Effect of acetylation on antioxidant and cytoprotective activity of polysaccharides isolated from pumpkin (Cucurbita pepo, lady godiva)

Acetylation of pumpkin (Cucurbita pepo, lady godiva variety) polysaccharide using acetic anhydride with pyridines as catalyst under different conditions was conducted to obtain different degrees of acetylation on a laboratory scale. Furthermore, antioxidant activities and cytoprotective effects of pumpkin polysaccharide and its acetylated derivatives were investigated employing various established in vitro systems. Results showed that addition of pyridine as catalyst could increase the degree of substitution, whereas volume of acetic anhydride had little effect. The acetylated polysaccharides in DPPH scavenging radical activity assay, superoxide anion radical activity assay and reducing power assay exhibited higher antioxidant activity than that of unmodified polysaccharide. H₂O₂-induced oxidative damages on rat thymic lymphocyte were also prevented by pumpkin polysaccharide and its acetylated derivatives and the derivatives presented higher protective effects. On the whole, acetylated polysaccharide showed relevant antioxidant activity both in vitro and in a cell system.     

Effect of acetylation on the material properties of glucuronoxylan from aspen wood

The effect of the degree of acetylation of glucuronoxylan on solubility, water content and thermal properties was investigated. Aspen glucuronoxylan, isolated by alkali extraction from wood chips, was acetylated to various degrees of substitution through reaction with acetic anhydride in formamide and pyridine by varying the reaction time. The degree of acetylation was determined by ¹H NMR spectroscopy. The molecular weight was decreased only to a small extent during the reaction, as seen by size exclusion chromatography. It was found that acetylation strongly affects the solubility properties as well as the equilibrium water content of the glucuronoxylans upon exposure to humidity. Samples with a high degree of acetylation are soluble only in aprotic solvents, whereas non-acetylated glucuronoxylan is partially soluble in hot water. In the same surrounding relative humidity, acetylated samples have lower water content than non-acetylated samples. Acetylation prevents thermal degradation, as shown by thermogravimetric analysis under nitrogen. Acetylation to a degree of substitution of 1.2 also results in a glass transition temperature, which we studied using differential scanning calorimetry, making it possible to thermoprocess acetylated glucuronoxylan.     

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.     

Acetyl substitution patterns of amylose and amylopectin populations in cowpea starch modified with acetic anhydride and vinyl acetate

To study the effect of reagent type on the distribution pattern of acetyl groups in acetylated cowpea starch, amylose and amylopectin populations were isolated from the starch granules after modification to a low degree of substitution (DS < 0.1) with acetic anhydride and vinyl acetate, respectively. Slowly reacting reagent vinyl acetate resulted in higher DS values for the amylopectin populations when compared to the rapidly reacting reagent acetic anhydride. The two reagents had similar effects on the acetylation level of amylose, suggesting that the amorphous regions of granules were easily accessible for both reagents. The acetyl substitution patterns were analyzed by enzymatic degradation followed by characterization of the obtained fragments using chromatographic and mass spectrometric techniques. The distributions of acetyl groups along the amylose and amylopectin chains were more clustered for modification with vinyl acetate as compared with modification with acetic anhydride. Between the two acetylation types, pronounced differences in the acetyl substitution patterns were observed for the large fragments obtained after -amylase digestion; only slight differences were exhibited for the small fragments obtained by exhaustive enzymatic digestion of amylose and amylopectin populations.     

Acetylation of wheat straw hemicelluloses in ionic liquid using iodine as a catalyst

Wheat straw hemicelluloses were acetylated with acetic anhydride using iodine as a novel catalyst in 1-butyl-3-methylimidzolium chloride ([C₄mim]Cl) ionic liquid (IL). Acetylated hemicelluloses with yield and degree of substitution (DS) from 70.5% to 90.8% and between 0.49 and 1.53, respectively, are accessible in a complete homogeneous procedure by changing the reaction temperature, reaction duration, the dosage of catalyst, and the dosage of acetic anhydride. The preferred reaction parameters that resulted in the highest DS were follows: 20:1 reactant molar ratio, 100 °C, 30 min, 15% iodine, in which about 83% hydroxyl groups in native hemicelluloses were esterified. The structural features of the acetylated hemicelluloses were characterized by ¹³C NMR and FT-IR spectroscopy. The thermal stability of the acetylated hemicelluloses increased upon chemical modification. It is the first time that we have demonstrated that ILs could be used as an environmentally friendly solvent for the chemical modification of hemicelluloses.     

