Structural investigation of hemicelluloses from Plantago ovata,Mimosa pudica and Lallemantia royleana by MALDI-ToF mass spectrometry

Abstract

This work concerns structural investigation of water-extractable hemicelluloses from seed husk of Plantago ovata and seeds of Mimosa pudica and Lallemantia royleana. The composition of the materials was determined by CHN elemental analysis, FT-IR spectrometry and monosaccharide analysis. The detailed structural analysis was carried out by MALDI-ToF mass spectrometry. The absence of nitrogen in the materials suggested that they were free from proteins. The isolated materials were found to be branched hemicelluloses. The mass spectrometric study showed presence of β-1,4-linked xylose with arabinose attached to main chain at β-1,3 positions (Plantago ovata), β-1,4-linked xylose with glucose attached to main chain at β-1,3 positions (Mimosa pudica), and β-1,2-linked rhamnose and β-1,3-linked arabinose units in the main chain with arabinose attached to the main chain through β-1,3-linkage (Lallemantia royleana).     

Novel polyurethanes from xylan and TDI: Preparation and characterization

Xylan is a hemicellulose, which is found abundantly in nature. In this work, a novel polyurethane was developed involving xylan and tolylene-2,4-diisocyanate (TDI). Polymer synthesis was achieved through conventional heat or microwave-assisted reaction in dimethyl sulfoxide. Because xylan has multiple OH groups on each polymer chain, the TDI/xylan molar ratio had to be adjusted to produce a soluble polymeric product. The reaction products were characterized by ¹³C NMR, FTIR, thermogravimetric analysis, and differential scanning calorimetry. The xylan polyurethane was shown to exhibit improved thermal stability over xylan.     

Xylan hydrolysis in zinc chloride solution

Xylan is the major component of hemicellulose, which consists of up to one-third of the lignocellulosic biomass. When the zinc chloride solution was used as a pretreatment agent to facilitate cellulose hydrolysis, hemicellulose was hydrolyzed during the pretreatment stage. In this study, xylan was used as a model to study the hydrolysis of hemicellulose in zinc chloride solution. The degradation of xylose that is released from xylan was reduced by the formation of zinc-xylose complex. The xylose yield was >90% (w/w) at 70°C. The yield and rate of hydrolysis were a function of temperature and the concentration of zinc chloride. The ratio of zinc chloride can be decreased from 9 to 1.3 (w/w). At this ratio, 76% of xylose yield was obtained. When wheat straw was pretreated with a concentrated zinc chloride solution, the hemicellulose hydrolysate contained only xylose and trace amounts of arabinose and oligosaccharides. With this approach, the hemicellulose hydrolysate can be separated from cellulose residue, which would be hydrolyzed subsequently to glucose by acid or enzymes to produce glucose. This production scheme provided a method to produce glucose and xylose in different streams, which can be fermented in separated fermenters.     

Extraction and characterization of native heteroxylans from delignified corn stover and aspen

Dimethylsulfoxide-solubilized polysaccharides from delignified corn stover and aspen were characterized. The biomass was delignified by two different techniques; a standard acid chlorite and a pulp and paper QPD technique comprising chelation (Q), peroxide (P), and acid-chlorite (D). Major polysaccharides in all fractions were diversely substituted xylan. Xylan acetylation was intact after chlorite delignification and, as expected, xylan from QPD-delignified fraction was de-acetylated by the alkaline peroxide step. The study of DMSO-extractable xylans from chlorite-delignified biomass revealed major differences in native acetylation patterns between corn stover and aspen xylan. Xylan from cell walls of corn stover contains 2-O- and 3-O-mono-acetylated xylan and [MeGlcA-α-(1 → 2)][3-OAc]-xylp units. In addition, aspen xylan also contains 2,3-di-O-acetylated xylose. 1,4-β-d-xylp residues substituted with MeGlcA at O-2 position are absent in chlorite-delignified aspen xylan. Sugar composition in accord with NMR-spectroscopic data indicated that corn stover xylan is arabinosylated while aspen xylan is not. We have shown that corn stover xylan has similar structure with xylans from other plants of Poales order. No evidence was found to indicate the presence of 1,4-β-d-[MeGlcA-α-(1 → 2)][Ara-α-(1 → 3)]-xylp in corn stover xylan fractions.     

