Immobilization of α-transglucosidase on silica-coated magnetic nanoparticles and its application for production of isomaltooligosaccharide from the potato peel.

In this study, the production of isomaltooligosaccharide from potato peel starch was carried out in three steps: liquefaction, saccharification, and transglucosylation. Further, cloning α-transglucosidase gene from Aspergillus niger (GH31 family), transforming into E. coli BL21 (DE3), overexpressing and purifying the resulting protein for the production of α-transglucosidase. The generated α-transglucosidase was then bound with magnetic nanoparticles, which improved reusability up to 5 cycles with more than 60% activity. All the modifications were characterized using the following methods: Fourier transform infra-red analysis, Transmission Electron Microscopy, Field Emission Scanning Electron Microscopy, Energy Dispersive X-ray spectroscopy, X-Ray Diffraction Spectroscopy, Thermogravimetric Analysis, and Dynamic Light Scattering (DLS) analysis. Further, the optimum conditions for transglucosylation were determined by RSM as follows: enzyme-to-substrate ratio 6.9 U g-1, reaction time 9 h, temperature 45 °C, and pH 5.5 with a yield of 70 g l-1 (± 2.1). MALDI-TOF-MS analysis showed DP of the IMOs in ranges of 2-10. The detailed structural characterization of isomaltooligosaccharide by GC-MS and NMR suggested the α-(1 → 4) and α-(1 → 6)-D-Glcp residues as major constituents along with minor α-(1 → 2) and α-(1 → 3) -D-Glcp residues.

Functional ß-mannooligosaccharides: Sources, enzymatic production and application as prebiotics.

One of the emerging non-digestible oligosaccharide prebiotics is ß-mannooligosaccharides (ß-MOS). ß-MOS are ß-mannan derived oligosaccharides, they are selectively fermented by gut microbiota, promoting the growth of beneficial microorganisms (probiotics), whereas the growth of enteric pathogens remains unaffected or gets inhibited in their presence, along with production of metabolites such as short-chain fatty acids. ß-MOS also exhibit several other bioactive properties and health-promoting effects. Production of ß-MOS using the enzymes such as ß-mannanases is the most effective and eco-friendly approach. For the application of ß-MOS on a large scale, their production needs to be standardized using low-cost substrates, efficient enzymes and optimization of the production conditions. Moreover, for their application, detailed in-vivo and clinical studies are required. For this, a thorough information of various studies in this regard is needed. The current review provides a comprehensive account of the enzymatic production of ß-MOS along with an evaluation of their prebiotic and other bioactive properties. Their characterization, structural-functional relationship and in-vivo studies have also been summarized. Research gaps and future prospects have also been discussed, which will help in conducting further research for the commercialization of ß-MOS as prebiotics, functional food ingredients and therapeutic agents.

Properties and antimicrobial activity of wheat-straw nanocellulose-arabinoxylan acetate composite films incorporated with silver nanoparticles.

In the current study, the novel eco-friendly and biodegradable nanocomposite films (NC-AXAc) were prepared from wheat-straw NC and AXAc with improved functional properties. NC derived from wheat-straw cellulose has a fibre-like structure with mean-particle size in the 340-520 nm range. AX derived AXAc was prepared with Degree of Substitution (DS) in the range of 1.85-1.89. Furthermore, to enhance antimicrobial properties, AgNPs were prepared via the reduction method using NaBH4 and added into the concentration of 4 × 10-4M into the emulsion forming composite films. The silver nanoparticles (AgNPs) incorporated in the composite exhibited an average size of 40-70 nm and a surface plasmon resonance (SPR) absorption peak at 395 nm. The high-resolution XPS spectrum of the Ag element showed that the two peaks at around 374.2 eV (Ag3d3/2) and 368.2 eV (Ag3d5/2) clearly revealed the metallic Ag existence in composite films. SEM analysis revealed the coarse and heterogeneous morphology of AgNPs incorporated films. The AgNPs incorporated composites exhibited good mechanical, thermal stability, and antimicrobial activity. The results suggested that AgNPs incorporated NC-AXAc composites could be used as a potential biodegradable antimicrobial nanocomposite in active food packaging systems for shelf-life extension of perishable commodities.

