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 feasibil-ity and usefulness of the combinatorial enzyme technique in the transforma-tion of plant biomass to value-added products.


Introduction
Combinatorial chemistry involves the synthesis of a large array of diverse structural variations of the molecule of interest and the constructed library is then screened for target variants exhibiting desirable activities and functions [1] [2] [3]. A simple organic molecule with a core structure carrying 3 scaffolds, each randomly comprised of 4 types of substituents would result in a combinatorial library of 4 3 = 64 structural variants. A hexapeptide library randomized with the 20 natural amino acids at each position would produce a population of 20 6 = 64 million distinct peptides. Chemical techniques used for creating combinatorial libraries involve solid-phase synthesis and its refinements for libraries of small organic molecules, peptides, and oligonucleotides. In contrast, the biological approach uses the genetic code as a precursor to express randomized libraries in microorganisms, such as phage, E. coli, and yeast, through systematic and repetitive selection, mutation and amplification. The recent development of dynamic combinatorial chemistry enables the design of "smart" materials libraries of molecules evolved under thermodynamic control by responding and re-equilibrating to external conditions [4].
Numerous reviews have discussed the technology and application of combinatorial chemistry for drug discovery and optimization [3] [5]. Receiving much less attention is the potential use of combinatorial chemistry concept in agrosciences [6], and in agriculture and food research [1] [2]. Plant cell wall polysaccharides, such as xylan, pectin, and xyloglucan, consist of polymeric backbones decorated with various side groups that are cleavage targets of specific enzymes.
The presence of these side groups as well as their positions, density, and types of linkages further influence the pattern of enzymatic degradation of the main chain polymer and vice versa. Alteration of the degradation pattern of the modified main chain would in turn lead to hydrolytic products of diverse structures.
Enzymatic removal of the side group moieties individually and/or sequentially, therefore, constitutes a combinatorial design. The structural variation expressed in the oligosaccharide products, will translate into changing reactivity and functional properties. Figure 1 presents schematically a few examples of potential oligosaccharide structures from enzymatic digestion of plant fibers.
The hemicellulose polymer xylan contains a β-1,4-xylosyl main chain decorated with at least four types of side groups viz acetyl groups, phenolic-ferulic acids, glucuronyl residues, and arabinofuranosyl residues [7]. The cleavage of these side groups requires acetylxylan esterase, feruloyl esterase, β-glucuronidase, α-L-arabinofuranosidase, respectively. These side groups can be removed in a combinatorial scheme by specific enzymes targeting each group individually or in various combinations under different reaction conditions. The present work

Hydrophobic-interaction chromatography
The enzyme hydrolysate was fractionated at room temperature using an Amberlite XAD-2 column (2.5 × 50 cm). The enzyme digest was first eluted with 470 ml H 2 O at a flow rate of 0.7 ml/min. Fractions of 12 ml were collected. After washing with H 2 O, 800 ml of CH 3 OH/H 2 O (1:1 v/v) was used to elute the adsorbed FOS from the column. The fractions were measured for ferulic acids by A 320 reading, for reducing sugars by the DNSA method [8], and for total carbohydrates by the phenol-sulfuric acid method [9] [10]. Fractions were pooled, concentrated by rotary evaporator, and reconstituted in H 2 O or suitable solvent.

Size-exclusion chromatography
The pooled fractions of FOS were dissolved in CH 3 OH/H 2 O (1:3 v/v), and further purified using a Sephadex LH20 column (2.5 × 70 cm). Separation was performed with 1:3 CH 3 OH/H 2 O as eluant at a flow rate of 0.6 ml/min. Fractions of 12 ml were collected with absorbance measured at 320 nm. Individual peaks were concentrated for further analysis of ferulic acids by HPLC and carbohydrate content as xylose equivalent by the phenol-sulfuric acid method as described below.