Dilute sulfuric acid pretreatment of agricultural and agro-industrial residues for ethanol production

The potential of dilute-acid prehydrolysis as a pretreatment method for sugarcane bagasse, rice hulls, peanut shells, and cassava stalks was investigated. The prehydrolysis was performed at 122 degrees C during 20, 40, or 60 min using 2% H(2)SO(4) at a solid-to-liquid ratio of 1:10. Sugar formation increased with increasing reaction time. Xylose, glucose, arabinose, and galactose were detected in all of the prehydrolysates, whereas mannose was found only in the prehydrolysates of peanut shells and cassava stalks. The hemicelluloses of bagasse were hydrolyzed to a high-extent yielding concentrations of xylose and arabinose of 19.1 and 2.2 g/L, respectively, and a xylan conversion of more than 80%. High-glucose concentrations (26-33.5 g/L) were found in the prehydrolysates of rice hulls, probably because of hydrolysis of starch of grain remains in the hulls. Peanut shells and cassava stalks rendered low amounts of sugars on prehydrolysis, indicating that the conditions were not severe enough to hydrolyze the hemicelluloses in these materials quantitatively. All prehydrolysates were readily fermentable by Saccharomyces cerevisiae. The dilute-acid prehydrolysis resulted in a 2.7- to 3.7-fold increase of the enzymatic convertibility of bagasse, but was not efficient for improving the enzymatic hydrolysis of peanut shells, cassava stalks, or rice hulls.     

Fractionation of Corn Fiber Treated by Soaking in Aqueous Ammonia (SAA) for Isolation of Hemicellulose B and Production of C5 Sugars by Enzyme Hydrolysis

A process was developed to fractionate and isolate the hemicellulose B component of corn fiber generated by corn wet milling. The process consisted of pretreatment by soaking in aqueous ammonia followed by enzymatic cellulose hydrolysis, during which the hemicellulose B was solubilized by cleavage into xylo-oligosaccharides and subsequently recovered by precipitation with ethanol. The pretreatment step resulted in high retention of major sugars and improvement of subsequent enzymatic hydrolysis. The recovered hemicellulose B was hydrolyzed by a cocktail of enzymes that consisted of β-glucosidase, pectinase, xylanase, and ferulic acid esterase (FAE). Xylanase alone was ineffective, demonstrating yields of less than 2% of xylose and arabinose. The greatest xylose and arabinose yields, 44% and 53%, respectively, were obtained by the combination of pectinase and FAE. A mass balance accounted for 87% of the initially present glucan, 91% of the xylan, and 90% of the arabinan. The developed process offered a means for production of corn fiber gum as a value-added co-product and C5 sugars, which could be converted to other valuable co-products through fermentation in a corn wet-milling biorefinery.     

High-performance liquid chromatography/electrospray ionization tandem mass spectrometry for characterization of enzymatic degradation of 2,2'-bis(2-oxazoline)-linked poly-epsilon-caprolactone

This paper describes a straightforward and rapid on-line characterization using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS(n)) of the enzymatic degradation products of 2,2'-bis(2-oxazoline)-linked poly-epsilon-caprolactone (PCL-O). These new PCL-O polymers are expected to be used in a variety of pharmaceutical and biomedical applications since they are degraded enzymatically by surface erosion. PCL-O was polymerized in a three-step reaction and characterized by (1)H-NMR and size-exclusion chromatography (SEC). Solvent cast polymer films were exposed to enzymatic degradation in phosphate buffer (pH 7.5, 1% pancreatin). The enzymatic degradation of the polymer produced a wide variety of water-soluble oligomers which were separated and identified by HPLC/ESI-MS(n). Optimization of the gradient HPLC method resulted in effective separation of the oligomers. Furthermore, specific structures of the oligomers were clearly identified by tandem mass spectrometry. According to these results, ester bonds seem to be most sensitive to enzymatic degradation and, correspondingly, pancreatic lipase seems to be mainly responsible for the enzymatic erosion of the PCL-O films. This novel mass spectrometric method provides important knowledge about the enzymatic degradation process and structure of the polymer which is difficult to ascertain by other conventional methods.     

桑叶多糖SJB的结构分析和蛋白酪氨酸磷酸酯酶PTP1B抑制活性

通过DEAE-纤维素和凝胶过滤柱色谱对桑叶碱提粗多糖进行分级分离, 获得均一多糖SJB, 进行结构鉴定. 采用蛋白酪氨酸磷酸酯酶PTP1B体外模型对SJB进行降血糖活性测定. 结果表明: SJB的相对分子质量为5.4×104, 由鼠李糖、阿拉伯糖、葡萄糖、半乳糖、半乳糖醛酸组成的酸性杂多糖; 主链由1,2-、1,2,4-连接的鼠李糖和1,4-、1,3,4-连接的半乳糖醛酸组成; 侧链包括末端、1,5-、1,3,5-连接的阿拉伯糖; 末端、1,4-连接的葡萄糖以及末端、1,3-、1,4-、1,6-连接的半乳糖, 主要通过鼠李糖的O4位和半乳糖醛酸的O3位与主链相连. 该多糖为首次从桑叶中获得的酸性杂多糖. 20 μg/mL SJB对PTP1B的抑制率为31.7%.     