Ultra high pressure (UHP)-assisted acetylation of corn starch

Potential roles of ultra high pressure (UHP) in starch granule reactivity and properties of acetylated starch were investigated. Corn starch was substituted with acetic anhydride at pressure range of 0.1–400 MPa for 15 min; also, conventional reaction (30 °C, 60 min) was conducted as reaction control. Native and acetylated corn starches were assessed with respect to degree of substitution (DS), X-ray diffraction pattern/relative crystallinity, starch solubility/swelling power, gelatinization, and pasting behavior. For the UHP-assisted acetylated starches, DS values increased along with increasing pressure levels from 200 to 400 MPa, and reaction at 400 MPa exhibited maximum reactivity (though lower than the DS value of the reaction control). Both UHP-assisted and conventional acetylation of starch likely occurred predominantly at amorphous regions within granules. Gelatinization and pasting properties of the UHP-assisted acetylated starches may be less influenced by UHP treatment in acetylation reaction, though restricted starch solubility/swelling were observed.     

Acetylation of wheat straw hemicellulose B in a new non-aqueous swelling system

The acetylation of wheat straw hemicellulose B was carried out in a homogeneous N,N-dimethylformamide and lithium chloride system with acetic anhydride using 4-dimethylaminopyridine as a catalyst. The degree of substitution of hemicellulose B acetates ranged between 0.59 and 1.25 as a function of experimental conditions. Under an optimum condition (85°C, 60 h), approximately 75% of the free hydroxyl groups in native hemicellulose B were acetylated. The molecular weight measurements (31,890–34,090 g mol⁻¹) showed a controllable degradation of hemicellulose B chains during the reactions at temperature 60–85°C and duration of 2–60 h. It was found that the thermal stability of the products was increased by chemical modification.     

Acetylation of sugarcane bagasse using NBS as a catalyst under mild reaction conditions for the production of oil sorption-active materials

Sugarcane bagasse was esterified with acetic anhydride using N-bromosuccinimide as a catalyst under mild conditions in a solvent free system. The extent of acetylation was measured by weight percent gain, which varied from 2.1% to 24.7% by changing the reaction temperature (25-130 degrees C) and duration (0.5-6.0 h). N-Bromosuccinimide was found to be a novel and highly effective catalyst for acetylation of hydroxyl groups in bagasse. At a concentration of 1% of the catalyst in acetic anhydride, a weight percent gain of 24.7% was achieved at 120 degrees C for 1 h, compared with 5.1% for the un-catalyst reaction under the same reaction condition. FT-IR and CP-MAS 13C-NMR studies produced evidence for acetylation. The thermal stability of the products decreased slightly upon chemical modification, but no significant decrease in thermal stability was observed for WPG > or = 24.7%. More importantly, the acetylation significantly increased hydrophobic properties of the bagasse. The oil sorption capacity of the acetylated bagasse obtained at 80 degrees C for 6 h, was 1.9 times higher than the commercial synthetic oil sorbents such as polypropylene fibres. Therefore, these oil sorption-active materials can be used to substitute non-biodegradable materials in oil spill cleanup.     

Effects of acetylation in vapor phase and mercerization on the properties of sugarcane fibers

Chemical modification of sugarcane bagasse fiber was achieved by mercerization reaction and esterification reaction with anhydride acetic vapor. This is a new acetylation procedure. The results show that the fiber length and diameter are reduced after the reactions. Fourier transform infrared spectroscopy (FT-IR) studies produced clear evidence of the partial acetylation reaction. Optical microscopy revealed fibrillation in the acetylated fiber attributed to hemicellulose dissolution. The thermal stability measured by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) increased after acetylation and decreased after mercerization. The higher thermal stability of the acetylated fiber as compared with modified fibers in liquid medium was attributed to the small quantity of water and acetic acid present for the reaction in vapor phase. The lesser tensile strength of the acetylated fiber was due to fibrillation. The porous structure obtained favors migration of the polymer chains into the fiber acetylated, and thus it should enhance the polymer–fiber adhesion in polymer composites.     

