Preparation of ß-galacto-oligosaccharides using a novel endo-1,4-ß-galactanase from Penicillium oxalicum.

Endo-1,4-ß-galactanase is an indispensable tool for preparing prebiotic ß-galacto-oligosaccharides (ß-GOS) from pectic galactan resources. In the present study, a novel endo-1,4-ß-galactanase (PoßGal53) belonging to glycoside hydrolase family 53 from Penicillium oxalicum sp. 68 was cloned and expressed in Pichia pastoris GS115. Upon purification by affinity chromatography, recombinant PoßGal53 exhibited a single band on SDS-PAGE with a molecular weight of 45.0 kDa. Using potato galactan as substrate, PoßGal53 showed optimal reaction conditions of pH 4.0, 40 °C, and was thermostable, retaining >80 % activity after incubating below 45 °C for 12 h. Significantly, PoßGal53 exhibited relatively conserved substrate specificity for (1 â†’ 4)-ß-D-galactan with an activity of 6244 ± 282 U/mg. In this regard, the enzyme is in effect the most efficient endo-1,4-ß-galactanase identified to date. By using PoßGal53, ß-GOS monomers were prepared from potato galactan and separated using medium pressure liquid chromatography. HPAEC-PAD, MALDI-TOF-MS and ESI-MS/MS analyses demonstrated that these ß-GOS species ranged from 1,4-ß-D-galactobiose to 1,4-ß-D-galactooctaose (DP 2-8) with high purity. This work provides not only a highly active tool for enzymatic degradation of pectic galactan, but an efficient protocol for preparing ß-GOS.

An efficient protocol for preparing linear ß-manno-oligosaccharides.

Linear ß-manno-oligosaccharides (l-ß-MOS) are widely used to investigate oligo- and poly-saccharide structures and mannanolytic enzyme activities. l-ß-MOS are also being used as prebiotic agents with potential bio-active properties. In this study, we developed an efficient protocol to prepare a series of l-ß-MOS by hydrolyzing cassia gum (CG) using mannanolytic enzymes (endo-1,4-ß-mannanase, α-galactosidases and ß-glucosidases). By using medium pressure liquid chromatography (MPLC), we purified l-ß-MOS with different degrees of polymerization (DPs). HPAEC-PAD, MALDI-TOF-MS and NMR studies confirmed that these l-ß-MOS species ranged from 1,4-ß-d-mannobiose to 1,4-ß-d-mannononaose (DP 2-9) with >95% purity. Our results provide a robust approach to preparing l-ß-MOS, thus enabling l-ß-MOS to be further used in the fields of chemistry, life science, and nutritional food.

Purification and identification of oligosaccharides from Cimicifuga heracleifolia Kom. rhizomes.

Even though Cimicifuga sp. is widely used in functional foods around the world, the content and structure of its oligosaccharides remain unclear. Here, we isolated a mixture of oligosaccharides from Cimicifuga heracleifolia Kom. rhizomes with a yield of 9.5% w/w. Twenty-six oligosaccharide monomers from the mixture were purified using optimized SEC and HILIC techniques. The oligosaccharides were identified as belonging to two groups by using HPAEC-PAD, MALDI-TOF-MS, NMR and GC-MS methylation analyses. One group belongs to sucrose and inulin type fructo-oligosaccharides (FOS) {ß-d-Fruf-(2 â†’ 1)-[ß-d-Fruf-(2 ↔ 1)]n=1-12-α-d-Glcp} with a 3-14 degree of polymerization (DP). Oligosaccharides in the other group belong to the inulo-n-ose type FOS {ß-d-Fruf-(2 â†’ 1)-[ß-d-Fruf-(2 â†’ 1)]m=0-12-ß-d-Frup} with a DP of 2-14. This appears to be the first time that these oligosaccharides have been purified from Cimicifuga heracleifolia Kom., thus providing useful information concerning the utilization of Cimicifuga heracleifolia Kom. in functional foods.

Determining Methyl-Esterification Patterns in Plant-Derived Homogalacturonan Pectins.

Homogalacturonan (HG)-type pectins are nutrient components in plants and are widely used in the food industry. The methyl-esterification pattern is a crucial structural parameter used to assess HG pectins in terms of their nutraceutical activity. To better understand the methyl-esterification pattern of natural HG pectins from different plants, we purified twenty HG pectin-rich fractions from twelve plants and classified them by their monosaccharide composition, Fourier transform-infrared spectroscopy (FT-IR) signatures, and NMR analysis. FT-IR shows that these HG pectins are all minimally esterified, with the degree of methyl-esterification (DM) being 5 to 40%. To examine their methyl-esterification pattern by enzymatic fingerprinting, we hydrolyzed the HG pectins using endo-polygalacturonase. Hydrolyzed oligomers were derivatized with 2-aminobenzamide and subjected to liquid chromatography-fluorescence-tandem mass spectrometry (HILIC-FLR-MSn). Twenty-one types of mono-/oligo-galacturonides having DP values of 1-10 were found to contain nonesterified monomers, dimers, and trimers, as well as oligomers with 1 to 6 methyl-ester groups. In these oligo-galacturonides, MSn analysis demonstrated that the number of methyl-ester groups in the continuous sequence was 2 to 5. Mono- and di-esterified oligomers had higher percentages in total methyl-esterified groups, suggesting that these are a random methyl-esterification pattern in these HG pectins. Our study analyzes the characteristics of the methyl-esterification pattern in naturally occurring plant-derived HG pectins and findings that will be useful for further studying HG structure-function relationships.

