Enzymatic Fingerprinting Research Articles

Characterization of (Glucurono)arabinoxylans from Oats Using Enzymatic Fingerprinting.

Cell wall material from whole oat grains was sequentially extracted to study the structural characteristics of individual arabinoxylan (AX) populations. Araf was singly substituted at both O-3 (mainly) and O-2 positions of Xylp, and no disubstitution of Xylp with Araf residues was found in oat AXs. Both highly substituted and sparsely substituted segments were found in AXs in Ba(OH)2 extracts, whereas AXs in 1 and 6 M NaOH extracts were rarely branched and easily aggregated. Both O-2-linked GlcA and 4-O-MeGlcA residues were present in oat AXs. A series of AX oligomers with galactose as a substituent was detected for the first time in oats. The present study suggested that the distribution of Araf was contiguous in oat AXs, different from the homogeneous distribution of Araf in wheat and barley AXs, which might result in different fermentation patterns in humans and animals.

Improvement in Saccharification Yield of Mixed Rumen Enzymes by Identification of Recalcitrant Cell Wall Constituents Using Enzyme Fingerprinting.

Identification of recalcitrant factors that limit digestion of forages and the development of enzymatic approaches that improve hydrolysis could play a key role in improving the efficiency of meat and milk production in ruminants. Enzyme fingerprinting of barley silage fed to heifers and total tract indigestible fibre residue (TIFR) collected from feces was used to identify cell wall components resistant to total tract digestion. Enzyme fingerprinting results identified acetyl xylan esterases as key to the enhanced ruminal digestion. FTIR analysis also suggested cross-link cell wall polymers as principal components of indigested fiber residues in feces. Based on structural information from enzymatic fingerprinting and FTIR, enzyme pretreatment to enhance glucose yield from barley straw and alfalfa hay upon exposure to mixed rumen-enzymes was developed. Prehydrolysis effects of recombinant fungal fibrolytic hydrolases were analyzed using microassay in combination with statistical experimental design. Recombinant hemicellulases and auxiliary enzymes initiated degradation of plant structural polysaccharides upon application and improved the in vitro saccharification of alfalfa and barley straw by mixed rumen enzymes. The validation results showed that microassay in combination with statistical experimental design can be successfully used to predict effective enzyme pretreatments that can enhance plant cell wall digestion by mixed rumen enzymes.

Strategy to identify and quantify polysaccharide gums in gelled food concentrates

A strategy for the unambiguous identification and selective quantification of xanthan gum and locust bean gum (LBG) in gelled food concentrates is presented. DNA detection by polymerase chain reaction (PCR) showed to be a fast, sensitive, and selective method that can be used as a first screening tool in intact gelled food concentrates. An efficient isolation procedure is described removing components that may interfere with subsequent analyses. NMR spectroscopy enabled the direct identification of xanthan gum and the discrimination between different galactomannans in the isolated polysaccharide fraction. An enzymatic fingerprinting method using endo-β-mannanase, in addition to being used to differentiate between galactomannans, was developed into a selective, quantitative method for LBG, whereas monosaccharide analysis was used to quantify xanthan gum. Recoveries for xanthan gum and LBG were 87% and 70%, respectively, with in-between day relative standard deviations below 20% for xanthan gum and below 10% for LBG.

Novel and diverse fine structures in LiCl–DMSO extracted apple hemicelluloses

Hemicelluloses are key polysaccharides in the regulation of the mechanical properties of plant cell walls during organ development and in fruit texture. Their diverse compositions and structures are partially known, in particular with regard to their function in cell walls. To that end, apple hemicelluloses were sequentially extracted by DMSO doped by LiCl followed by potassium hydroxide. The weakly bounded hemicelluloses in the LiCl–DMSO soluble extract were fractionated by ion exchange (AEC) and size exclusion (SEC) chromatographies. The structure of all the extracts and fractions was established by enzymatic fingerprinting using β-glucanase, β-mannanase and β-xylanase. Molecular weight of the fraction was established by HPSEC. MS as well as HPAEC analyses of the enzyme digests revealed the remarkable diversity of apple hemicelluloses. Different xyloglucan (XyG), galactoglucomannan (GgM) and glucuronoarabinoxylan were isolated along the extraction and fractionation process. All LiCl–DMSO soluble fractions were acetyl-esterified. Besides, the LiCl–DMSO soluble XyG differed from the 4M KOH extracted one essentially on the basis of its molecular weight. At least two populations differing in their content and distribution of glucose and mannose composed GgM. Moreover, galactose ramifications occurred on mannose blocks in the glucose rich fraction. These results open the way for future studies on the complex structure–function relationship of hemicelluloses in plant cell walls.

