Preparation and Antioxidant Activity of Acetylated Mung Bean Peel Polysaccharides

Mung bean peel polysaccharides are one of the main active components in mung bean peel. Acetylated mung bean peel polysaccharides were prepared by extracting and acetylating them, and characterized by infrared and ultraviolet methods to preliminarily understand the structural characteristics and activity of acetylated mung bean peel polysaccharides. Acetylation modification can improve the structure of polysaccharides, thereby causing changes in their properties. The product obtained after acetylation modification exhibited new characteristic absorption peaks at 1732 cm⁻¹, and the scavenging ability of hydroxyl radicals was improved. Therefore, acetylation modification of mung bean peel polysaccharides could enhance the activity by improving the structure, which provided an experimental basis for the application of mung bean peel polysaccharides.     

Preparation of Acetylated Grapefruit Peel Polysaccharide

Grapefruit peel polysaccharide has antioxidant, antitumor, hypoglycemic and other biological activities, and chemical modification can further improve the properties of the polysaccharide. Acetylation modification of polysaccharides has the advantages of simple operation, low cost and little pollution, and is widely used at present. Different degrees of acetylation modification have different effects on the properties of polysaccharides, so it is necessary to optimize the preparation technology of acetylated grapefruit peel polysaccharides. In this article, acetylated grapefruit peel polysaccharide was prepared by acetic anhydride method. With the degree of acetyl substitution as the evaluation index, combined with the analysis of sugar content and protein content in the polysaccharide before and after modification, the effects of three feeding ratios of 1:0.6, 1 : 1.2 and 1 : 1.8 (polysaccharide: acetic anhydride, mass/volume) on acetylation modification were explored through single factor experiments. The results showed that the optimum ratio of material to liquid for acetylation modification of grapefruit peel polysaccharide was 1:0.6. Under these conditions, the degree of substitution of acetylated grapefruit peel polysaccharide was 0.323, the sugar content was 59.50 % and the protein content was 1.038 %. The results provide some reference for the study of acetylated grapefruit peel polysaccharide.     

Structural analysis of alfa grass (Stipa tenacissima L.) lignin obtained by acetic acid/formic acid delignification

Alfa grass lignin obtained by the acetic acid/formic acid/water CIMV pulping process was characterized by FTIR and (1)H, (13)C-(1)H 2D HSQC, and (31)P NMR spectroscopies. Lignin samples purified by further dissolution/precipitation or basic hydrolysis steps were also analyzed. The CIMV alfa lignin is a mixture of low molar mass compounds (M(n) = 1500 g/mol) of SGH type with β-O-4 ether bonds as the major interunit linkage. The crude lignin contains fatty acids and residual polysaccharides. It also contains large amounts of acetate and hydroxycinnamates, mostly in the γ-position of β-O-4 interunit linkages. Although partial acetylation induced by the process cannot be excluded, the absence of aromatic acetates and acetylated polysaccharides in crude lignin demonstrates the mildness of the process. By combining smooth alkaline hydrolysis and dissolution/precipitation steps to the CIMV pulping, it is possible to produce a purified lignin with a composition and a structure quite analogous to that of the native polymer in the plant.     

多糖修饰物及其抗肿瘤作用机制研究

多糖是指一类由十个以上单糖分子通过糖苷键连接而成的糖类物质,因其具有许多重要的生物学活性而备受关注,这其中最为突出的是其在抗肿瘤方面所表现出的效用。目前多糖抗肿瘤方面的主要研究课题集中在如何进一步提高多糖的抗肿瘤活性上。对多糖分子进行结构上的修饰能够使其抗肿瘤的活性得到一定程度地提高。本文对常见的多糖修饰方法,如硫酸化、羧甲基化、磷酸化、硒化、乙酰化等进行了总结,对经修饰后所得产物的抗肿瘤作用的不同机制进行了阐述,并对多糖化学修饰物的应用前景进行了展望,为未来多糖的开发与利用提供一定的理论依据。     

Synergistic interactions between the genetically modified bacterial polysaccharide P2 and carob or konjac mannan

