increased porosity of the materials, and enhanced the accessibility of the hydrolytic enzymes. Hot water pretreatment has been shown to produce dissolution of the carbohydrates with minimum degradation (Kim et al. 2009). With pretreated CF, the synergistic effect of adding XYN resulted in ca. 60 mg FA/100 mg CF (5 nmole XYN, 0.5 nmole FAE), a 4-fold increase compared with the use of the untreated CF under similar reaction conditions.

Synergistic Action of Endo-xylanase and FAE on Main Chain Hydrolysis

The addition of FAE resulted in improving the endo-xylanase action on the hydrolysis of the xylan main chain. The synergistic effect was shown by the increase in the amount of reducing sugar (measured as xylose equivalent) in the reactions. The effect was more noticeable when pretreated CF was used. With 0.5 nmole FAE-C3 added, 5 nmole of XYN was produced, 319 μg xylose equivalent per 100 mg pretreated CF, compared to 84 μg for untreated CF.

CONCLUSIONS

1. A feruloyl esterase (FAE) gene was isolated from rumen microbial metagenome, expressed in E. coli, and the enzyme protein (fae-C3) was purified in active form. It consists of an esterase domain with an / hydrolase fold, composed of a catalytic triad Ser₈₀His₂₀₅Asp₁₇₇.

2. The FAE-C3 was characterized using corn fiber as substrate. Its synergistic action with endo-xylanase was studied. Under optimum pH and temperature (pH 7.0, 40 °C), 0.5 nmole FAE-C3 with 5 nmoles XYN were able to catalyze the release of 59.0 ± 0.2 mg ferulic acid and 319 g xylose equivalent from 100 mg of pretreated CF.

3. It was determined that a combination of selected feruloyl esterase and endo-xylanase can lead to simultaneous increase in the hydrolysis of ferulic acid and the xylan main chain. It consists of an esterase domain with an / hydrolase fold, composed of a catalytic triad Ser₈₀His₂₀₅Asp₁₇₇.

ACKNOWLEDGEMENTS

Reference to a company and/or product is only for purposes of information and does not imply approval of recommendation of the product to the exclusion of others that may also be suitable. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap. The authors declare that there is no conflict of interest regarding the publication of this paper.

REFERENCES CITED

Agger, J., Vikso-Nielsen, A., and Meyer, A. S. (2010). “Enzymatic xylose release from pretreated corn bran arabinoxylan: Differential effects of deacetylation and deferuloylation on insoluble and soluble substrate fractions,” J. Agric. Food Chem. 58(10), 6141-6148. DOI: 10.1021/jf100633f

Akin, D. E. (2007). “Grass lignocellulose,” Appl. Biochem. Biotechnol. 137-140, 3-15.

Appeldoorn, M. M., Kabel, M. A., and van Eylen, D. (2010). “Characterization of oligomeric xylan structures from corn fiber resistant to pretreatment and simultaneous saccharification and fermentation,” J. Agric. Food Chem. 58(21), 11294-11301. DOI: 10.1021/jf102849x.

Appeldoorn, M. M., de Waard, P., Kabel, M. A., Cruppen, H., and Schols, H. A. (2013). “Enzyme resistant feruloylated xylooligomer analogues from thermochemically treated corn fiber contain large side chains, ethyl glycosides and novel sites of acetylation,” Carbohydr. Res. 381, 33-42. DOI: 10.1016/j.carres.2013.08.024

Biely, P., Singh, S., and Puchart, V. (2016). “Towards enzymatic breakdown of complex plant xylan structures: State of the art,” Biotechnol. Adv. 34(7), 1260-1274. DOI: 10.1016/j.biotechadv.2016.09.001

Brenner, S. (1988). “The molecular evolution of genes and proteins: A tale of two serines,” Nature 334, 528-530. DOI: 10.1038/334528a0

Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins, D. G., and Thompson, J. D. (2003). “Multiple sequence alignment with the Clustal series of programs,” Nucleic Acids Res. 31(13), 3497-3500. DOI: 10.1093/nar/gkg500

Faulds, C. B., and Williamson, G. (1993). “Ferulic acid esterase from Aspergillus niger: Purification and partial characterization of two forms from a commercial source of pectinase,” Biotechnol. Appl. Biochem. 17(3), 249-259. DOI: 10.1111/j.1470-8744.1993.tb00249.x

Faulds, C. B., and Williamson, G. (1995). “Release of ferulic acid from wheat bran by a ferulic acid esterase (FAE-III) from Aspergillus niger,” Appl. Microbiol. Biotechnol. 43, 1082-1087.