Two-step enzymatic fingerprinting of sugar beet pectin

A two-step enzymatic fingerprinting method was introduced to analyze a highly methylesterified and acetylated sugar beet pectin having a degree of methylesterification (DM) of 62 and acetylation of 30. A cocktail of pectolytic enzymes, including endo-polygalacturonase II (endo-PGII) and pectin lyase (PL), was used for the first digestion. The endo-PGII and PL resistant pectin fragments were isolated and subjected to a second digestion using fungal pectin methylesterase and endo-PGII. After the two sequential digestions, 78% of the total GalA residues present in the parental pectin were recovered as mono- and oligomers, which were used to quantitatively describe the parental SBP. For this reason, the descriptive parameters degree of blockiness (DBabs), degree of hydrolysis by PG (DHPG) and degree of hydrolysis by PL (DHPL) were established for both digestions. The first digestion revealed the presence of short blocks of nonesterified GalA residues and blocks of partly methylesterified and acetylated GalA residues in the parental SBP, in addition to blocks of highly methylesterified and acetylated GalA residues. The second digestion revealed the presence of blocks of methylesterified, partly methylesterified and/or acetylated GalA residues in a sequence not to be degradable by neither endo-PGII nor by PL. The acetyl groups were present in an blockwise manner. Application of the method to two differently prepared DM 50 SBPs showed that the two pectins differ in the ratio of blocks of nonesterified and blocks of partly methylesterified and acetylated GalA residues.

Enzymatic fingerprinting of structurally similar homologous proteins using polyion complex library constructed by tuning PEGylated polyamine functionalities.

Human plasma proteins and even structurally similar homologous albumins were fingerprinted and discriminated by a sensor array consisting of a polyion complex library with artificial differentiation constructed by facile tuning of PEGylated polyamine functionalities.

Descriptive parameters for revealing substitution patterns of sugar beet pectins using pectolytic enzymes

Enzymatic fingerprinting was applied to sugar beet pectins (SBPs) modified by either plant or fungal pectin methyl esterases and alkali catalyzed de-esterification to reveal the ester distributions over the pectin backbone. A simultaneous pectin lyase (PL) treatment to the commonly used endo-polygalacturonase (endo-PG) degradation showed to be effective in degrading both high and low methylesterified and/or acetylated homogalaturonan regions of SBP simultaneously. Using LC-HILIC–MS/ELSD, we studied in detail all the diagnostic oligomers present, enabling us to discriminate between differently prepared sugar beet pectins having various levels of methylesterification and acetylation. Furthermore, distinction between commercially extracted and de-esterified sugar beet pectin having different patterns of substitution was achieved by using novel descriptive pectin parameters. In addition to DBabs approach for nonmethylesterified sequences degradable by endo-PG, the “degree of hydrolysis” (DHPG) representing all partially saturated methylesterified and/or acetylated galacturonic acid (GalA) moieties was introduced as a new parameter. Consequently, the description DHPL has been introduced to quantify all esterified unsaturated GalA oligomers.

