Rhamnogalacturonan I (Potato)

Content: 2 g
Shipping Temperature: Ambient
Storage Temperature: Ambient
Physical Form: Powder
Stability: > 2 years under recommended storage conditions
CAS Number: 39280-21-2
Source: Potato fiber
Purity: > 90%
Monosaccharides (%): Galacturonic Acid: Rhamnose: Arabinose: Xylose: Galactose: Other Sugars = 61.0: 6.2: 2.5: 0.5: 23.1: 6.7
Treatment: Enzyme Hydrolysis
Substrate For (Enzyme): Rhamnogalacturonan Hydrolase, Rhamnogalacturonan Lyase

High purity Rhamnogalacturonan I (Potato) for use in research, biochemical enzyme assays and in vitro diagnostic analysis.

Prepared from pectic galactan from potato fiber. An excellent substrate for the assay of rhamnogalacturonan hydrolase and rhamnogalacturonan lyase.

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Data Sheet
Publications
Publication

Acetylation in Ionic Liquids Dramatically Increases Yield in the Glycosyl Composition and Linkage Analysis of Insoluble and Acidic Polysaccharides.

Black, I. M., Ndukwe, I. E., Vlach, J., Backe, J., Urbanowicz, B. R., Heiss, C. & Azadi, P. (2023). Analytical Chemistry, 95(34), 12851-12858.

Glycosyl composition and linkage analyses are important first steps toward understanding the structural diversity and biological importance of polysaccharides. Failure to fully solubilize samples prior to analysis results in the generation of incomplete and poor-quality composition and linkage data by gas chromatography-mass spectrometry (GC-MS). Acidic polysaccharides also do not give accurate linkage results, because they are poorly soluble in DMSO and tend to undergo β-elimination during permethylation. Ionic liquids can solubilize polysaccharides, improving their derivatization and extraction for analysis. We show that water-insoluble polysaccharides become much more amenable to chemical analysis by first acetylating them in an ionic liquid. Once acetylated, these polysaccharides, having been deprived of their intermolecular hydrogen bonds, are hydrolyzed more readily for glycosyl composition analysis or methylated more efficiently for glycosyl linkage analysis. Acetylation in an ionic liquid greatly improves composition analysis of insoluble polysaccharides when compared to analysis without acetylation, enabling complete composition determination of normally recalcitrant polysaccharides. We also present a protocol for uronic acid linkage analysis that incorporates this preacetylation step. This protocol produces partially methylated alditol acetate derivatives in high yield with minimal β-elimination and gives sensitive linkage results for acidic polysaccharides that more accurately reflect the structures being analyzed. We use important plant polysaccharides to show that the preacetylation step leads to superior results compared to traditional methodologies.

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Substrate size-dependent conformational changes of bacterial pectin-binding protein crucial for chemotaxis and assimilation.

Anamizu, K., Takase, R., Hio, M., Watanabe, D., Mikami, B., & Hashimoto, W. (2022). Scientific Reports, 12(1), 1-15.

Gram-negative Sphingomonas sp. strain A1 exhibits positive chemotaxis toward acidic polysaccharide pectin. SPH1118 has been identified as a pectin-binding protein involved in both pectin chemotaxis and assimilation. Here we show tertiary structures of SPH1118 with six different conformations as determined by X-ray crystallography. SPH1118 consisted of two domains with a large cleft between the domains and substrates bound to positively charged and aromatic residues in the cleft through hydrogen bond and stacking interactions. Substrate-free SPH1118 adopted three different conformations in the open form. On the other hand, the two domains were closed in substrate-bound form and the domain closure ratio was changed in response to the substrate size, suggesting that the conformational change upon binding to the substrate triggered the expression of pectin chemotaxis and assimilation. This study first clarified that the solute-binding protein with dual functions recognized the substrate through flexible conformational changes in response to the substrate size.

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Symbiont Digestive Range Reflects Host Plant Breadth in Herbivorous Beetles.