Structural insight into a glucomannan-type extracellular polysaccharide produced by a marine Bacillus altitudinis SORB11 from Southern Ocean.

Extracellular polysaccharide (EPS) produced by a deep-sea, psychrotolerant Bacillus altitudinis SORB11 was evaluated by considering physiochemical nature and structural constituents. The productivity of crude EPS was measured ~ 13.17 g L-1. The surface topography of the crude EPS showed a porous, webbed structure along with a branched coil-like configuration. The crystalline crude EPS contained a high amount of sulfur. Further, the crude EPS was subjected for purification. The molecular weight of purified EPS was determined ~ 9.8 × 104 Da. The purified EPS was appeared to show glucomannan-like configuration that is composed of → 4)-ß-Manp-(1 → and → 4)-ß-Glcp-(1 → residues. So, this polysaccharide was comparable to the structure of plant-derived glucomannan. Subsequently, EPS biosynthesis protein clusters like EpsC, EpsD, EpsE, and glycosyltransferase family proteins were predicted from the genome of strain SORB11, which may provide an insight into the production of glucomannan-type of polysaccharide. This low molecular weight linear form of glucomannan-type EPS might be involved to form a network-like unattached aggregation, and helps in cell-to-cell interaction in deep-sea microbial species.

Wheat inositol pyrophosphate kinase TaVIH2-3B modulates cell-wall composition and drought tolerance in Arabidopsis.

BACKGROUND: Inositol pyrophosphates (PP-InsPs) are high-energy derivatives of inositol, involved in different signalling and regulatory responses of eukaryotic cells. Distinct PP-InsPs species are characterized by the presence of phosphate at a variable number of the 6-carbon inositol ring backbone, and two distinct classes of inositol phosphate kinases responsible for their synthesis have been identified in Arabidopsis, namely ITPKinase (inositol 1,3,4 trisphosphate 5/6 kinase) and PP-IP5Kinase (diphosphoinositol pentakisphosphate kinases). Plant PP-IP5Ks are capable of synthesizing InsP8 and were previously shown to control defense against pathogens and phosphate response signals. However, other potential roles of plant PP-IP5Ks, especially towards abiotic stress, remain poorly understood. RESULTS: Here, we characterized the physiological functions of two Triticum aestivum L. (hexaploid wheat) PPIP5K homologs, TaVIH1 and TaVIH2. We demonstrate that wheat VIH proteins can utilize InsP7 as the substrate to produce InsP8, a process that requires the functional VIH-kinase domains. At the transcriptional level, both TaVIH1 and TaVIH2 are expressed in different wheat tissues, including developing grains, but show selective response to abiotic stresses during drought-mimic experiments. Ectopic overexpression of TaVIH2-3B in Arabidopsis confers tolerance to drought stress and rescues the sensitivity of Atvih2 mutants. RNAseq analysis of TaVIH2-3B-expressing transgenic lines of Arabidopsis shows genome-wide reprogramming with remarkable effects on genes involved in cell-wall biosynthesis, which is supported by the observation of enhanced accumulation of polysaccharides (arabinogalactan, cellulose, and arabinoxylan) in the transgenic plants. CONCLUSIONS: Overall, this work identifies a novel function of VIH proteins, implicating them in modulation of the expression of cell-wall homeostasis genes, and tolerance to water-deficit stress. This work suggests that plant VIH enzymes may be linked to drought tolerance and opens up the possibility of future research into using plant VIH-derived products to generate drought-resistant plants.

Application and toxicity studies of arabinoxylan and ß-D-glucan stearic acid ester composite coatings in extending postharvest storage of peach.