XAX1 from glycosyltransferase family 61 mediates xylosyltransfer to rice xylan

Xylan is the second most abundant polysaccharide on Earth and represents an immense quantity of stored energy for biofuel production. Despite its importance, most of the enzymes that synthesize xylan have yet to be identified. Xylans have a backbone of β-1,4-linked xylose residues with substitutions that include α-(1→2)-linked glucuronosyl, 4-O-methyl glucuronosyl, and α-1,2- and α-1,3-arabinofuranosyl residues. The substitutions are structurally diverse and vary by taxonomy, with grass xylan representing a unique composition distinct from dicots and other monocots. To date, no enzyme has yet been identified that is specific to grass xylan synthesis. We identified a xylose-deficient loss-of-function rice mutant in Os02g22380, a putative glycosyltransferase in a grass-specific subfamily of family GT61. We designate the mutant xax1 for xylosyl arabinosyl substitution of xylan 1. Enzymatic fingerprinting of xylan showed the specific absence in the mutant of a peak, which was isolated and determined by (1)H-NMR to be (β-1,4-Xyl)(4) with a β-Xylp-(1→2)-α-Araf-(1→3). Rice xax1 mutant plants are deficient in ferulic and coumaric acid, aromatic compounds known to be attached to arabinosyl residues in xylan substituted with xylosyl residues. The xax1 mutant plants exhibit an increased extractability of xylan and increased saccharification, probably reflecting a lower degree of diferulic cross-links. Activity assays with microsomes isolated from tobacco plants transiently expressing XAX1 demonstrated xylosyltransferase activity onto endogenous acceptors. Our results provide insight into grass xylan synthesis and how substitutions may be modified for increased saccharification for biofuel generation.

Enzymatic fingerprinting of arabinoxylan and β-glucan in triticale, barley and tritordeum grains

Enzymatic fingerprinting of arabinoxylan (AX) and β-glucan using endo-xylanase and lichenase, respectively, helps determine the structural heterogeneity between different cereals and within genotypes of the same cereal. This study characterised the structural features of AX and β-glucan in whole grains of eight triticale cultivars grown at two locations, 20 barley cultivars/lines with wide variation in composition and morphology and five tritordeum breeding lines. Principal component analysis (PCA) resulted in clear clustering of these cereals. In general, barley and tritordeum had a higher relative proportion of highly branched arabinoxylan oligosaccharides (AXOS) than triticale. Subsequent analysis of triticale revealed two clusters based on growing region along principal component (PC) 1, while PC2 explained the genetic variability and was based on mono-substitution and di-substitution in AX fragments. PCA of β-glucan features separated the three cereals based on β-glucan content. The molar ratio of trisaccharide to tetrasaccharide was 2.5–3.4 in triticale, 2.3–3.3 in barley and 2.8–3.4 in tritordeum. Barley showed a strong positive correlation (r=0.86) between β-glucan content and relative proportion of trisaccharide. The results show that structural features of AX and β-glucan vary between and within triticale, barley and tritordeum grains which might be important determinants of end-use quality of grains.

Enzymatic fingerprints of polysaccharides of Dendrobium officinale and their application in identification of Dendrobium species

Enzymatic fingerprinting of polysaccharides from Dendrobium officinale was studied and applied to authenticate Dendrobium species. Results showed that Dendrobium officinale species from Anhui province, Fujian province, Yunnan province, Guangdong province and Guangxi province of China, could be identified by polysaccharide analysis using carbohydrate gel electrophoresis (PACE). However, the fingerprints of Dendrobium officinale from Jiangxi province, Hu'nan province and Wenzhou, Yandangshan and Fuyang in Zhejiang province were very similar. As far as the fingerprints of different Dendrobium species were concerned, the differences between Dendrobium officinale, Dendrobium huoshanense, Dendrobium moniliforme, Dendrobium devonianum, Dendrobium aphyllum, Dendrobium wilsonii and Dendrobium crystallinum were obvious. Moreover, the genetic relationships between different samples were analyzed by using principal component analysis and unweighted pair group method with arithmetic mean cluster analysis. Results suggested that polysaccharide fingerprint analysis by PACE has the potential to become a valuable new method for the identification and control of quality of herbal medicines in future.

