Enzymatic Fingerprinting Research Articles

Fast insights into chitosan-cleaving enzymes by simultaneous analysis of polymers and oligomers through size exclusion chromatography

The thorough characterization of chitosan-cleaving enzymes is crucial to unveil structure–function relationships of this promising class of biomolecules for both, enzymatic fingerprinting analyses and to use the enzymes as biotechnological tools to produce tailor-made chitosans for diverse applications. Analyzing polymeric substrates as well as oligomeric products has been established as an effective way to understand the actions of enzymes, but it currently requires separate, rather laborious methods to obtain the full picture. Here, we present ultra high performance size exclusion chromatography coupled to refractive index and mass spectrometry detection (UHPSEC-RI-MS) as a straightforward method for the semi-quantitative analysis of chitosan oligomers of up to ten monomers in length. Additionally, the method allows to determine the average molecular weight of the remaining polymers and its distribution. By sampling live from an ongoing enzymatic reaction, UHPSEC-RI-MS offers the unique opportunity to analyze polymers and oligomers simultaneously—i.e., to monitor the molecular weight reduction of the polymeric substrate over the course of the digestion, while at the same time analyzing the emerging oligomeric products in a semi-quantitative manner. In this way, a single simple analysis yields detailed insights into an enzyme’s action on a given substrate.

Comprehensive structural characterization of water-soluble and water-insoluble homoexopolysaccharides from seven lactic acid bacteria

Several lactic acid bacteria are able to produce water-soluble and water-insoluble homoexopolysaccharides (HoEPS) from sucrose. In this study, structures of all HoEPS which were fermentatively produced by Leuconostoc mesenteroides subsp. dextranicum NRRL B-1121 and B-1144, Leuconostoc mesenteroides subsp. mesenteroides NRRL B-1149, B-1438 and B-1118, Leuconostoc suionicum DSM 20241, and Liquorilactobacillus satsumensis DSM 16230 were systematically analyzed. Monosaccharide analysis, methylation analysis, NMR spectroscopy, size-exclusion chromatography, and different enzymatic fingerprinting methods were used to obtain detailed structural information. All strains produced water-soluble dextrans and/or levans as well as water-insoluble glucans. Levans showed different degrees of branching and high molecular weights, whereas dextrans had comparable structures and broader size distributions. Fine structures of water-soluble HoEPS were analyzed after endo-dextranase and endo-levanase hydrolysis. Water-insoluble glucans were composed of different portions of 1,3-linkages (5 to 40 %). Hydrolysis with endo-dextranase and endo-mutanase yielded further information on block sizes and varying fine structures. Overall, clear differences between HoEPS yields and structures were observed.

A wide diversity exists in pectin structure from thirteen apple cultivars

To emphasize that differences in pectin structure among cultivars play a crucial role in the texture and quality of fruits and vegetables, the sugar content and methyl-esterification of pectin fractions from 13 apple cultivars was studied. Cell wall polysaccharides were isolated as alcohol-insoluble solids (AIS) and subsequently extracted to yield water-soluble solids (WSS) and chelating-soluble solids (ChSS). All fractions contained significant amounts of galacturonic acid, while sugar compositions varied between cultivars. AIS and WSS pectins showed a degree of methyl-esterification (DM) > 50 %, while ChSS pectins had either a medium (∼50 %) or low (<30 %) DM. Homogalacturonan as major structure was studied using enzymatic fingerprinting. Methyl-ester distribution of pectin was described by degrees of blockiness and -hydrolysis. Novel descriptive parameters were obtained by measuring the levels of methyl-esterified oligomers released by endo-PG (DBPGme) and PL (DBPLme). Pectin fractions differed in relative amounts of non-, moderately-, and highly methyl-esterified segments. WSS pectins were mostly lacking non-esterified GalA sequences, while ChSS pectins had medium DM and many non-methyl-esterified blocks or a low DM with many intermediate methyl-esterified GalA blocks. These findings will be of help to better understand physicochemical properties of apple and its products.