Rheological studies have confirmed that the bacterial polysaccharide P2, a genetically modified variant of the Acetobacter xylinum polysaccharide acetan, undergoes synergistic gelation with either of the plant polysaccharides carob or konjac mannan. X-ray fibre diffraction data shows that P2 can form a 5-fold helical structure of pitch 4.7nm and an axial rise per disaccharide repeat of 0.92nm. Optical rotation data demonstrate that P2 undergoes a coil-helix transition in solution and that deacylation enhances the stability of the helical structure in solution. Studies made on mixtures prepared at different temperatures and ionic strengths suggest that denaturation of the P2 helix favours interaction and gelation. Deacetylation of P2 enhances gelation. X-ray diffraction data for oriented fibres prepared from deacetylated P2-konjac mannan mixed films reveal a 6-fold helical structure of pitch 5.54nm with an axial rise per disaccharide repeat also of 0.92nm. This mixed helix provides direct evidence for binding between the two polysaccharides. P2 contains two sites of acetylation: one on the backbone and one on the sidechain. The former site of acetylation inhibits helix formation for P2. It is suggested that this site of acetylation also inhibits formation of the mixed helix, explaining the enhanced gelation of mixtures on deacetylation.     

Acetylesterase-mediated deacetylation of pectin impairs cell elongation, pollen germination, and plant reproduction

Pectin is a major component of the primary cell wall of higher plants. Some galacturonyl residues in the backbone of pectinaceous polysaccharides are often O-acetylated at the C-2 or C-3 position, and the resulting acetylesters change dynamically during the growth and development of plants. The processes involve both enzymatic acetylation and deacetylation. Through genomic sequence analysis, we identified a pectin acetylesterase (PAE1) from black cottonwood (Populus trichocarpa). Recombinant Pt PAE1 exhibited preferential activity in releasing the acetate moiety from sugar beet (Beta vulgaris) and potato (Solanum tuberosum) pectin in vitro. Overexpressing Pt PAE1 in tobacco (Nicotiana tabacum) decreased the level of acetyl esters of pectin but not of xylan. Deacetylation engendered differential changes in the composition and/or structure of cell wall polysaccharides that subsequently impaired the cellular elongation of floral styles and filaments, the germination of pollen grains, and the growth of pollen tubes. Consequently, plants overexpressing PAE1 exhibited severe male sterility. Furthermore, in contrast to the conventional view, PAE1-mediated deacetylation substantially lowered the digestibility of pectin. Our data suggest that pectin acetylesterase functions as an important structural regulator in planta by modulating the precise status of pectin acetylation to affect the remodeling and physiochemical properties of the cell wall's polysaccharides, thereby affecting cell extensibility.     

Comparative genomics of pectinacetylesterases: Insight on function and biology

Pectin acetylation influences the gelling ability of this important plant polysaccharide for the food industry. Plant apoplastic pectinacetylesterases (PAEs) play a key role in regulating the degree of pectin acetylation and modifying their expression thus represents one way to engineer plant polysaccharides for food applications. Identifying the major active enzymes within the PAE gene family will aid in our understanding of this biological phenomena as well as provide the tools for direct trait manipulation. Using comparative genomics we propose that there is a minimal set of 4 distinct PAEs in plants. Possible functional diversification of the PAE family in the grasses is also explored with the identification of 3 groups of PAE genes specific to grasses.     

Mise en évidence de polysaccharides par le noir Soudan B après estérification

Résumé Les effets de l'acétylation réversible et de l'estérification sulfurique sur la coloration de polysaccharides par le noir Soudan B sont étudiés après inclusion à la paraffine; les faits suivants sont à noter. 1° Une affinité nette pour le noir Soudan B en solution alcoolique existe, dans le cas des polysaccharides très acides, sans aucun prétraitement des coupes. 2° Tous les polysaccharides présentent, après acétylation ou estérification sulfurique, une forte affinité pour le noir Soudan B, le bleu alcian et le bleu de toluidine; la teinte est souvent métachromatique avec ce dernier colorant. 3° L'affinité des polysaccharides pour les trois colorants disparaît après saponification des coupes acétylées. 4° Les teintes les plus intenses sont obtenues avec les solutions vieillies ou acétifiées de noir Soudan B. — Ces faits plaident en faveur de l'hypothèse suivant laquelle le noir Soudan B possède certaines propriétés des colorants basiques.