Rheological properties of Ca2+-gels of partially methylesterified polygalacturonic acid: Effect of “mixed” patterns of methylesterification

Abstract Subjecting homogalacturonan to a combination of processes which modify the extent of pectin's methylesterification results in the generation of distinct patterns of methylesterification on the polymer. In this study, the influence of these “mixed” patterns of methylesterification on the rheological characteristics of Ca 2+ -gels is extensively examined, using partially methylesterified poly- d -galacturonic acid (mPGA) as a model for homogalacturonan. The uncontrolled pattern of methylesterification induced on mPGA was combined with that resulting from subsequent controlled chemical or enzymatic de-esterification. Afterwards, the produced mPGAs were characterised in terms of degree and pattern of methylesterification. The latter structural feature was quantified as “absolute degree of blockiness” through enzymatic fingerprinting. Characterised mPGAs were used for the preparation of gels with various calcium ion (Ca 2+ ) concentrations. All Ca 2+ -pectin systems formed “true” gels with a strong elastic behaviour, but vary in gel stiffness. The gels’ rheological characteristics not only depend on the extent of mPGA de-esterification, but also on the “mixed” patterns of methylesterification. The experimental results also indicate that extremely short non-cooperative junction zones contribute to the stiffness of Ca 2+ -mPGA gels.

Wheat endosperm cell walls: Spatial heterogeneity of polysaccharide structure and composition using micro-scale enzymatic fingerprinting and FT-IR microspectroscopy

Micro-scale enzymatic fingerprinting and FT-IR microspectroscopy were used to investigate changes of polysaccharide structure and composition in cell walls from wheat endosperm. These methods were applied to transverse and longitudinal sections of wheat grains harvested at maturity and 270°D. Principal component analysis treatment of the data revealed marked differences in the (1,3)-(1,4)-beta-glucans (BG)/arabinoxylans (AX) ratio and in the structure of AX depending on both cell position in the grain and stage of development. Cell walls close to the germ were enriched in BG in both developing and mature grain. AX in developing grain were characterized by a higher proportion of di-substitution by arabinose as compared to mature grain AX. In addition, AX in mature grains exhibited a different structure depending on prismatic or central origin of cell walls in the grain. These results indicated a high spatial and temporal control of the biosynthesis of AX and BG in wheat endosperm.

Overexpression of pectin methylesterase inhibitors in Arabidopsis restricts fungal infection by Botrytis cinerea.

Pectin, one of the main components of plant cell wall, is secreted in a highly methylesterified form and is demethylesterified in muro by pectin methylesterase (PME). The action of PME is important in plant development and defense and makes pectin susceptible to hydrolysis by enzymes such as endopolygalacturonases. Regulation of PME activity by specific protein inhibitors (PMEIs) can, therefore, play a role in plant development as well as in defense by influencing the susceptibility of the wall to microbial endopolygalacturonases. To test this hypothesis, we have constitutively expressed the genes AtPMEI-1 and AtPMEI-2 in Arabidopsis (Arabidopsis thaliana) and targeted the proteins into the apoplast. The overexpression of the inhibitors resulted in a decrease of PME activity in transgenic plants, and two PME isoforms were identified that interacted with both inhibitors. While the content of uronic acids in transformed plants was not significantly different from that of wild type, the degree of pectin methylesterification was increased by about 16%. Moreover, differences in the fine structure of pectins of transformed plants were observed by enzymatic fingerprinting. Transformed plants showed a slight but significant increase in root length and were more resistant to the necrotrophic fungus Botrytis cinerea. The reduced symptoms caused by the fungus on transgenic plants were related to its impaired ability to grow on methylesterified pectins.