Cost-effective and controllable synthesis of isomalto/malto-polysaccharides from β-cyclodextrin by combined action of cyclodextrinase and 4,6-α-glucanotransferase GtfB

Isomalto/malto-polysaccharides (IMMPs) derived from malto-oligosaccharides such as maltoheptaose (G7) are elongated non-branched gluco-oligosaccharides produced by 4,6-α-glucanotransferase (GtfB). However, G7 is expensive and cumbersome to produce commercially. In this study, a cost-effective enzymatic process for IMMPs synthesis is developed that utilizes the combined action of cyclodextrinase from Palaeococcus pacificus (PpCD) and GtfB-ΔN from Limosilactobacillus reuteri 121 to convert β-cyclodextrin into IMMPs with a maximum yield (16.19 %, w/w). The purified IMMPs synthesized by simultaneous or sequential treatments, designated as IMMP-Sim and IMMP-Seq, possess relatively high contents of α-(1 → 6) glucosidic linkages. By controlling the release of G7 and smaller malto-oligosaccharides by PpCD, IMMP-Seq was obtained of DP varying from 12.9 to 29.5. Enzymatic fingerprinting revealed different linkage-type distribution of α-(1 → 6) linked segments with α-(1 → 4) segments embedded at the reducing end and middle part. The proportion of α-(1 → 6) segments containing the non-reducing end was 56.76 % for IMMP-Sim but 28.98 % for IMMP-Seq. Addition of G3 or G4 as specific acceptors resulted in IMMPs exhibiting low polydispersity. This procedure can be applied as a novel bioprocess that does not require costy high-purity malto-oligosaccharides and with control of the average DP of IMMPs by adjusting the substrate composition.

Combined FRET-FLIM and NAD(P)H FLIM to Analyze B Cell Receptor Signaling Induced Metabolic Activity of Germinal Center B Cells In Vivo.

Affinity maturation of B cell clones within germinal centers constitutes an important mechanism for immune memory. During this process, B cell receptor signaling capacity is tested in multiple rounds of positive selection. Antigen stimulation and co-stimulatory signals mobilize calcium to switch on gene expression leading to proliferation and survival and to differentiation into memory B cells and plasma cells. Additionally, all these processes require adaption of B cell metabolism, and calcium signaling and metabolic pathways are closely interlinked. Mitochondrial adaption, ROS production, and NADPH oxidase activation are involved in cell fate decisions, but it remains elusive to what extent, especially because the analysis of these dynamic processes in germinal centers has to take place in vivo. Here, we introduce a quantitative intravital imaging method for combined measurement of cytoplasmic calcium concentration and enzymatic fingerprinting in germinal center B cells as a possible tool in order to further examine the relationship of calcium signaling and immunometabolism.

TLR 2/1 interaction of pectin depends on its chemical structure and conformation

Citrus pectins have demonstrated health benefits through direct interaction with Toll-like receptor 2. Methyl-ester distribution patterns over the homogalacturonan were found to contribute to such immunomodulatory activity, therefore molecular interactions with TLR2 were studied. Molecular-docking analysis was performed using four GalA-heptamers, GalA7Me0, GalA7Me1,6, GalA7Me1,7 and GalA7Me2,5. The molecular relations were measured in various possible conformations. Furthermore, commercial citrus pectins were characterized by enzymatic fingerprinting using polygalacturonase and pectin-lyase to determine their methyl-ester distribution patterns. The response of 12 structurally different pectic polymers on TLR2 binding and the molecular docking with four pectic oligomers clearly demonstrated interactions with human-TLR2 in a structure-dependent way, where blocks of (non)methyl-esterified GalA were shown to inhibit TLR2/1 dimerization. Our results may be used to understand the immunomodulatory effects of certain pectins via TLR2. Knowledge of how pectins with certain methyl-ester distribution patterns bind to TLRs may lead to tailored pectins to prevent inflammation.

Enzymatic fingerprinting reveals specific xyloglucan and pectin signatures in the cell wall purified with primary plasmodesmata.