Peaches are good source of nutrients and known for their taste and aroma. The highly perishable nature of the peaches tends to decay rapidly during transportation and storage is a serious constraint for efficient transportation and storage. Therefore, the effect of arabinoxylan (AX) and ß-D-glucan stearic acid ester (SABG) composite coating material was examined for the postharvest storage quality of peach under storage at 22 ± 2 °C with 85% relative humidity (RH). Both, AX-SABG and shellac (1-2%) coatings significantly reduced the change in the quality attributes like weight loss (1.2-1.4 fold), respiration rate (1.1-1.2 fold), ripening index (1.3-1.5 fold) and firmness (1.3-1.5 fold) during 6 days storage as compared to the uncoated peaches. In addition, AX-SABG (1-2%) coating was more effective in retaining aroma volatiles and reducing disease incidence compared to shellac. Further, acute and chronic toxicological studies have shown no tissue related toxicity and mortality in mice. Our results suggest that AX-SABG as an edible coating has the potential to preserve the fruit quality during 6 days storage at 22 ± 2 °C and extend the postharvest shelf life of peach during storage.

Effect of cellulose and starch fatty acid esters addition on microstructure and physical properties of arabinoxylan films.

Arabinoxylan (AX) and cellulose were extracted from wheat straw, whereas starch was extracted from potato peel. Thereafter, cellulose and starch were esterified with lauric, myristic, palmitic and stearic acids to prepare corresponding cellulose (CFAs) and starch fatty acid esters (SFAs) with DS 2.1-2.8. XRD study revealed remarkable loss of crystallinity in cellulose and starch due to fatty acid esterification. The addition of palmitate and stearate esters of cellulose and starch to AX formed laminar film microstructures which limited water vapor permeability whereas films prepared by blending AX with laurate and myristate esters of starch and cellulose were less effective as water vapor barrier due to their non-layer microstructures. The laminar structures also resulted significant reduction in mechanical strength of the composite films. Furthermore, all AX-CFAs and AX-SFAs films were thermally more stable than native composite films. These films might be used to produce industrially useful coating material for food products.

Improved postharvest quality of apple (Rich Red) by composite coating based on arabinoxylan and ß-glucan stearic acid ester.

Apples (Rich Red) were coated with wheat straw arabinoxylan (AX) and ß-glucan stearic acid ester (SABG) composite coating material in the concentration range of 1-2%. The postharvest storage life of coated apples was studied at 22 °C (±2) with 65% and 85% relative humidity for 45 days. Application of both AX-SABG (1-2%) and shellac (1-2%) coatings material significantly reduced the weight loss, respiration process, color degradation, process of fruit softening and ripening index as compared to the uncoated apples up to 30 days storage. However, the AX-SABG coatings were found more effective in preventing the aroma loss, reducing microbial spoilage and maintaining sensorial attributes as compared to shellac and uncoated apples during storage.

Effect of arabinoxylan and ß-glucan stearic acid ester coatings on post-harvest quality of apple (Royal Delicious).

The effect of wheat straw arabinoxylan (AX) and ß-glucan stearic acid ester (SABG) composite coating on the quality and storage life of apple (Royal Delicious) was studied at 22 °C (±2) with relative humidity of 65% and 85% for 60 days. Fresh fruits were coated with surface coatings of AX-SABG, shellac in the concentration range of 1-4%. Application of both AX-SABG (1-4%) and shellac (1-4%) coatings was found to significantly reduce weight loss, respiration rate, fruit softening process, ripening index, color degradation and polyphenol oxidase activity compared to control during the storage period of more than 30 days. However, an AX-SABG coating was more effective in reducing fruit decay and loss of aroma volatiles followed by shellac coated apples; the un-coated apples being showing maximum quality deterioration. These findings confirmed the potential benefits of applying AX-SABG coating to extend the shelf life and quality of apples especially during transportation and storage.

Structural and Functional Properties of Exopolysaccharide Excreted by a Novel Bacillus anthracis (Strain PFAB2) of Hot Spring Origin.

Exopolysaccharide produced by a unique avirulent Bacillus anthracis strain PFAB2 of hot spring origin has been characterized and its functional properties are investigated which is a first report. Maximum yield of EPS is 7.66 g/l with 2% glucose and 1% peptone as optimum carbon and nitrogen source respectively. The EPS is found to be a homopolymer consisting of only glucose as principle monosaccharide component. Through 1H NMR study, different dextran-like proton peaks are observed. Molecular weight of the EPS resembles low molecular weight bacterial origin polysaccharides. Melting transition of the EPS has started after 276 °C which indicates good thermal stability. The EPS also shows potent antioxidant activity in terms of DPPH and ABTS mediated free radical scavenging property compared to standard ascorbic acid. Emulsifying property of the EPS is also observed and has shown good emulsification of vegetable oils. The polysaccharide forms a thermo resistant gel during the heating phase, with G' higher than G″ indicating excellent shear-thinning behaviour and viscoelastic nature of the EPS.

