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 Ser154-Asp263His295 and a classical Gly-X-Ser154-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.

Combinatorial Enzyme Approach to Convert Wheat Insoluble Arabinoxylan to Bioactive Oligosaccharides

Combinatorial enzyme technology was applied for the conversion of wheat insoluble arabinoxylan to oligosaccharide structural variants. The digestive products were fractionated by Bio-Gel P4 column and screened for bioactivity. One fraction pool was observed to exhibit antimicrobial property resulting in the suppression of cell growth of the test organism ATCC 8739 E. coli. It has a MIC value of 1.5% (w/v, 35°C, 20 hr) and could be useful as a new source of prebiotics or preservatives. The present results further confirm the science and useful application of combinatorial enzyme approach.

Release of Arabinose from Wheat Insoluble Arabinoxylan by the Action of α-L-Arabinofuranosidase in Synergism with Endo-Xylanases

Wheat arabinoxylan (water-insoluble fraction) contains ~36% arabinose which may include both singly or doubly substitutions at C2/C3 of the Xylp units. α-L-Arabinofuranosidses (ABFs) of two GH families were analyzed for their respective activities on the hydrolysis of Xylp-Araf. BaABF (GH43) produced twice the yield of arabinose residues from the heteroxylan compared to AnABF (GH51) under the same reaction conditions. Two endo-xylanases (of GH10 and 11) also showed differential hydrolytic activities on the Xylp chain, with the GH10 XYN-ATM double the amount of reducing sugar yield (as xylose equivalent) than using the GH11 XYN-M3. When the ABF and XYN were combined in optimial ratios, a synergistic increase of 73.8% in arabinose yield was observed.

Combinatorial Enzyme Approach for Production and Screening of Libraries of Feruloyl Oligosaccharides

Combinatorial chemistry involves the chemical or biological synthesis of diverse variation of the structures of a target molecule and the library is then screened for variants of desirable target properties. The approach has been a focus of research activity in drug discovery and biotechnology. This report is to demonstrate the application of enzyme technology using the concept of combinatorial chemistry as a novel approach for the bioconversion of plant fibers. Wheat insoluble fiber was subjected to combinatorial enzyme digestion to create structural variants of feruloyl oligosaccharides (FOS). Fractionation and screening resulted in the isolation of a fraction of bioactive FOS species showing antimicrobial activity. These results demonstrate the feasibility and usefulness of the combinatorial enzyme technique in the transformation of plant biomass to value-added products.

Cloning of an α-L-Arabinofuranosidase and Characterization of Its Action on Mono- and Di-Substituted Xylopyranosyl Units

An α-L-arabinofuranosidase (ARF) gene of 1503 bp was synthesized, subcloned into pET26b vector, and expressed in Escherichia coli. The enzyme was purified in active form, and consisted of 500 amino acid residues, corresponding to 55 kD based on SDS-PAGE. The affinity-purified protein was characterized using arabinofuranosyl xylooligosaccharides (AXOS) as substrates. The pH effect was investigated showing an optimum at pH 5.5. XaARF catalyzed the cleavage of arabinose at C3 of the xylopyranosyl unit efficiently if the arabinofuranosyl substitution was at the terminal compared to internal xylose units. The enzyme was able to act on di-substituted xylopyranosyl units with the first cleavage at C3 followed by C2 linkages.

Combinatorial Enzyme Approach to Produce Oligosaccharides of Diverse Structures for Functional Screen

Combinatorial chemistry has been a focus of research activity in modern drug discovery and biotechnology. It is a concept by which a vast library of molecular diversity is synthesized and screened for target properties. This report is to illustrate the application of enzyme technology using the concept of combinatorial chemistry as a novel approach for the bioconversion of plant fibers. Citrus pectin was subjected to combinatorial enzyme digestion to create libraries of pectic oligosaccharides with diverse structural variants. Repeated cycles of fractionation and screening resulted in the isolation and identification of an active oligoGalA species with antimicrobial activity.

Preparative Fractionation of Feruloyl Oligosaccharides Produced by Combinatorial Enzyme Digestion of Arabinoxylan

Pretreated wheat insoluble arabinoxylan was converted to oligosaccharides of structural variants using combinatorial enzyme approach. The digestive products were separated by preparative scale chromatographic Amberlite XAD-2 column. Fractions containing feruloyl oligosaccharides (FOS) were isolated, pooled, freeze-dried, and demonstrated to possess antimicrobial activity. The FOS suppressed cell growth of the test organism ATCC 8739 E. coli with a MIC value of 0.028% (w/v, 35˚C, 24 hr). The antimicrobial action was observed exceeding 72 hr of culture incubation. The FOS product could be a useful source of prebiotics or preservatives. The present results further confirm the science and application of the concept of combinatorial enzyme technique.