Salem, H., Kirsch, R., Pauchet, Y., Berasategui, A., Fukumori, K., Moriyama, M., et al. (2020). Current Biology, 30(15), 2875-2886

Numerous adaptations are gained in light of a symbiotic lifestyle. Here, we investigated the obligate partnership between tortoise leaf beetles (Chrysomelidae: Cassidinae) and their pectinolytic Stammera symbionts to detail how changes to the bacterium’s streamlined metabolic range can shape the digestive physiology and ecological opportunity of its herbivorous host. Comparative genomics of 13 Stammera strains revealed high functional conservation, highlighted by the universal presence of polygalacturonase, a primary pectinase targeting nature’s most abundant pectic class, homogalacturonan (HG). Despite this conservation, we unexpectedly discovered a disparate distribution for rhamnogalacturonan lyase, a secondary pectinase hydrolyzing the pectic heteropolymer, rhamnogalacturonan I (RG-I). Consistent with the annotation of rhamnogalacturonan lyase in Stammera, cassidines are able to depolymerize RG-I relative to beetles whose symbionts lack the gene. Given the omnipresence of HG and RG-I in foliage, Stammera that encode pectinases targeting both substrates allow their hosts to overcome a greater diversity of plant cell wall polysaccharides and maximize access to the nutritionally rich cytosol. Possibly facilitated by their symbionts’ expanded digestive range, cassidines additionally endowed with rhamnogalacturonan lyase appear to utilize a broader diversity of angiosperms than those beetles whose symbionts solely supplement polygalacturonase. Our findings highlight how symbiont metabolic diversity, in concert with host adaptations, may serve as a potential source of evolutionary innovations for herbivorous lineages.

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Isolation of a rhamnogalacturonan lyase expressed during ripening of the Chilean strawberry fruit and its biochemical characterization.

Méndez-Yañez, A., González, M., Carrasco-Orellana, C., Herrera, R. & Moya-León, M. A. (2020). Plant Physiology and Biochemistry, 146, 411-419.

Fragaria chiloensis (L.) Mill. fruit has exotic organoleptic properties however commercialization is a challenge due to its fast and intensive softening. Texture modifications associated to ripening are related to cell wall metabolism. Main cell wall polysaccharides metabolized in F. chiloensis fruit are pectins, being rhamnogalacturonan I (RG-I) an abundant pectin domain in strawberry. Several enzymes belonging to the fruit molecular machinery have been described to act on different cell wall polysaccharides in F. chiloensis, but none acting on the main chain of RG-I until now. A gene sequence coding for a rhamnogalacturonan endolyase (RG-lyase) (EC 4.2.2.23) was isolated from F. chiloensis. The FchRGL1 sequence belongs to Polysaccharide Lyase family 4 and contains the three functional domains of RG-lyases: RGL4 domain, fibronectin type III and the carbohydrate binding module. In addition, it contains key amino acid residues for activity and Ca2+ coordination. qRT-PCR analyses indicate that FchRGL1 transcripts increase in fruit throughout ripening. RG-lyase activity evidences a remarkable increase as the fruit ripens. The heterologous expression of FchRGL1 in Pichia pastoris provided an active protein that allows its biochemical characterization. RG-lyase activity is optimum at pH 5.0, 25-30°C and 2 mM Ca2+. A KM of 0.086 mg mL-1 was determined for potato RG-I, and the enzyme undergoes inhibition at high substrate concentration. The enzyme is also able to degrade the mucilage of germinating A. thaliana's seeds. Finally, the properties of FchRGL1 and its expression pattern are congruent with a crucial role in cell wall re-organization during softening of F. chiloensis fruit.

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Kinetics and regioselectivity of three GH62 α-L-arabinofuranosidases from plant pathogenic fungi.

Sarch, C., Suzuki, H., Master, E. R. & Wang, W. (2019). Biochimica et Biophysica Acta (BBA)-General Subjects, 1863(6), 1070-1078.