Structure-activity relationship study of WSS25 derivatives with anti-angiogenesis effects

WGEW, an α(1-4) linked glucan with an α(1-4) linked branch attached to C-6, was isolated from the rhizoma of Gastrodia elata Bl. WSS25, a sulfated derivative of WGEW, was reported to inhibit angiogenesis by disrupting BMP2/Smad/Id1 signaling pathway. However, the structure-activity relationship (SAR) for WSS25 is not known. To study the SAR, seven sulfated saccharides derived from WGEW degradation products, six sulfated polysaccharides with varying degrees of substitution, and four aminopropylated, carboxymethylated, phosphorylated, and acetylated derivatives of WGEW were prepared. A sulfated, unbranched product of polysaccharide was also obtained. The structural features of these derivatives were characterized by infrared spectroscopy and nuclear magnetic resonance spectroscopy. An HMEC-1 cell tube formation assay was employed to measure the antiangiogenic effect of the derivatives. The results indicated that only sulfated polysaccharides with molecular weights of more than 41,000?Da could inhibit HMEC-1 cell tube formation. The inhibition effect was dependent on the presence of a sulfate group, since the tube formation was not blocked by aminopropylated, carboxymethylated, phosphorylated, or acetylated WGEW. A higher degree of sulfate substitution on the polysaccharide led to a stronger inhibitory effect, and the degree of sulfate substitution between 0.173 and 0.194 was found to be optimal. Interestingly, the WGEW side chain was not required for anti-tube formation activity. All these preliminary results may provide a clue for further modification of the core structure of WSS25 to discover polysaccharide derivatives as novel anti-angiogenic inhibitors.     

Modification of histone binding in calf thymus chromatin and in the chromatin-protamine complex by acetic anhydride.

A relationship between side-chain modification of histones and their displaceability from DNA has been investigated using calf thymus chromatin which was chemically acetylated with acetic anhydride. When the chromatin is treated with increasingly higher concentrations of the reagent, histones become acetylated to an increasingly greater extent, attaining the modification at 23-24 sites for histone I, 5-6 for IIb1, 9-10 for IIb2, 5-6 for III and 3-4 for IV. As the chromatin becomes more acetylated, NaCl concentrations required for histone removal are lowered. Saturation binding of protamine does not bring about either an increase in the number of acetylation sites of histones in chromatin or a decrease of the NaCl requirement for dissociation of the acetylated chromatins. A comparison of the present results with the extents of histone acetylation known to occur enzymatically in vivo indicates that the complete removal of somatic histones during transformation of chromatin in spermiogenesis cannot be explained on the basis of decreased binding of the histone to DNA by acetylation or by a combination of acetylation and protamine binding, suggesting that the displacement process may require some additional processes.     

Acetylation of αA-crystallin in the human lens: effects on structure and chaperone function

α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N(ε)-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.     

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.     

Acetylation of αA-crystallin in the human lens: Effects on structure and chaperone function

α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N^ε-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.     

植物组织中低聚糖乙酰化及毛细管气相色谱分析

建立一种用乙酰化衍生处理低聚糖并用毛细管气相色谱-FID进行分析的方法。以1-甲基咪唑为催化剂并以乙酸酐为乙酰化试剂,同时对植物样品中蔗糖、棉子糖和水苏糖等低聚糖乙酰化产物进行毛细管气相色谱分离和FID检测。确定了低聚糖乙酰化衍生物的毛细管气相色谱分析条件,并对低聚糖乙酰化反应条件及色谱分离条件进行了优化。结果表明,在80–1000ng·μL–1范围内线性关系良好,蔗糖、棉子糖和水苏糖的相关系数（R）分别为0.9952、0.9957和0.9877,并且精准度与回收率均较高。使用该方法对低聚糖进行乙酰化反应重现性好、所需样品材料及试剂量少且污染毒害小,能够得到理想的分离、检测和定量分析效果,适用于少量植物组织中低聚糖的定量分析。该方法在食品、医药检测和基础科学研究领域均具有广泛的适用性及参考价值。     

