Breakdown of Corn Fiber by a Metagenomic Ferulolyl Esterase in Combination with Glycosyl Hydrolases

A feruloyl esterase (FAE-C6) gene of 957 bp was isolated from rumen microbial metagenome, subcloned into pET32b vector, and expressed in Escherichia coli. The enzyme purified in active form, consisted of 319 amino acid residues, with a molecular weight of 43.7 based on SDS-PAGE. Homology modeling showed that the FAE contained the catalytic triad composed of Ser 154- Asp 263 His 295 and a classical Gly-X-Ser 154 -X-Gly nucleophile motif commonly found in esterases. The FAE-C6 was characterized using corn fiber as substrate. Its combining action with glycoside hydrolases (C, X, A) individually and in various combinations was studied with focus on the difference in the effects on FA and sugar release. Glycoside hydrolases with endo-xylanase included in the enzyme mixture showed significant impact on increasing the FA yield. For the release of sugar, FAE enhanced the yield in all hydrolase combinations moderately and endo-xylanase was not the key factor in the enzyme formulation.

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 arabinofuranosyl side groups of the xylan main chain. It has been proposed that enzymes, such as FAE, can be used to augment the hydrolytic efficiency of cellulases and hemicellulases in biomass conversion, thus requiring less severe pretreatment conditions [8] [9]. Direct cloning of metagenomes provides high efficiency exploration of the sequence space of unculturable microbial communities for novel gene discovery and biocatalyst development. Most studies on the enzymology of feruloyl esterases focus on the reaction of hydrolyzing FA from the substrate. In a previous study, combinatorial enzyme approach has been applied for production and screening of libraries of feruloyl oligosaccharides [10]. The objective of this paper is to report the effect of cellulolytic and xylanolytic enzymes individually or in combination acting synergistically with FAE for the hydrolysis of CF with focus on sugar release.

Experimental
Cloning of Feruloyl Esterase Metagenomic DNA was isolated from the microflora of a cow's rumen, and was used to construct a λZAP library. Library screening for FAE enzyme activity identified the FAE-C6 gene. It was subcloned in pET vector, transformed in BL21, and the protein was purified to homogeneity, using previously reported procedures [11] [12].

Results and Discussion
Isolation, Cloning, and Bioinformatics of the FAE Gene The genomic insert isolated from metagenomic library contained an esterase      1.55, GH51), labeled as C, X and A in the figures, respectively. All enzymes are from Aspergillus niger, and exhibit pH and temperature optima of 4.0 -4.5 and 60˚C, respectively (specifications from Megazyme, Ireland). The enzymes (2U each) were added individually and in combinations to 100 mg CF in pH 5.0 buffer, incubated for 2 hr at 37˚C. Figure 4 shows that combinations of the enzymes substantially increased the sugar yield compared to single enzymes. A combination of CXA increased the sugar yield by 93% over that by cellulase alone. Likewise, combination of XA increased the yield by 167% and 245%, respectively, over that by X or A alone.

Effect of FAE added to Glycoside Hydrolases
FAE alone had little effect on the sugar yield ( Figure 5). It is evident that the sugar yield was largely attributed to the actions of glycoside hydrolases. Adding FAE-C6 to CXA increased the yield by 6.5% and 17.6%, respectively at pH 5 and 6. Apparently, it was the non-covalent sugar molecules in the fiber complex that was released by the action of FAE. The sugar released in this case may not represent entirely hydrolytic products. The sugar yield decreased with increasing pH, with a 40% reduction when the reaction was performed at pH 7 instead of pH 5 ( Figure 5). This result suggests further evidence that the hydrolase enzymes (CXA) provided key catalytic actions in the release of sugar from the CF substrates.

Conclusion
A feruloyl esterase (FAE) gene was isolated from rumen microbial metagenome, expressed in E. coli, and the enzyme protein (fae-C6) was purified in active form. It consists of an esterase domain with an α/β hydrolase fold, composed of a catalytic triad Ser 154 Asp 263 His 295 . The FAE-C6 was characterized using corn fiber as substrate. Its combining action with glycoside hydrolases (C, X, A) individually and in various combinations was studied with focus on the difference in the hydrolytic effects on FA and sugar release. Glycoside hydrolases with endoxylanase included in the enzyme mixture show significant impact on increasing the FA yield. For the release of sugar, FAE enhanced the xylose yield in all hydrolases moderately with 11.9% the highest increase in CXAF. Endo-xylanase was not the key factor in the enzyme formulation.