Measurement of xylose equivalent and ferulic acid content
The ferulic acid content in the concentrated samples was quantified by HPLC after alkaline hydrolysis (1N NaOH, 37˚C, 16 hr) [11]. The carbohydrate con-

Results and Discussion
Feruloyl oligosaccharides have been studied using various degradation methods, including acid hydrolysis, hydrothermal processing, microwave-assisted auto- The FOS was applied to a Sephadex LH20 column, which further separated into The FOS showed an inhibitory effect on the growth of the E. coli strain ATCC 8379 in an initial testing. The enzyme-derived FOS was screened with the test microorganism E. coli ATCC 8379 for bioactive properties. In each set of experiments, the initial inoculation of the microorganism was carefully controlled to 1 × 10 3 cfu/ml titer, so that the comparisons were performed under same starting conditions. Figure 3(a) shows that the inhibitory effect increased with the concentration, and a complete suppression of cell growth was achieved at 0.07% w/v, which was the MIC (minimum inhibitory concentration) value. The antimicrobial effect was sustainable for three days and possibly longer (Figure 3(b)).
The FOS major peak from the LH20 column was analyzed by HPLC gel permeation chromatography showing a peak of MW  1.3 kD (based on a pullulan standard curve) ( Figure 4). This size range is generally considered in the category of low molecular weight oligosaccharides. The small size of the oligosaccharide may be a factor in facilitating its passage through the cell membrane.
Reports of antimicrobial oligosaccharides generally refer to low molecular weight molecules, consisting of tri-, tetra-, and pentamers. Oligosaccharides in the high molecular weight range have been shown to prevent efficient in vivo utilization and generally are not effective in functional and biological activities [21] [22]. The present results seem to support the suggestion that small molecular  The mechanism of inhibition may be linked to the unique structural properties of the active species. The antimicrobial activity of some classes of phenolic compounds has been attributed to the presence of reactive double bonds and the association of the acid moiety [23]. Double bonds are electrophilic and can participate in a variety of reactions, resulting in crosslinking and inactivation of biomolecules. Antimicrobial activities of phenolic compounds are found in hydrolysis of lignocellulosic materials, comparable to the common preservative sodium benzoate [24]. Feruloyl oligosaccharides derived from feruloyl polysaccharides have been shown to have biological activities. A feruloylated arabinoxylan trisaccharide FAXX prepared from cell walls of bamboo shoots inhibits axin-stimulated cell growth of rice plants [25]. The study also indicates that the feruloyl substituent is necessary for the inhibitory effect, but the glycosyl portion of FAXX is also important for exerting full activity. Feruloyl oligosaccharides from corn bran and wheat aleurone have been shown to inhibit sucrase and maltase activities of α-glucosidase and glucose absorption [26]. Wheat bran FOS has shown a higher antioxidant activity than free ferulic acid, and in vivo protection against oxidative stress in animal studies [27].  [29]. Acidic (glucuronic acid-containing) xylo-oligosaccharides, particularly aldopentauronic acids, are effective inhibitors of certain gram-positive bacteria [21]. The pectic hydrolysate obtained by the digestion of citrus pectin with endo-polygalacturonase and pectate lyase resulted in active pectic oligo species with antimicrobial properties [30]. The mechanism of inhibition may be attributed to the unsaturated structure (reactive double bonds) due to the elimination reaction of pectate lyase, the acidic nature (carboxylic side groups) of the substituents, and the small size range of the molecule.
Investigation will be necessary to characterize the structure and function connectivity as well as health cause-effects of functional oligosaccharides using molecular, biochemical, microbiological, and physiological studies.
The activity of FOS species in the present study can be considered comparable to those of some common food preservatives, which are generally applied in the range of 0.1%. The use of non-digestible oligosaccharides (NDO) has become a thriving industry providing various prebiotics for food uses. Another potential use may be found in the animal feed industry. Functional oligosaccharides have been promoted as alternatives for antibiotics in animal production. These so-called natural antimicrobial growth promoters (AGP) have gained increasing attention in recent years [31]. The health cause-effect of these products is generally linked to the promotion of beneficial bacteria in the gut microbiome, and modification of the physiological environment of the intestinal digestive system [32]. In practical applications, functional oligosaccharides are used in sub-minimum inhibitor concentrations acting to modulate the microbiota composition.

Conclusion
Active FOS species has been derived from combinatorial enzyme digestion and isolated and shown to inhibit the growth of the E. coli test organism ATCC 8739.
The MIC value was estimated to be comparable favorably to those reported for other active oligosaccharides and some common food preservatives. The FOS species may find useful application as a new source of high-value prebiotics or as alternatives to antimicrobial growth promoters.