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Staining of polysaccharides with Sudan black B after esterification

Summary This study centers on the effects of acetylation, deacetylation, and sulphation on the staining of polysaccharides with Sudan black B in paraffin sections; it brings up the following facts: A definite staining of strongly acidic polysaccharides with alcoholic Sudan black B is obtained without any pretreatment of the sections. — After acetylation or sulphation, all the investigated polysaccharides exhibit a strong affinity to Sudan black B, alcian blue and toluidine blue; a distinct metachromasia is obtained with the third of these stains. — Any affinity of polysaccharides to the three dyestuffs is suppressed by deacetylation of the acetylated sections. — The most intense staining with Sudan black B is given by old or acetified solutions. — These data are consistent with the hypothesis according to which Sudan black B is provided with some characteristics of a basic stain.

     

Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen employing 1H and 13C NMR spectroscopy

Water-soluble hemicelluloses were extracted from milled aspen wood (Populus tremula) employing microwave oven treatment at 180 degrees C for 10 min. The final pH of this extract was 3.5. From this extract oligo- and polysaccharides were isolated and subsequently fractionated by size-exclusion chromatography. The structures of the saccharides in three of the fractions obtained were determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. The polysaccharides present in the two fractions eluted first were O-acetyl-(4-O-methylglucurono)xylans. The average degree of acetylation of the xylose residues in these compounds was 0.6. The structural element -->4)[4-O-Me-alpha-D-GlcpA-(1-->2)][3-O-Ac]-beta-D-Xylp-(1 --> could also be identified. On the average, these two xylans were composed of the following (1-->4)-linked beta-D-xylopyranosyl structural elements: unsubstituted (50 mol%), 2-O-acetylated (13 mol%), 3-O-acetylated (21 mol%), 2,3-di-O-acetylated (6 mol%) and [MeGlcA alpha-(1-->2)][3-O-acetylated] (10 mol%). Most of the 4-O-methylglucuronyl and acetyl substituents in the isolated polysaccharides survived the microwave oven treatment. The third fraction, eluted last, contained acetylated xylo-oligosaccharides, with minor contamination by an acetylated mannan. In the case of these xylo-oligosaccharides, the average degree of acetylation was 0.3.     

Acetylation of glucosyltransferases regulates Streptococcus mutans biofilm formation and virulence

Lysine acetylation is a frequently occurring post-translational modification (PTM), emerging as an important metabolic regulatory mechanism in prokaryotes. This process is achieved enzymatically by the protein acetyltransferase (KAT) to specifically transfer the acetyl group, or non-enzymatically by direct intermediates (acetyl phosphate or acetyl-CoA). Although lysine acetylation modification of glucosyltransferases (Gtfs), the important virulence factor in Streptococcus mutans, was reported in our previous study, the KAT has not been identified. Here, we believe that the KAT ActG can acetylate Gtfs in the enzymatic mechanism. By overexpressing 15 KATs in S. mutans, the synthesized water-insoluble extracellular polysaccharides (EPS) and biofilm biomass were measured, and KAT (actG) was identified. The in-frame deletion mutant of actG was constructed to validate the function of actG. The results showed that actG could negatively regulate the water-insoluble EPS synthesis and biofilm formation. We used mass spectrometry (MS) to identify GtfB and GtfC as the possible substrates of ActG. This was also demonstrated by in vitro acetylation assays, indicating that ActG could increase the acetylation levels of GtfB and GtfC enzymatically and decrease their activities. We further found that the expression level of actG in part explained the virulence differences in clinically isolated strains. Moreover, overexpression of actG in S. mutans attenuated its cariogenicity in the rat caries model. Taken together, our study demonstrated that the KAT ActG could induce the acetylation of GtfB and GtfC enzymatically in S. mutans, providing insights into the function of lysine acetylation in bacterial virulence and pathogenicity.     