The effect of barley husk arabinoxylan adsorption on the properties of cellulose fibres

This study investigates the adsorption of (glucurono)arabinoxylan (GAX) on cellulose fibres and the properties thereof. A water-soluble GAX, from barley husks (Hordeum vulgare), was isolated using chlorite delignification and alkaline extraction followed by enzymatic purification. The isolated GAX fraction showed an arabinose to xylose ratio of 0.22 and a weight average molar mass of 20,200 g/mol, as determined by size exclusion chromatography (SEC) in DMSO:H₂O. The GAX was adsorbed on cellulose fibres under well controlled conditions, where temperature and initial concentration of GAX proved to be important parameters in controlling the level of adsorption. The adsorption process was also dependent on xylan molecular structure. Carbohydrate analysis on the modified fibres showed a preferential adsorption of low substituted xylans (arabinose to xylose ratio of ∼0.10). During the adsorption process the GAX solution was analyzed using SEC-RI-MALLS in aqueous solvent, which demonstrated a molecular xylan adsorption on cellulose fibres. Additionally, a decrease in light scattering responses, which corresponds to an adsorption of aggregated xylan and/or xylan with a great tendency towards self-association, could be observed during the adsorption process. This was demonstrated by adsorption of GAX on regenerated cellulose fibres (Lyocell), which compared to native fibres possesses a relatively smooth fibre surface. Atomic force microscopy analysis visualised a heterogeneous decoration of the Lyocell fibres with xylan agglomerates. The effect of GAX adsorption on paper strength was also investigated. A GAX modified kraft pulp showed an evident increase in tensile strength, which might be due to a retained fibre–fibre bonding ability for xylan coated fibrils after drying and rewetting.     

一种刺松藻来源的硫酸阿拉伯聚糖的制备及特征结构表征

刺松藻(Codium fragile)经水提-醇沉获得粗多糖, 进一步将刺松藻粗多糖(CFP) 通过Q-Sepharose Fast Flow(QFF) 阴离子交换柱纯化得到6个多糖组分CFP1CFP6, 其中, 在CFP6中发现纯度较高的阿拉伯聚糖. 采用高效凝胶渗透色谱与十八角激光散射仪联用法和1-苯基-3-甲基-5-吡唑啉酮(PMP)柱前衍生高效液相色谱法对CFP6的分子量及单糖组成进行了分析. 结果表明, CFP6是一种分子量为79290的多糖, 由阿拉伯糖(Ara)和半乳糖(Gal)组成, 二者摩尔比为14.8:1.0. 通过多维核磁共振波谱、 液相色谱-质谱联用及二级质谱等方法对CFP6的糖苷键连接方式及其寡糖序列结构进行表征, 进一步阐明了该复杂多糖的特征结构. 经判断, CFP6主链由Ara组成, 通过 β-(1→3)糖苷键连接, 在Ara的C2位存在分支结构, 硫酸基位于Ara的C4或C2位.     

不同酸解聚麦秸产物分析及其动力学

以麦秸为研究对象,解聚剂为HCl、HNO₃和H₃PO₄,对解聚产物进行定性和定量分析,并利用动力学模型描述木糖及糠醛的产生过程。 结果表明,解聚液中的产物有葡萄糖、木糖、阿拉伯糖、纤维二糖、乙酸、糠醛、5-羟甲基糠醛。 通过引入变量(α,木糖/木聚糖的比值)利用Saeman动力学模型获得了不同温度下,木聚糖的水解速率常数、木糖的转化速率常数以及糠醛的生成速率常数。 HCl、HNO₃和H₃PO₄解聚麦秸,木糖的生成活化能分别为55.5、46.3和59.8 kJ/mol。 结合反应温度、反应时间、反应速率以及木糖和糠醛的浓度,确定最佳解聚条件为:硝酸作解聚剂,在130 ℃下水解95 min。     