Plasmodesmata (PD) pores connect neighbouring plant cells and enable direct transport across the cell wall. Understanding the molecular composition of these structures is essential to address their formation and later dynamic regulation. Here we provide a biochemical characterisation of the cell wall co-purified with primary PD of Arabidopsis thaliana cell cultures. To achieve this result we combined subcellular fractionation, polysaccharide analyses and enzymatic fingerprinting approaches. Relative to the rest of the cell wall, specific patterns were observed in the PD fraction. Most xyloglucans, although possibly not abundant as a group, were fucosylated. Homogalacturonans displayed short methylated stretches while rhamnogalacturonan I species were remarkably abundant. Full rhamnogalacturonan II forms, highly methyl-acetylated, were also present. We additionally showed that these domains, compared to the broad wall, are less affected by wall modifying activities during a time interval of days. Overall, the protocol and the data presented here open new opportunities for the study of wall polysaccharides associated with PD.

Determining Methyl-Esterification Patterns in Plant-Derived Homogalacturonan Pectins.

Homogalacturonan (HG)-type pectins are nutrient components in plants and are widely used in the food industry. The methyl-esterification pattern is a crucial structural parameter used to assess HG pectins in terms of their nutraceutical activity. To better understand the methyl-esterification pattern of natural HG pectins from different plants, we purified twenty HG pectin-rich fractions from twelve plants and classified them by their monosaccharide composition, Fourier transform-infrared spectroscopy (FT-IR) signatures, and NMR analysis. FT-IR shows that these HG pectins are all minimally esterified, with the degree of methyl-esterification (DM) being 5 to 40%. To examine their methyl-esterification pattern by enzymatic fingerprinting, we hydrolyzed the HG pectins using endo-polygalacturonase. Hydrolyzed oligomers were derivatized with 2-aminobenzamide and subjected to liquid chromatography-fluorescence-tandem mass spectrometry (HILIC-FLR-MSn). Twenty-one types of mono-/oligo-galacturonides having DP values of 1–10 were found to contain nonesterified monomers, dimers, and trimers, as well as oligomers with 1 to 6 methyl-ester groups. In these oligo-galacturonides, MSn analysis demonstrated that the number of methyl-ester groups in the continuous sequence was 2 to 5. Mono- and di-esterified oligomers had higher percentages in total methyl-esterified groups, suggesting that these are a random methyl-esterification pattern in these HG pectins. Our study analyzes the characteristics of the methyl-esterification pattern in naturally occurring plant-derived HG pectins and findings that will be useful for further studying HG structure-function relationships.

Directed Mutation of Two Key Amino Acid Residues Alters the Product Structure of the New 4,6-α-Glucanotransferase from Bacillus sporothermodurans.

4,6-α-Glucanotransferases (4,6-α-GTs) convert amylose V into two types of differently structured products: a linear product connected by continuous α,1 → 6 bonds, such as isomalto/malto-polysaccharide (IMMP), and a highly branched product connected by alternating α,1 → 4 and α,1 → 6 bonds, such as reuteran-like polysaccharide (RLP). The synthesis process of 4,6-α-GT products is unclear, and exploring this process is significant for producing dietary fibers with potential applications. This study identified and expressed Geobacillus sp. 12AMOR1 GtfD-ΔC and Bacillus sporothermodurans GtfC-ΔC. After characterizing their products through 1H NMR and enzymatic fingerprinting, we found that GtfD-ΔC synthesized RLP with 29% α,1 → 6 bonds, and GtfC-ΔC synthesized IMMP with 71% α,1 → 6 bonds. The maltoheptaose incubation experiment showed different chain-length transfer patterns of two 4,6-α-GTs, GtfC-ΔC and GtfD-ΔC, transferring single and multiple glucose residues in each transglycosylation reaction, respectively. Site-directed mutagenesis confirmed that positions S345 and I347 influence the product structure of GtfC-ΔC, and the S345T/I347V mutation changed the GtfC-ΔC product to a linear product connected by alternating α,1 → 4 and α,1 → 6 bonds (pullulan-like polysaccharide) and altered the chain-length transfer pattern of GtfC-ΔC. We proposed that different chain-length transfer patterns between GtfD-ΔC and GtfC-ΔC may explain their differences in product structures. These findings are significant for obtaining the desired dietary fiber by engineering 4,6-α-GT.