Loss-of-function mutation of REDUCED WALL ACETYLATION2 in Arabidopsis leads to reduced cell wall acetylation and increased resistance to Botrytis cinerea

Nearly all polysaccharides in plant cell walls are O-acetylated, including the various pectic polysaccharides and the hemicelluloses xylan, mannan, and xyloglucan. However, the enzymes involved in the polysaccharide acetylation have not been identified. While the role of polysaccharide acetylation in vivo is unclear, it is known to reduce biofuel yield from lignocellulosic biomass by the inhibition of microorganisms used for fermentation. We have analyzed four Arabidopsis (Arabidopsis thaliana) homologs of the protein Cas1p known to be involved in polysaccharide O-acetylation in Cryptococcus neoformans. Loss-of-function mutants in one of the genes, designated REDUCED WALL ACETYLATION2 (RWA2), had decreased levels of acetylated cell wall polymers. Cell wall material isolated from mutant leaves and treated with alkali released about 20% lower amounts of acetic acid when compared with the wild type. The same level of acetate deficiency was found in several pectic polymers and in xyloglucan. Thus, the rwa2 mutations affect different polymers to the same extent. There were no obvious morphological or growth differences observed between the wild type and rwa2 mutants. However, both alleles of rwa2 displayed increased tolerance toward the necrotrophic fungal pathogen Botrytis cinerea.     

The acetylation degree of alginates in Azotobacter vinelandii ATCC9046 is determined by dissolved oxygen and specific growth rate: studies in glucose-limited chemostat cultivations

Alginates are polysaccharides that may be used as viscosifiers and gel or film-forming agents with a great diversity of applications. The alginates produced by bacteria such as Azotobacter vinelandii are acetylated. The presence of acetyl groups in this type of alginate increases its solubility, viscosity, and swelling capability. The aim of this study was to evaluate, in glucose-limited chemostat cultivations of A. vinelandii ATCC9046, the influence of dissolved oxygen tension (DO) and specific growth rate (μ) on the degree of acetylation of alginates produced by this bacterium. In glucose-limited chemostat cultivations, the degree of alginate acetylation was evaluated under two conditions of DO (1 and 9 %) and for a range of specific growth rates (0.02–0.15 h^?1). In addition, the alginate yields and PHB production were evaluated. High DO in the culture resulted in a high degree of alginate acetylation, reaching a maximum acetylation degree of 6.88 % at 9 % DO. In contrast, the increment of μ had a negative effect on the production and acetylation of the polymer. It was found that at high DO (9 %) and low μ, there was a reduction of the respiration rate, and the PHB accumulation was negligible, suggesting that the flux of acetyl-CoA (the acetyl donor) was diverted to alginate acetylation.     

Reduced Wall Acetylation Proteins Play Vital and Distinct Roles in Cell Wall O-Acetylation in Arabidopsis