Comparison of enzymatic de-esterification of strawberry and apple pectin at elevated pressure by fungal pectinmethylesterase

Abstract Water soluble pectin was isolated from strawberries. Sugar composition, degree of esterification and molar mass were compared with commercial apple pectin. Both pectins were subjected to enzymatic de-esterification by recombinant Aspergillus aculeatus pectinmethylesterase (PME). Rate of enzymatic de-esterification at elevated pressures (0.1–500 MPa) and temperatures (20–60 °C) was assayed by measuring the release of methanol as a function of time. Optimal activity was observed at 200–300 MPa combined with 45–55 °C. At all conditions investigated, both pectins were de-esterified at similar initial rates. However, after prolonged enzymatic treatment at atmospheric pressure and 30 °C, apple pectin was de-esterified to a significantly lower degree of esterification (7%) than strawberry pectin (32%). The mode of action of A. aculeatus PME was investigated by enzymatic fingerprinting of de-esterified pectin chains. The enzyme de-esterified according to a “multiple chain, multiple attack” mechanism, irrespective of the substrate. Industrial relevance This article demonstrates that both strawberry and apple pectin de-esterification by recombinant Aspergillus aculeatus PME is accelerated by high hydrostatic pressure. Since de-esterification of pectin in fruits gives rise to a texture improvement, this enzyme can be used to minimize texture damage during high-pressure processing of fruits and fruit-based products. It was also shown that this enzyme de-esterifies strawberry and apple pectin according to a “multiple chain, multiple attack” mechanism. The resulting pattern of esterification might have an influence on textural properties of fruits.

Effect of Temperature and High Pressure on the Activity and Mode of Action of Fungal Pectin Methyl Esterase

Pectin was de-esterified with purified recombinant Aspergillus aculeatus pectin methyl esterase (PME) during isothermal-isobaric treatments. By measuring the release of methanol as a function of treatment time, the rate of enzymatic pectin conversion was determined. Elevated temperature and pressure were found to stimulate PME activity. The highest rate of PME-catalyzed pectin de-esterification was obtained when combining pressures in the range 200-300 MPa with temperatures in the range 50-55 degrees C. The mode of pectin de-esterification was investigated by characterizing the pectin reaction products by enzymatic fingerprinting. No significant effect of increasing pressure (300 MPa) and/or temperature (50 degrees C) on the mode of pectin conversion was detected.

Enzymatic fingerprinting of Arabidopsis pectic polysaccharides using polysaccharide analysis by carbohydrate gel electrophoresis (PACE)

Plant cell wall polysaccharides vary in quantity and structure between different organs and during development. However, quantitative analysis of individual polysaccharides remains challenging, and relatively little is known about any such variation in polysaccharides in organs of the model plant Arabidopsis thaliana. We have analysed plant cell wall pectic polysaccharides using polysaccharide analysis by carbohydrate gel electrophoresis. By highly specific enzymatic digestion of a polysaccharide in a cell wall preparation, a unique fingerprint of short oligosaccharides was produced. These oligosaccharides gave quantitative and structural information on the original polysaccharide chain. We analysed enzyme-accessible polygalacturonan (PGA), linear beta(1,4) galactan and linear alpha(1,5) arabinan in several organs of Arabidopsis: roots, young leaves, old leaves, lower and upper inflorescence stems, seeds and callus. We found that this PGA constitutes a high proportion of cell wall material (CWM), up to 15% depending on the organ. In all organs, between 60 and 80% of the PGA was highly esterified in a blockwise fashion, and surprisingly, dispersely esterified PGA was hardly detected. We found enzyme-accessible linear galactan and arabinan are both present as a minor polysaccharide in all the organs. The amount of galactan ranged from ~0.04 to 0.25% of CWM, and linear arabinan constituted between 0.015 and 0.1%. Higher levels of galactan correlated with expanding tissues, supporting the hypothesis that this polysaccharide is involved in wall extension. We show by analysis of mur4 that the methods and results presented here also provide a basis for studies of pectic polysaccharides in Arabidopsis mutants.

Pectin: new insights into an old polymer are starting to gel

Pectin is a high value functional food ingredient widely used as a gelling agent and stabilizer. It is also an abundant, ubiquitous and multifunctional component of the cell walls of all land plants. Food scientists and plant scientists therefore share a common goal to better understand the structure and functionalities of pectic polymers at the molecular level. The basic properties of pectin have been known for nearly 200 years, but recently there has been tremendous progress in our understanding of the very complex fine structure of pectic polymers and pectinolytic enzymes. This has been made possible by synergies between plant and food research and by the application of a range of state-of-the-art techniques including enzymatic fingerprinting, mass spectrometry, NMR, molecular modelling, and monoclonal antibodies. With this increased knowledge comes the prospect of novel applications. Producers are beginning to develop a new generation of sophisticated designer pectins with specific functionalities. Moreover, the ability to manipulate pectin in planta would have a major impact on fruit and vegetable quality and processing, as well as on pectin production.