Incorporation of carboxyl groups into xylan for improved absorbency

The objective of this research was to investigate green, renewable reaction systems for xylan that introduce crosslinking and carbonyl group for improved performance in water absorption applications. Xylan was modified separately with three different reaction agents, citric acid, succinic anhydride and sodium monochloracetate (SMCA). The xylan was reacted with citric acid in the presence or absence of sodium hypophosphite (SHP) as a catalyst, both in a solution form and in a semi-dry form in an oven. Acid–base titrations, FTIR, TGA, and DSC were used to confirm the composition of the reactant products. Reacted xylan had significantly increased carboxyl content, degree of esterification and degree of substitution with citric acid, succinic anhydride, and sodium monochloracetate. Effective reaction conditions were determined for high yield products. For the xylan reacted with citric acid, the catalyst (SHP) and the use of a semi-dry reaction condition increased the yield significantly. Succinic anhydride and SMCA had high yields. The reaction trends observed were consistent with similar results for starch. Increases in water absorption, saline solution absorption, and decreases in water contact angle for the xylan citrate relative to the xylan indicate that high carboxyl content materials can be generated with xylan and that the resulting materials have enhanced water affinity.     

Potential of Agricultural Residues and Hay for Bioethanol Production

Production of bioethanol from agricultural residues and hays (wheat, barley, and triticale straws, and barley, triticale, pearl millet, and sweet sorghum hays) through a series of chemical pretreatment, enzymatic hydrolysis, and fermentation processes was investigated in this study. Composition analysis suggested that the agricultural straws and hays studied contained approximately 28.62-38.58% glucan, 11.19-20.78% xylan, and 22.01-27.57% lignin, making them good candidates for bioethanol production. Chemical pretreatment with sulfuric acid or sodium hydroxide at concentrations of 0.5, 1.0, and 2.0% indicated that concentration and treatment agent play a significant role during pretreatment. After 2.0% sulfuric acid pretreatment at 121 degrees C/15 psi for 60 min, 78.10-81.27% of the xylan in untreated feedstocks was solubilized, while 75.09-84.52% of the lignin was reduced after 2.0% sodium hydroxide pretreatment under similar conditions. Enzymatic hydrolysis of chemically pretreated (2.0% NaOH or H2SO4) solids with Celluclast 1.5 L-Novozym 188 (cellobiase) enzyme combination resulted in equal or higher glucan and xylan conversion than with Spezyme(R) CP- xylanase combination. The glucan and xylan conversions during hydrolysis with Celluclast 1.5 L-cellobiase at 40 FPU/g glucan were 78.09 to 100.36% and 74.03 to 84.89%, respectively. Increasing the enzyme loading from 40 to 60 FPU/g glucan did not significantly increase sugar yield. The ethanol yield after fermentation of the hydrolyzate from different feedstocks with Saccharomyces cerevisiae ranged from 0.27 to 0.34 g/g glucose or 52.00-65.82% of the theoretical maximum ethanol yield.     

The Effect of Lignin Removal by Alkaline Peroxide Pretreatment on the Susceptibility of Corn Stover to Purified Cellulolytic and Xylanolytic Enzymes

Pretreatment of corn stover with alkaline peroxide (AP) at pH 11.5 resulted in reduction of lignin content in the residual solids as a function of increasing batch temperature. Scanning electron microscopy of these materials revealed notably more textured surfaces on the plant cell walls as a result of the delignifying pretreatment. As expected, digestion of the delignified samples with commercial cellulase preparations showed an inverse relationship between the content of lignin present in the residual solids after pretreatment and the extent of both glucan and xylan conversion achievable. Digestions with purified enzymes revealed that decreased lignin content in the pretreated solids did not significantly impact the extent of glucan conversion achievable by cellulases alone. Not until purified xylanolytic activities were included with the cellulases were significant improvements in glucan conversion realized. In addition, an inverse relationship was observed between lignin content after pretreatment and the extent of xylan conversion achievable in a 24-h period with the xylanolytic enzymes in the absence of the cellulases. This observation, coupled with the direct relationship between enzymatic xylan and glucan conversion observed in a number of cases, suggests that the presence of lignins may not directly occlude cellulose present in lignocelluloses but rather impact cellulase action indirectly by its association with xylan.     