The Reduced Wall Acetylation (RWA) proteins are involved in cell wall acetylation in plants. Previously, we described a single mutant, rwa2, which has about 20% lower level of O-acetylation in leaf cell walls and no obvious growth or developmental phenotype. In this study, we generated double, triple, and quadruple loss-of-function mutants of all four members of the RWA family in Arabidopsis (Arabidopsis thaliana). In contrast to rwa2, the triple and quadruple rwa mutants display severe growth phenotypes revealing the importance of wall acetylation for plant growth and development. The quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco)mannan, and xyloglucan as well as overall cell wall acetylation is affected differently in different combinations of triple mutants, suggesting their diversity in substrate preference. The overall degree of wall acetylation in the rwa quadruple mutant was reduced by 63% compared with the wild type, and histochemical analysis of the rwa quadruple mutant stem indicates defects in cell differentiation of cell types with secondary cell walls.Plant cell walls are multifunctional viscoelastic networks mainly composed of polysaccharides. Many of these polysaccharides, including xylans, (gluco)mannans, xyloglucans (XyGs), and pectins, have various degrees and patterns of acetyl esterification (Gille and Pauly, 2012; Pawar et al., 2013). The biological role of cell wall acetylation is not well understood, but it is believed to be important for pathogen resistance and plant development, and the acetylation of pectin also impacts upon the mechanical properties of cell walls (Manabe et al., 2011; Orfila et al., 2012; Pogorelko et al., 2013). In vitro, acetyl groups influence susceptibility to enzymatic degradation of pectin and xylan (Selig et al., 2009; Chen et al., 2012; Gou et al., 2012; Orfila et al., 2012; Pogorelko et al., 2013), and therefore acetylation may constitute a barrier to cell wall deconstruction. Alkali treatment of wall materials, which hydrolyzes the ester bonds, is broadly used to make polysaccharides more extractable. The treatment does not only facilitate the degradation of xylan and pectins, but also improves the deconstruction of cellulose, as the depolymerization of noncellulosic polymers results in a better accessibility to cellulose by degrading enzymes (Selig et al., 2009). Low levels of acetylated polysaccharides in plant feedstocks would be desirable for downstream processing in biorefineries, firstly, because the cell wall material of plant feedstocks with low level of acetylation is expected to be more easily extracted and, secondly, because less acetate, which is highly toxic to microorganisms such as yeast (Saccharomyces cerevisiae), would be released during extraction (Manabe et al., 2011; Gille and Pauly, 2012; Pawar et al., 2013). However, although reducing the O-acetylation level of xylan by approximately 60%, as observed in the walls of the Arabidopsis (Arabidopsis thaliana) eskimo1 mutant, enhances enzymatic degradation of isolated xylan (Yuan et al., 2013), enzymatic hydrolysis yields of whole wall materials have been reported to actually be decreased (Xiong et al., 2013). This presumably results from a tighter association between these now lowly substituted xylan polymers and cellulose (Xiong et al., 2013).Recently, we reported REDUCED WALL ACETYLATION2 (RWA2), the first protein to be involved in cell wall acetylation in planta (Manabe et al., 2011). RWA2 is a member of a small family consisting of four proteins in Arabidopsis, and its loss-of-function mutants display 20% reduction of acetylation in a range of polysaccharides that include XyG and pectins. We have hypothesized, based on phylogenetic analysis, expression pattern, moderate reduction in acetylation, and the absence of morphological phenotype, that RWA proteins have redundant functions in a biochemical reaction that occurs prior to the actual acetylation of specific polysaccharides. Independently to our research, a quadruple mutant of RWA has been reported to display reduction in xylan acetylation, secondary cell wall thickness, and mechanical strength of the stem (Lee et al., 2011). Meanwhile, Gille et al. (2011) have discovered a new family of proteins involved in the acetylation of specific polysaccharides: the plant-specific DOMAIN OF UNKNOWN FUNCTION (DUF) 231 family (also known as TRICHOME BIREFRINGENCE-LIKE [TBL] family). The loss-of-function mutants altered xyloglucan4 (axy4)/tbl27 and axy4L/tbl22 lack O-acetylation specifically of XyG in certain tissues, while eskimo1/tbl29 mutants contain reduced O-acetylation of xylan (Xiong et al., 2013; Yuan et al., 2013). The TBL/DUF231 family proteins and the RWA proteins have sequence similarity to the N-terminal and C-terminal regions of the fungal protein Cas1p, respectively (Anantharaman and Aravind, 2010). This could suggest that the TBL and RWA proteins function in protein complexes where the determinants of substrate specificity reside in the TBL partner (Manabe et al., 2011). However, because there are many more TBL proteins than RWA proteins (e.g. 46 TBL proteins versus four RWA proteins in the genome of Arabidopsis), it is likely that they do not form discrete and invariable complexes. Crossing of rwa2-3 and a leaky allele of axy4, axy4-1, resulted in a double mutant with partially additive phenotype (Gille et al., 2011). Its XyG acetylation is lower compared with either single mutant. From this analysis, RWA2 and AXY4 have been hypothesized to work in synergy, although the function of RWA2 might be substituted by other RWAs (Gille et al., 2011). Here, we have generated all the combinations of double, triple, and quadruple mutants of all four members of RWA family to further investigate the functional diversity and redundancy and to explore the function of cell wall acetylation and the role of RWAs in the network of acetylation-related enzymes. The triple and quadruple mutants we have obtained displayed severe and distinct phenotypes such as extreme dwarfism. This contrasts with the very mild phenotypes reported by Lee et al. (2011). Taken together, RWAs have partially redundant functions in the process of cell wall acetylation and show distinct impacts upon different cell wall polysaccharides.     