Backgound: Xylan is the second most abundant plant cell wall polysaccharide after cellulose with α-L-arabinofuranose (L-Araf) as one of the major side substituents. Capacity to degrade xylan is characteristic of many plant pathogens; and corresponding enzymes that debranch arabinoxylan provide tools to tailor xylan functionality or permit its full hydrolysis. Method: Three GH62_2 family α-arabinofuranosidases (Abfs) from plant pathogenic fungi, NhaAbf62A from Nectria haematococca, SreAbf62A from Sporisorium reilianum and GzeAbf62A from Gibberella zeae, were recombinantly produced in Escherichia coli. Their biochemical properties and substrate specificities were characterized in detail. Particularly with 1H NMR, the regioselectivity and debranching preference of the three Abfs were directly compared. Results: The activities of selected Abfs towards arabinoxylan were all optimal at pH 6.5. Their preferred substrates were wheat arabinoxylan, followed by soluble oat spelt xylan. The Abfs displayed selectivity towards either α-(1 → 2) or α-(1 → 3)-L-Araf mono-substituents in arabinoxylan. Specifically, SreAbf62A and GzeAbf62A removed m-α-(1 → 3)-L-Araf and m-α-(1 → 2)-L-Araf substituents with a similar rates, whereas NhaAbf62A released m-α-(1→ 3)-L-Araf 1.9 times faster than m-α-(1 → 2)-L-Araf. Major conclusions: Building upon the known selectivity of GH62 family α-arabinofuranosidases towards L-Araf mono-substituents in xylans, the current study uncovers enzyme-dependent preferences towards m-α-(1 → 3)-L-Araf and m-α-(1 → 2)-L-Araf substitutions. Comparative sequence-structure analyses of Abfs identified an arginine residue in the xylose binding +2R subsite that was correlated to the observed enzyme-dependent L-Araf debranching preferences. General significance: This study expands the limited pool of characterized GH62 Abfs particularly those from plant pathogenic fungi, and provides biochemical details and methodology to evaluate regioselectivity within this glycoside hydrolase family.

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Paenibacillus amylolyticus 27C64 has a diverse set of carbohydrate-active enzymes and complete pectin deconstruction system.

Keggi, C. & Doran-Peterson, J. (2019). Journal of Industrial Microbiology & Biotechnology, 46(1), 1-11.

A draft genome of Paenibacillus amylolyticus 27C64 was assembled and a total of 314 putative CAZymes in 108 diferent families were identifed. Comparison to well-studied polysaccharide-degrading organisms revealed that P. amylolyticus 27C64 has as many or more putative CAZymes than most of these organisms. Four different pectic substrates and xylan supported growth but cellulose was not utilized. Measurement of enzyme activities in culture supernatants revealed low levels of cellulase activity, high levels of xylanase activity, and pectinase activities that adapted to the specifc polysaccharides provided. Relative expression levels of each putative pectinase in cells grown with and without three diferent pectic substrates were evaluated with RT-qPCR and distinct sets of genes upregulated in response to homogalacturonan, methylated homogalacturonan, and rhamnogalacturonan I were identifed. It is also noted that this organism’s pectinolytic system difers from other well-studied systems and contains enzymes which are of value for further study.

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A rapid-throughput adaptable method for determining the monosaccharide composition of polysaccharides.

Amicucci, M. J., Galermo, A. G., Nandita, E., Vo, T. T. T., Liu, Y., Lee, M., Xu, G. & Lebrilla, C. B. & Lebrilla, C. B. (2019). International Journal of Mass Spectrometry, 438, 22-28.

Polysaccharides make up the largest non-water component of plant-based foods. Their ability to manipulate the gut microbiome and modulate the immune system has increased interest in the rapid elucidation of their structures. A necessary component for the structural characterization of polysaccharides is the determination of their monosaccharide composition. Current methods of monosaccharide analysis are not suitable for analyzing large sample-sets and are limited by their inability to analyze polysaccharides. We have developed a 96-well plate hydrolysis and derivatization procedure followed by a rapid and sensitive 10-min ultra-high performance liquid chromatography triple quadrupole mass spectrometry analysis capable of the absolute quantitation of 14 plant monosaccharides. Four polysaccharide standards, inulin, xyloglucan, arabinogalactan, and rhamnogalacturonan-I, which are commonly found in plants, were used to optimize and validate the method. The optimized conditions were applied to eight foods to show the method’s reproducibility and ability to analyze complicated and insoluble polysaccharide mixtures. This approach will allow researchers to obtain accurate and absolute quantitation of monosaccharides in the large sample-sets that are required for agricultural, food, clinical, and nutrition-based studies.

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Crystal structure of exo‐rhamnogalacturonan lyase from Penicillium chrysogenum as a member of polysaccharide lyase family 26.

Kunishige, Y., Iwai, M., Nakazawa, M., Ueda, M., Tada, T., Nishimura, S. & Sakamoto, T. (2018). FEBS Letters, 592(8), 1378-1388.