大豆戊聚糖的化学结构及对面团流变学特性的影响

用1molk/L NaOH提取并经层析柱的分级与提纯, 从大豆细胞壁物质中提取出的戊聚糖纯组分(PRF5-1, PRF5-2)属于多糖-多肽复合物。由β(1→4)-糖苷键连的木聚糖构成多糖部分的主链结构, 主链上部分Xyl残基分别通过3-C与2-C分支点连有Ara和GlcA作为侧链。大戊聚糖对面团流变学特性(粉质特性与拉伸特性)有明显的改良作用, 是一种潜在的面粉品质改良剂。     

Xylanase supplementation on enzymatic saccharification of dilute acid pretreated poplars at different severities

Three pairs of solid substrates from dilute acid pretreatment of two poplar wood samples were enzymatically hydrolyzed by cellulase preparations supplemented with xylanase. Supplementation of xylanase improved cellulose saccharification perhaps due to improved cellulose accessibility by xylan hydrolysis. Total xylan removal directly affected enzymatic cellulose saccharification. Furthermore, xylan removal by pretreatment and xylanase are indifferent to enzymatic cellulose saccharification. However, more enzymatic xylose and glucose yields were obtained for a substrate with lower xylan content after a severer pretreatment at the same xylanase dosage. The effectiveness of xylanase at increased dosages depended on the substrates structure or accessibility. High xylanase dosages were more effective on well pretreated substrates than on under-pretreated substrates with high xylan content. The application sequence of xylanase and cellulase affected cellulose saccharification. This effect varied with substrate accessibility, perhaps due to competition between xylanase and cellulase binding to the substrate.     

Cationic Xylan Derivatives with High Degree of Functionalization

Xylan from birch wood was characterized regarding both the supramolecular structure (X-ray, CP/MAS ¹³C-NMR) and the sugar composition. The reaction of the birch wood xylan with 2,3-epoxypropyltrimethylammonium chloride in 1,2-dimethoxyethane as slurry medium yields water-soluble, cationic 2-hydroxypropyltrimethylammonium xylan derivatives with high degree of substitution (DS). The DS values up to 1.6 can be controlled by adjusting the molar ratio in a one step synthesis. The structure of the cationic xylan derivatives was confirmed by means of DEPT(135) NMR spectroscopy. Film forming properties of cationic xylan derivatives were investigated with SEM measurements.     

A concise synthesis of arabinogalactan with β-(1→6) galactopyranose backbone and α-(1→2) arabinofuranose side chains

Penta- and octasaccharides composed of β-(1→6)-linked galactose backbone with α-(1→2)-linked arabinose branches were synthesized through coupling of α-(1→5)-linked arabinofuranosyl disaccharide donor with a tri- and tetrasaccharide backbone at C-2, respectively.     

Oxidative Lime Pretreatment of Dacotah Switchgrass

Oxidative lime pretreatment increases the enzymatic digestibility of lignocellulosic biomass primarily by removing lignin. In this study, recommended pretreatment conditions (reaction temperature, oxygen pressure, lime loading, and time) were determined for Dacotah switchgrass. Glucan and xylan overall hydrolysis yields (72 h, 15 FPU/g raw glucan) were measured for 105 different reaction conditions involving three different reactor configurations (very short term, short term, and long term). The short-term reactor was the most productive. At the recommended pretreatment condition (120 °C, 6.89 bar O₂, 240 min), it achieved an overall glucan hydrolysis yield of 85.2 g glucan hydrolyzed/100 g raw glucan and an overall xylan yield of 50.1 g xylan hydrolyzed/100 g raw xylan. At this condition, glucan oligomers (1.80 g glucan recovered/100 g glucan in raw biomass) and xylan oligomers (25.20 g xylan recovered/100 g xylan in raw biomass) were recovered from the pretreatment liquor, which compensate for low pretreatment yields.     

Pretreatment of corn stover by soaking in aqueous ammonia at moderate temperatures

Soaking in aqueous ammonia at moderate temperatures was investigated as a method of pretreatment for enzymatic hydrolysis as well as simultaneous saccharification and cofermentation (SSCF) of corn stover. The method involves batch treatment of the feedstock with aqueous ammonia (15-30 wt%) at 40-90 degrees C for 6-24 h. The optimum treatment conditions were found to be 15 wt% of NH(3), 60 degrees C, 1:6 of solid-to-liquid ratio, and 12 h of treatment time. The treated corn stover retained 100% glucan and 85% of xylan, but removed 62% of lignin. The enzymatic digestibility of the glucan content increased from 17 to 85% with 15 FPU/g-glucan enzyme loading, whereas the digestibility of the xylan content increased to 78%. The treated corn stover was also subjected to SSCF test using Spezyme-CP and recombinant Escherichia coli (KO11). The SSCF of the soaking in aqueous ammonia treated corn stover resulted in an ethanol concentration of 19.2 g/L from 3% (w/v) glucan loading, which corresponds to 77% of the maximum theoretical yield based on glucan and xylan.