Antigenic polysaccharides of bacteria: 40. The structures of O-specific polysaccharides from Shigella dysenteriae types 3 and 9 and S. boydii type 4 revised by NMR spectroscopy

The reported structures of O-specific polysaccharides from three standard strains of Shigella bacteria were corrected by modern NMR techniques. The revisions concerned the configuration of the O-glycoside linkage (S. dysenteriae type 3, structure 1), the positions of monosaccharide residue glycosylation and acetylation by pyruvic acid (S. dysenteriae type 9, structure 2), and the attachment position of the side monosaccharide chain (S. boydii type 4, structure 3) [struxture in text].     

Degree of acetylation of heteropolysaccharides

The acetyl groups in polysaccharides and glycoproteins have been determined using 4 N HCl at 120 degrees C for acid hydrolysis. Acetic acid and hexosamine were determined by high-performance cation-exchange chromatography with UV detection and high-performance anion-exchange chromatography with pulsed amperomeric detection, respectively. The method compares well with other procedures and shows an additional advantage of being able to analyze for hexosamine in the same hydrolyzate, thus permitting the degree of acetylation of hexosamine-containing biopolymers to be determined directly without correction for additional components in the material of interest.     

Ginseng root water-extracted pectic polysaccharides originate from secretory cavities

A range of molecular probes for cell wall polysaccharides has been used to explore the structure and location of water-extracted pectic polysaccharides occurring in fractions isolated from ginseng roots. The LM19 homogalacturonan (HG) epitope was abundant in an HG fraction and analysis of LM19 binding to a rhamnogalacturonan-I (RG-I) rich-fraction indicated that the LM19 epitope is sensitive to acetylation. A specific RG-I epitope (LM16), four arabinogalactan-protein (AGP) epitopes (LM2, LM14, JIM16, MAC207) and an extensin epitope (JIM20) were found to be abundant and co-located in several isolated polysaccharide fractions including an arabinogalactan fraction and two RG-I fractions. Detection of the RG-I, AGP and extensin epitopes identified in isolated polysaccharide fractions in sections of ginseng roots indicated that they were most abundant in secretory cavities found in the cortical regions of ginseng roots. In addition, the immunocytochemical study indicated that polysaccharide epitope masking is a widespread phenomenon in the primary cell walls of ginseng roots.     

Expression of mung bean pectin acetyl esterase in potato tubers: effect on acetylation of cell wall polymers and tuber mechanical properties

A mung bean (Vigna radiata) pectin acetyl esterase (CAA67728) was heterologously expressed in tubers of potato (Solanum tuberosum) under the control of the granule-bound starch synthase promoter or the patatin promoter in order to probe the significance of O-acetylation on cell wall and tissue properties. The recombinant tubers showed no apparent macroscopic phenotype. The enzyme was recovered from transgenic tubers using a high ionic strength buffer and the extract was active against a range of pectic substrates. Partial in vivo de-acetylation of cell wall polysaccharides occurred in the transformants, as shown by a 39% decrease in the degree of acetylation (DA) of tuber cell wall material (CWM). Treatment of CWM using a combination of endo-polygalacturonase and pectin methyl esterase extracted more pectin polymers from the transformed tissue compared to wild type. The largest effect of the pectin acetyl esterase (68% decrease in DA) was seen in the residue from this extraction, suggesting that the enzyme is preferentially active on acetylated pectin that is tightly bound to the cell wall. The effects of acetylation on tuber mechanical properties were investigated by tests of failure under compression and by determination of viscoelastic relaxation spectra. These tests suggested that de-acetylation resulted in a stiffer tuber tissue and a stronger cell wall matrix, as a result of changes to a rapidly relaxing viscoelastic component. These results are discussed in relation to the role of pectin acetylation in primary cell walls and its implications for industrial uses of potato fibres.     

Bacterial protein acetylation: the dawning of a new age

Protein acetylation has historically been considered a predominantly eukaryotic phenomenon. Recent evidence, however, supports the hypothesis that acetylation broadly impacts bacterial physiology. To explore more rapidly the impact of protein acetylation in bacteria, microbiologists can benefit from the strong foundation established by investigators of protein acetylation in eukaryotes. To help advance this learning process, we will summarize the current understanding of protein acetylation in eukaryotes, discuss the emerging link between acetylation and metabolism and highlight the best‐studied examples of protein acetylation in bacteria.