N-乙酰化反应制备水溶性壳聚糖研究

将高度脱乙酰化的壳聚糖在均相介质中进行N-乙酰化反应,制备水溶性壳聚糖。研究了制备工艺条件对脱乙酰度及水溶性的影响。结果表明,在乙酸—乙醇均相体系中进行乙酰化反应时,壳聚糖与乙酸酐的质量比为1∶0.6,反应温度控制在20℃,反应时间为8 h时,产品的脱乙酰度在50%左右,获得了水溶性良好的N-乙酰化壳聚糖。     

Mass spectrometric identification of lysine residues of heme oxygenase-1 that are involved in its interaction with NADPH-cytochrome P450 reductase

The lysine residues of rat heme oxygenase-1 (HO-1) were acetylated by acetic anhydride in the absence and presence of NADPH-cytochrome P450 reductase (CPR) or biliverdin reductase (BVR). Nine acetylated peptides were identified by MALDI-TOF mass spectrometry in the tryptic fragments obtained from HO-1 acetylated without the reductases (referred to as the fully acetylated HO-1). The presence of CPR prevented HO-1 from acetylation of lysine residues, Lys-149 and Lys-153, located in the F-helix. The heme degradation activity of the fully acetylated HO-1 in the NADPH/CPR-supported system was significantly reduced, whereas almost no inactivation was detected in HO-1 in the presence of CPR, which prevented acetylation of Lys-149 and Lys-153. On the other hand, the presence of BVR showed no protective effect on the acetylation of HO-1. The interaction of HO-1 with CPR or BVR is discussed based on the acetylation pattern and on molecular modeling.     

Microbial carbohydrate esterases deacetylating plant polysaccharides

Several plant polysaccharides are partially esterified with acetic acid. One of the roles of this modification is protection of plant cell walls against invading microorganisms. Acetylation of glycosyl residues of polysaccharides prevents hydrolysis of their glycosidic linkages by the corresponding glycoside hydrolases. In this way the acetylation also represents an obstacle of enzymatic saccharification of plant hemicelluloses to fermentable sugars which appears to be a hot topic of current research. We can eliminate this obstacle by alkaline extraction or pretreatment leading to saponification of ester linkages. However, this task has been accomplished in a different way in the nature. The acetyl groups became targets of microbial carbohydrate esterases that evolved to overcome the complexity of the plant cell walls and that cooperate with glycoside hydrolases in plant polysaccharide degradation. This article concentrates on enzymes deacetylating plant hemicelluloses excluding pectin. They are currently grouped in at least 8 families, specifically in CE families 1–7 and 16, originally assigned as acetylxylan esterases, the enzymes acting on hardwood acetyl glucuronoxylan and its fragments generated by endo-β-1,4-xylanases. There are esterases deacetylating softwood galactoglucomannan, but they have not been classified yet. The enzymes present in CE families 1–7 differ in structure and substrate and positional specificity. There are families behaving as endo-type and exo-type deacetylates, i.e. esterases deacetylating internal sugar residues of partially acetylated polysaccharides and also esterases deacetylating non-reducing end sugar residues in oligosaccharides. With one exception, the enzymes of all mentioned CE families belong to serine type esterases. CE family 4 harbors enzymes that are metal-dependent aspartic esterases. Three-dimensional structures have been solved for members of the first seven CE families, however, there is still insufficient knowledge about their substrate specificity and real physiological role. Current knowledge on catalytic properties of the selected families of CEs is summarized in this review. Some of the families are emerging also as new biocatalysts for regioselective acylation and deacylation of carbohydrates.     

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.