Exo‐rhamnogalacturonan lyase from Penicillium chrysogenum 31B (PcRGLX) was recently classified as a member of polysaccharide lyase (PL) family 26 along with hypothetical proteins derived from various organisms. In this study, we determined the crystal structure of PcRGLX as the first structure of a member of this family. Based on the substrate‐binding orientation and substrate specificity, PcRGLX is an exo‐type PL that cleaves rhamnogalacturonan from the reducing end. Analysis of PcRGLX‐complex structures with reaction products indicate that the active site possesses an L‐shaped cleft that can accommodate galactosyl side chains, suggesting side‐chain‐bypassing activity in PcRGLX. Furthermore, we determined the residues critical for catalysis by analyzing the enzyme activities of inactive variants.

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Characterisation of pectin-xylan complexes in tomato primary plant cell walls.

Broxterman, S. E. & Schols, H. A. (2018). Carbohydrate Polymers, 197, 269-276.

The primary plant cell wall is composed of a complex network of pectin, hemicellulose and cellulose. Potential interactions between these polysaccharides were studied for carrot, tomato and strawberry, with a focus on the role of pectin. The Chelating agent Unextractable Solids (ChUS), the residue after water- and EDTA extraction, was ball milled and subsequently water extracted. For tomato and strawberry, pectin and substantial amounts of hemicellulose were solubilised. Anion exchange chromatography (AEC) showed co-elution of pectin and acetylated glucuronoxylan in tomato, representing 18% of solubilised uronic acid and 48% of solubilised xylose by ball milling from ChUS. The existence of a covalently linked pectin-xylan complex was proposed since xylan co-precipitated with pectin under mild alkali conditions. It was proposed that pectin links with xylan through the RG-I region since degradation of HG did not alter AEC elution patterns for RG-I and xylan, suggesting RG-I - xylan interactions.

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Protopectinase production by Paenibacillus polymyxa Z6 and its application in pectin extraction from apple pomace.

Zhang, J., Zhao, L., Gao, B., Wei, W., Wang, H. & Xie, J. (2018). Journal of Food Processing and Preservation, 42(1), e13367.

Paenibacillus polymyxa Z6 was screened as protopectinase (PPase) producing strain and its PPase activity was 44.4 U/mL. The factors influencing PPase production were identified by a two-level Plackett-Burman design with seven variables. The results indicated that Ca2+ concentration, fermentation time, and temperature were the most influential factors on the PPase production, which were applied in the Box-Behnken design. The predicted maximum PPase activity was 219 U/mL and the experimental maximum PPase activity was 221 U/mL, under the predicted optimum conditions, 170 mg/L Ca2+, 27°C, and 29 hr of fermentation. The present PPase was composed of both type-A PPase, polygalacturonase; and type-B PPase, arabinanase, and rhamnogalacturonase. Finally, the PPase was applied for the pectin extraction from apple pomace and achieved an average yield of 11.9% with properties like 8.5% moisture content, 1.6% ash content, 3.8 mPa.S viscosity, and pH 6.1 of 1% solution.

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Biochemical characterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall.

Uehara, Y., Tamura, S., Maki, Y., Yagyu, K., Mizoguchi, T., Tamiaki, H., Imai, T., Ishii, T., Ohashi, T., Fujiyama, K. & Ishimizu, T. (2017). Biochemical and Biophysical Research Communications, 486(1), 130-136.

The pectin in plant cell walls consists of three domains: homogalacturonan, rhamnogalacturonan (RG)-I, and RG-II. It is predicted that around 50 different glycosyltransferases are required for their biosynthesis. Among these, the activities of only a few glycosyltransferases have been detected because pectic oligosaccharides are not readily available for use as substrates. In this study, fluorogenic pyridylaminated RG-I-backbone oligosaccharides (PA-RGs) with 3–14 degrees of polymerization (DP) were prepared. Using these oligosaccharides, the activity of RG-I:rhamnosyltransferase (RRT), involved in the biosynthesis of the RG-I backbone diglycosyl repeating units (-4GalUAα1-2Rhaα1-), was detected from the microsomes of azuki bean epicotyls. RRT was found to prefer longer acceptor substrates, PA-RGs with a DP > 7, and it does not require any metal ions for its activity. RRT is located in the Golgi and endoplasmic reticulum. The activity of RRT coincided with epicotyl growth, suggesting that RG-I biosynthesis is involved in plant growth.

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