Guex, N., Peltsch, M. C., and Schwede, T. (2009). “Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: A historical perspective,” Electrophoresis 30(S1), S162-S173. DOI: 10.1002/elps.200900140

Kim, Y., Hendrickson, R., Mosier, N. S., and Ladisch, M. R. (2009). “Liquid hot water pretreatment of cellulosic biomass,” in: Biofuels Methods and Protocols, J. R. Mielenz (ed.), Humana Press, Totowa, NJ, USA, pp. 83-102.

Lai, K. K., Stogios, P. J., Vu, C., Xu, X., Cui, H., Molloy, S., Savchenko, A., Yakunin, A., and Gonzalez, C. F. (2011). “An inserted / subdomain shapes the catalytic pocket of Lactobacillus johnsonii cinnamoyl esterase,” PLOS One 6(8), e23269. DOI: 10.1371/journal.pone.0023269

Mosier, N., Wyman, C., Dalee, B., Elander, R., Lee, Y. Y., Holtzapple, M., and Ladisch, M. (2005). “Features of promising technologies for pretreatment of lignocellulosic biomass,” Bioresource Technol. 96(6), 673-686. DOI: 10.1016/j.biortech.2004.06.025

National Agricultural Statistics Service (NASS) (2018). Grain Crushings and Co-Products Production (USDA NASS Report), NASS, Washington D.C., USA.

Pell, G., Taylor, E. J., Gloster, T. M., Turkenburg, J. P., Fontes, C. M. G. A., Ferrrira, L. M. A., Nagy, T., Clark, S. J., Davies, G. J., and Gilbert, H. J. (2004). “The mechanisms by which family 10 glycoside hydrolases bind decorated substrates,” J. Biol. Chem. 289, 9597-9605. DOI: 10.1074/jbc.M312278200

Shang, M. L., Chan, V. J., Wong, D. W. S., and Liao, H. (2018). “A novel method for rapid and sensitive metagenomic activity screening,” MethodsX 5, 669-675. DOI: 10.1016/j.mex.2018.06.011

Vardakou, M., Katapodis, P., Topakas, E., Kekos, D., Macris, B. J., and Christakopoulos, P. (2004). “Synergy between enzymes involved in the degradation of insoluble wheat flour arabinoxylan,” Innov. Food Sci. Emerg. Technol. 5(1), 107-112. DOI: 10.1016/S1466-8564(03)00044-4

Wong, D. W. S. (2006). “Feruloyl esterase – A key enzyme in biomass degradation,” Appl. Biochem. Biotechnol. 133, 87-112.

Wong, D. W. S., Chan, V. J., Liao, H., and Zidwick, M. J. (2013). “Cloning of a novel feruloyl esterase gene from rumen microbial metagenome and enzyme characterization in synergism with endoxylanases,” J. Ind. Microbiol. Biotechnol. 40, 287-295. DOI: 10.1007/s10295-013-1234-1

Wong, D. W. S., Takeoka, G., Chan, V. J., Liao, H., and Murakami, M. T. (2015). “Cloning of a novel feruloyl esterase from rumen microbial metagenome for substantial yield of mono- and diferuic acids from natural substrates,” Protein Pept. Lett. 22, 681-688.

Article submitted: September 10, 2020; Peer review completed: November 7, 2020; Revised version received and accepted: December 4, 2020; Published: December 10, 2020.

DOI: 10.15376/biores.16.1.825-834