Finger millet arabinoxylan protects mice from high-fat diet induced lipid derangements, inflammation, endotoxemia and gut bacterial dysbiosis.

Arabinoxylan (AX), a non-starch polysaccharide extracted from cereals such as wheat, rice and millets, is known to impart various health promoting effects. Our earlier study suggested that finger millet (FM) could ameliorate high fat diet (HFD)-induced metabolic derangements. The present study is aimed to evaluate the effect of FM-AX supplementation, a key bioactive from finger millet, on HFD-induced metabolic and gut bacterial derangements. Male Swiss albino mice were fed with normal chow diet (NPD) or HFD (60%kcal from fat) for 10 weeks. FM-AX was orally supplemented at doses of 0.5 and 1.0g/kg bodyweight on every alternate day for 10 weeks. Glucose tolerance, serum hormones, hepatic lipid accumulation and inflammation, white adipose tissue marker gene expression, adipocyte size and inflammation; metagenomic alterations in cecal bacteria; cecal short chain fatty acids and colonic tight junction gene expressions were studied. FM-AX supplementation prevented HFD-induced weight gain, alerted glucose tolerance and serum lipid profile, hepatic lipid accumulation and inflammation. Hepatic and white adipose tissue gene expressions were beneficially modulated. Further, AX supplementation prevented metagenomic alterations in cecum; improved ileal and colonic health and overall prevented metabolic endotoxemia. Present work suggests that AX from finger millet can be developed as a nutraceutical for the management of HFD- induced obesity.

Effect of ß-glucan-fatty acid esters on microstructure and physical properties of wheat straw arabinoxylan films.

Arabinoxylans (AX) was isolated from wheat straw, whereas ß-glucan (BG) was extracted from oat flour. The compositional analysis indicated wheat straw AX contained arabinose and xylose as major constituent sugars whereas higher ß-glucan content (77%) was found in the extracted material from oat flour. The BG was conjugated with lauric (LA), myristic (MA), palmitic (PA), stearic (SA) and oleic (OA) acid to prepare corresponding ß-glucan-fatty acid esters (BGFAs) with nearly similar degree of substitution. The effect of BGFAs to AX films on the water barrier, optical and mechanical properties were investigated. The addition of LABG and MABG to AX formed laminar structures in the composite films which limited water vapor permeability, giving rise to more opacity. Films prepared by blending AX with SABG and OABG were less effective as water vapor barrier due to their non-layer film microstructures; however they were less opaque. The laminar structures also imparted less mechanical strength and flexibility in the composite films. Furthermore, thermogravimetric analysis (TGA) revealed that all AX-BGFAs composite films were thermally more stable than pure AX and AX-BG films.

Hydroxycinnamic acid bound arabinoxylans from millet brans-structural features and antioxidant activity.

Hydroxycinnamic acid bound arabinoxylans (HCA-AXs) were extracted from brans of five Indian millet varieties and response surface methodology was used to optimize the extraction conditions. The optimal condition to obtain highest yield of millet HCA-AXs was determined as follows: time 61min, temperature 66°C, ratio of solvent to sample 12ml/g. Linkage analysis indicated that hydroxycinnamic acid bound arabinoxylan from kodo millet (KM-HCA-AX) contained comparatively low branched arabinoxylan consisting of 14.6% mono-substituted, 1.2% di-substituted and 41.2% un-substituted Xylp residues. The HPLC analysis of millet HCA-AXs showed significant variation in the content of three major bound hydroxycinnamic acids (caffeic, p-coumaric and ferulic acid). The antioxidant activity of millet HCA-AXs were evaluated using three in vitro assay methods (DPPH, FRAP and ß-carotene linoleate emulsion assays) which suggested both phenolic acid composition and structural characteristics of arabinoxylans could be correlated to their antioxidant potential, the detailed structural analysis revealed that low substituted KM-HCA-AX exhibited relatively higher antioxidant activity compared to other medium and highly substituted HCA-AXs from finger (FM), proso (PM), barnyard (BM) and foxtail (FOXM) millet.