Classification of Wheat Varieties Based on Structural Features of Arabinoxylans As Revealed by Endoxylanase Treatment of Flour and Grain

Arabinoxylans (AX) are cell wall polysaccharides of complex structure involved in many aspects of wheat flour end uses. The study of the variations of AX structure can lead to the identification of genes involved in their biosynthesis, and thus in the control of the various aspects of grain quality related to their presence. A method is proposed to identify AX variations directly in whole grain by enzymatic degradation. An endoxylanase from Trichoderma viride was used to extract AX from a collection of 20 wheat cultivars (Triticum aestivum L.). Enzymatic degradation products were analyzed by HPAEC and multivariate analysis techniques (principal component analysis, canonical correlation analysis, and cluster analysis) were applied to analyze chromatographic data. The method evidenced variations in the proportion of mono- and disubstitution of the xylan backbone by arabinose side chains, allowing classification of the different varieties according to the structural features of AX. A similar classification was obtained starting from flour or whole grain, indicating that the method was specific of AX from endosperm tissues. In conclusion, the method combining endoxylanase treatment of wheat grain and the analysis of degradation products, e.g., enzymatic fingerprinting, can be applied to collections of wheat cultivars, and possibly other cereals in order to establish quantitative trait loci related to the biosynthesis of AX.

Preparation and properties of enzymatically and chemically modified sugar beet pectins

Sugar beet pectin was extracted from fresh sugar beet pulp and further deesterified by base, plant or fungal pectin esterase or esterified in acid methanol. The modified sugar beet pectins were characterised by chemical analyses, macromolecular and rheological properties, capillary electrophoresis and enzymatic fingerprinting. The majority of the modified sugar beet pectins had the same molar mass and neutral sugar content except for loss of arabinose by prolonged acid hydrolysis. The ferulic acid content was almost stable at about 0.8%. It was found that the enzymatically modified sugar beet pectins were more inhomogeneous due to the presence of acetyl groups hindering the attack by pectin esterases.

Rapid structural phenotyping of plant cell wall mutants by enzymatic oligosaccharide fingerprinting.

Various biochemical, chemical, and microspectroscopic methods have been developed throughout the years for the screening and identification of mutants with altered cell wall structure. However, these procedures fail to provide the insight into structural aspects of the cell wall polymers. In this paper, we present various methods for rapidly screening Arabidopsis cell wall mutants. The enzymatic fingerprinting procedures using high-performance anion-exchange-pulsed-amperometric detection liquid chromatography, fluorophore-assisted carbohydrate electrophoresis, and matrix-assisted laser-desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) were exemplified by the structural analysis of the hemicellulose xyloglucan. All three techniques are able to identify structural alterations of wall xyloglucans in mur1, mur2, and mur3, which in comparison with the wild type have side chain defects in their xyloglucan structure. The quickest analysis was provided by MALDI-TOF MS. Although MALDI-TOF MS per se is not quantitative, it is possible to reproducibly obtain relative abundance information of the various oligosaccharides present in the extract. The lack of absolute quantitation by MALDI-TOF MS was compensated for with a xyloglucan-specific endoglucanase and simple colorimetric assay. In view of the potential for mass screening using MALDI-TOF MS, a PERL-based program was developed to process the spectra obtained from MALDI-TOF MS automatically. Outliers can be identified very rapidly according to a set of defined parameters based on data collected from the wild-type plants. The methods presented here can easily be adopted for the analysis of other wall polysaccharides. MALDI-TOF MS offers a powerful tool to screen and identify cell wall mutants rapidly and efficiently and, more importantly, is able to give initial insights into the structural composition and/or modification that occurs in these mutants.