Structural characterization and immunomodulatory activity of a heterogalactan from Panax ginseng flowers.

A neutral polysaccharide fraction (WGFPN) was isolated from Panax ginseng flowers. Monosaccharide composition and HPSEC-MALLS-RI (high-performance size exclusion chromatography coupled with multi-angle laser light scattering detector and refractive index detector) analyses showed WGFPN was a heterogalactan with a molecular weight of 11.0 kDa. Methylation, 1D/2D NMR (nuclear magnetic resonance) spectra and enzymatic hydrolysis methods were used to characterize the structure of WGFPN. It possessed a less branched (1 â†’ 4)-ß-D-galactan and a significantly branched (1 â†’ 6)-ß-D-galactan. The side chains of (1 â†’ 6)-ß-D-galactan were branched with α-L-1,5-Araf and t-α-L-Araf residues at O-3. Trace amount of 1,4-linked Glcp, terminal Galp, terminal Glcp and terminal Manp residues might attached to the 1,6-linked galactan through O-3 or 1,4-linked galactan through O-6 as side chains. WGFPN could activate RAW264.7 macrophages through increasing macrophage phagocytosis, releasing NO and secreting TNF-α, IL-6, IFN-γ and IL-1ß in vitro. Moreover, WGFPN could enhance the immunity of cyclophosphamide (CTX)-induced immunosuppressed mice in vivo. Hence, WGFPN might be a potential natural immunomodulatory agent.

Analysis of pectin from Panax ginseng flower buds and their binding activities to galectin-3.

Water-soluble pectic polysaccharides isolated from Panax ginseng flower buds (WGFPA) were completely fractionated into six homogeneous fractions (WGFPA-1a, WGFPA-2a, WGFPA-3a, WGFPA-1b, WGFPA-2b and WGFPA-3b) by a combination of ion-exchange and size exclusion chromatographies. Monosaccharide composition, enzymatic hydrolysis and 13C nuclear magnetic resonance (NMR) spectra analysis were combined to characterize their structural features. Furthermore, the interactions between these polysaccharides and galectin-3 were evaluated by biolayer interferometry assay. The results showed that WGFPA-1a, WGFPA-2a and WGFPA-3a were rhamnogalacturonan I (RG-I) type pectin with abundant side chains, including α-L-1,5-arabinan, ß-D-1,4-galactan, arabinogalactan I (AG-I) and arabinogalactan II (AG-II), exhibiting strong binding activities to galectin-3 with apparent KD values 4.9 µM, 0.71 µM and 0.24 µM, respectively. WGFPA-1b, WGFPA-2b and WGFPA-3b were homogalacturonan (HG) type pectin covalently linked with different ratios of rhamnogalacturonan II (RG-II) domains, showing weaker or no interactions with galectin-3. This study provides useful structural information for further investigation on the structure-activity relationship of ginseng flower buds pectin.

Structural characterization of rhamnogalacturonan domains from Panax ginseng C. A. Meyer.

Rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II) domains were isolated from ginseng pectin by alkali saponification and endo-polygalacturonase hydrolysis, then purified by anion-exchange and size-exclusion chromatography. Monoclonal antibody detection indicated that ginseng RG-I contained →4)-α-GalpA-(1→2)-α-Rhap-(1→ repeating units as backbone, with arabinan, galactan and type II arabinogalactan (AG-II) as side chains. The use of galactose- and arabinose-releasing enzymes, mass spectrometry analysis of the oligosaccharides generated by rhamnogalacturonan hydrolase, and glycosidic linkage analyses provided evidence that ginseng RG-I contains both single galactose-branched subunits and highly branched subunits with arabinan and AG-II side chains. RG-II was analyzed by sequential acid hydrolysis followed by mass spectrometry. Ginseng RG-II contains 9 galacturonic acid units as backbone. Side chain A is an octasaccharide with 0 ∼ 1 methyl ether group. Side chain B is a nonasaccharide with 0 ∼ 1 acetyl group. These results provide useful information for further investigation of structure-activity relationship of ginseng pectin.

Preparing rhamnogalacturonan II domains from seven plant pectins using Penicillium oxalicum degradation and their structural comparison.

Rhamnogalacturonan II (RG-II) is a complex pectin with diverse pharmaceutical activities. To assess how RG-II functions, the development of methods for its preparation is required. In this paper, pectin from Codonopsis pilosula was used to evaluate the ability of fungi and bacteria to degrade the pectin. We discovered that the fungus Penicillium oxalicum could efficiently lead to the recovery of RG-II domains by degrading the other pectic domains. Further, six pectin fractions from different medical plants were used as the sole carbon source for the growth of Penicillium oxalicum. The major polymeric products remaining after fungus degradation was RG-II domains. Depending of plant source, side chains A differed with respect to their proportion of L-Gal and L-Fuc and to their degree of methyletherification. Side chains B were made of 8-10 sugar residues and up to 2 acetyl groups. Overall, our method provides an effective way to prepare RG-II pectin domains for investigating their structure-function relationships.