Structural characterization of the heteroxylans from poplar and switchgrass.

Heteroxylans are polysaccharides with a backbone composed of 1,4-linked ß-D-xylosyl residues. In hardwoods some of these xylosyl residues are substituted at O-2 with 4-O-methyl α-D-glucuronic and occasionally with α-D-glucuronic acid. In grasses, the xylan backbone is predominantly substituted with α-L-arabinofuranosyl residues (most often at O-3, but sometimes at O-2). Grass heteroxylan backbone residues may also have small amounts of α-D-glucuronic acid and/or 4-O-methyl α-D-glucuronic acid at O-2. Heteroxylans have a role in maintaining the structural integrity of the cell walls that comprise the bulk of lignocellulosic biomass. Moreover, differences in the molecular features of these hemicellulosic polysaccharides, including their degree of polymerization, degree of branching and spatial arrangement of side chains along the xylan backbone, have been correlated to altered cell wall properties (Izydorczyk MS, Biliaderis CG, Carbohydr Polym 28:33-48, 1995) and the ease with which biomass can be enzymatically converted to fermentable sugars. Thus, understanding the relationship between heteroxylan structure and biomass properties is required to engineer bioenergy crops with improved processing characteristics. In this chapter we describe some of the analytical methods we routinely use to perform in-depth structural analysis of heteroxylans from poplar and switchgrass biomass.

The ability of land plants to synthesize glucuronoxylans predates the evolution of tracheophytes.

Glucuronoxylans with a backbone of 1,4-linked ß-D-xylosyl residues are ubiquitous in the secondary walls of gymnosperms and angiosperms. Xylans have been reported to be present in hornwort cell walls, but their structures have not been determined. In contrast, the presence of xylans in the cell walls of mosses and liverworts remains a subject of debate. Here we present data that unequivocally establishes that the cell walls of leafy tissue and axillary hair cells of the moss Physcomitrella patens contain a glucuronoxylan that is structurally similar to glucuronoxylans in the secondary cell walls of vascular plants. Some of the 1,4-linked ß-D-xylopyranosyl residues in the backbone of this glucuronoxylan bear an α-D-glucosyluronic acid (GlcpA) sidechain at O-2. In contrast, the lycopodiophyte Selaginella kraussiana synthesizes a glucuronoxylan substituted with 4-O-Me-α-D-GlcpA sidechains, as do many hardwood species. The monilophyte Equisetum hyemale produces a glucuronoxylan with both 4-O-Me-α-D-GlcpA and α-D-GlcpA sidechains, as does Arabidopsis. The seedless plant glucuronoxylans contain no discernible amounts of the reducing-end sequence that is characteristic of gymnosperm and eudicot xylans. Phylogenetic studies showed that the P. patens genome contains genes with high sequence similarity to Arabidopsis CAZy family GT8, GT43 and GT47 glycosyltransferases that are likely involved in xylan synthesis. We conclude that mosses synthesize glucuronoxylan that is structurally similar to the glucuronoxylans present in the secondary cell walls of lycopodiophytes, monilophytes, and many seed-bearing plants, and that several of the glycosyltransferases required for glucuronoxylan synthesis evolved before the evolution of tracheophytes.

Structure and function of an arabinoxylan-specific xylanase.

The enzymatic degradation of plant cell walls plays a central role in the carbon cycle and is of increasing environmental and industrial significance. The enzymes that catalyze this process include xylanases that degrade xylan, a ß-1,4-xylose polymer that is decorated with various sugars. Although xylanases efficiently hydrolyze unsubstituted xylans, these enzymes are unable to access highly decorated forms of the polysaccharide, such as arabinoxylans that contain arabinofuranose decorations. Here, we show that a Clostridium thermocellum enzyme, designated CtXyl5A, hydrolyzes arabinoxylans but does not attack unsubstituted xylans. Analysis of the reaction products generated by CtXyl5A showed that all the oligosaccharides contain an O3 arabinose linked to the reducing end xylose. The crystal structure of the catalytic module (CtGH5) of CtXyl5A, appended to a family 6 noncatalytic carbohydrate-binding module (CtCBM6), showed that CtGH5 displays a canonical (α/ß)(8)-barrel fold with the substrate binding cleft running along the surface of the protein. The catalytic apparatus is housed in the center of the cleft. Adjacent to the -1 subsite is a pocket that could accommodate an l-arabinofuranose-linked α-1,3 to the active site xylose, which is likely to function as a key specificity determinant. CtCBM6, which adopts a ß-sandwich fold, recognizes the termini of xylo- and gluco-configured oligosaccharides, consistent with the pocket topology displayed by the ligand-binding site. In contrast to typical modular glycoside hydrolases, there is an extensive hydrophobic interface between CtGH5 and CtCBM6, and thus the two modules cannot function as independent entities.

Structural analysis of arabinoxylans isolated from ball-milled switchgrass biomass.

Ball-milled alcohol-insoluble residue (AIR) was prepared from switchgrass (Panicum virgatum var Alamo) and sequentially extracted with 50 mM ammonium oxalate buffer, 50 mM sodium carbonate, 1 M KOH containing 1% NaBH(4), and 4 M KOH containing 1% NaBH(4). Arabinoxylan was the most abundant component of the 1 M KOH-extracted fraction, which was treated with endoxylanase to generate oligosaccharides. Gel-permeation chromatography of these oligosaccharides produced three size-homogeneous oligosaccharide fractions with molecular weights of 678, 810, and 1074 Da, corresponding to 5, 6, and 8 pentose units, respectively. Detailed structural analysis of these oligosaccharides was performed using methylation analysis, multiple-step mass spectrometry (ESIMS(n)), and 1D and 2D NMR spectroscopy. The preferred gas-phase fragmentation pathways were identified for these oligosaccharides, providing extensive sequence information that was completely consistent with structures determined by ab initio NMR analysis. These results demonstrate the high information content of ESIMS(n) analysis when applied to cell-wall-derived oligosaccharides and provide standard data that will facilitate the analysis of cell-wall polysaccharide fragments with a sensitivity that is sufficient for the analysis of samples obtained from dissected tissues as well as other small samples.

Virus-induced gene silencing offers a functional genomics platform for studying plant cell wall formation.

Virus-induced gene silencing (VIGS) is a powerful genetic tool for rapid assessment of plant gene functions in the post-genomic era. Here, we successfully implemented a Tobacco Rattle Virus (TRV)-based VIGS system to study functions of genes involved in either primary or secondary cell wall formation in Nicotiana benthamiana plants. A 3-week post-VIGS time frame is sufficient to observe phenotypic alterations in the anatomical structure of stems and chemical composition of the primary and secondary cell walls. We used cell wall glycan-directed monoclonal antibodies to demonstrate that alteration of cell wall polymer synthesis during the secondary growth phase of VIGS plants has profound effects on the extractability of components from woody stem cell walls. Therefore, TRV-based VIGS together with cell wall component profiling methods provide a high-throughput gene discovery platform for studying plant cell wall formation from a bioenergy perspective.

Identification of a novel sugar 5,7-diacetamido-8-amino-3,5,7,8,9-pentadeoxy-D-glycero-D-galacto-non-2-ulosonic acid present in the lipooligosaccharide of Vibrio parahaemolyticus O3:K6.

A novel sugar, 5,7-diacetamido-8-amino-3,5,7,8,9-pentadeoxy-D-glycero-D-galacto-non-2-ulosonic acid (NonlA), has been identified as a component of the oligosaccharide (OS) isolated from the lipooligosaccharide (LOS) of the emerging strain of Vibrio parahaemolyticus O3:K6 associated with a global pandemic. In the present study we report the identification and characterization of this novel sugar present in the OS of V. parahaemolyticus O3:K6, using chemical analysis, NMR spectroscopy and mass spectrometry.