Revealing methyl-esterification patterns of pectins by enzymatic fingerprinting: Beyond the degree of blockiness

Citrus pectins were studied by enzymatic fingerprinting using a simultaneous enzyme treatment with endo-polygalacturonase (endo-PG) from Kluyveromyces fragilis and pectin lyase (PL) from Aspergillus niger to reveal the methyl-ester distribution patterns over the pectin backbone. Using HILIC-MS combined with HPAEC enabled the separation and identification of the diagnostic oligomers released. Structural information on the pectins was provided by using novel descriptive parameters such as degree of blockiness of methyl-esterified oligomers by PG (DBPGme) and degree of blockiness of methyl-esterified oligomers by PL (DBPLme). This approach enabled us to clearly differentiate citrus pectins with various methyl-esterification patterns. The simultaneous use of PG and PL showed additional information, which is not revealed in digests using PG or PL alone. This approach can be valuable to differentiate pectins having the same DM and to get specific structural information on pectins and therefore to be able to better predict their physical and biochemical functionalities.

Structural study of a pectic polysaccharide fraction isolated from “mountain tea” (Sideritis scardica Griseb.)

The present study was conducted to investigate the structural characteristics of an acid-extracted polysaccharide fraction from mountain tea. The monosaccharide composition revealed that uronic acids (72.4 mol%) considerably predominated in the fraction, followed by smaller amounts of galactose (14.5 mol%) and glucose (6.2 mol%). The fraction contained mostly a highly methyl-esterified homogalacturonan (HG) – 71 mol%. The pectin had a high molecular weight population (∼60−100 kDa). Enzymatic fingerprinting was employed with a combination of HG degrading enzymes and LC-HILIC-MS, HPAEC, HPSEC to examine the structure in greater detail. Unsaturated oligomers released indicated the presence of large blocks of highly methyl-esterified GalA residues. Furthermore, the presence of blocks of non-esterified GalA residues and partly methyl-esterified and acetylated GalA residues in HG domain was demonstrated. The research findings provide a basis for further investigations regarding biological activity and commercial exploitation of mountain tea.

Structural, rheological and functional properties of galactose-rich pectic polysaccharide fraction from leek

An acid-extracted polysaccharide from alchohol-insoluble solids of leek was obtained. The sugar composition indicated that galactose and galacturonic acid were the major sugars, followed by small amounts of rhamnose and arabinose. The fraction contained a relatively high methyl-esterified homogalacturonan next to rhamnogalacturonan type I decorated with galactose-rich side chains. The fraction consisted of three high Mw populations, covering the range of 10–100 kDa. Enzymatic fingerprinting was performed with HG/RG-I degrading enzymes to elucidate the structure. The oligomers were analysed using LC-HILIC-MS, HPAEC, and MALDI-TOF MS. The data revealed the presence of GalA sequences, having different patterns of methyl-esterification, RG-I composed of unbranched segments and segments heavily substituted with β-(1→4)-linked galactan chains of varying length. The rheological study showed the shear-thinning, weak thixotropic, anti-thixotropic, and non-Newtonian behavior of the polysaccharide. The pectin exhibited higher water holding capacity than oil-holding capacity and the fraction did form stable foams at high concentration.

Challenging the putative structure of mannan in wheat (Triticum aestivum) endosperm

In wheat endosperm, mannan, is poorly documented. Nevertheless, this hemicellulosic polysaccharide might have a determinant role in wheat grain development since, in Arabidopsis thaliana, mutants with a reduced amount of mannan show an altered seed development. In order to gain knowledge about mannan in wheat, we have determined its biochemical structure in wheat endosperm where mannose content is about 0.2% (dry weight basis). We developed a method of enzymatic fingerprinting and isolated mannan-enriched fractions to decipher its fine structure. Although it is widely accepted that the class of mannan present in grass cell walls is glucomannan, our data indicate that, in wheat endosperm, this hemicellulose is only represented by short unsubstituted chains of 1,4 linked D-mannose residues and is slightly acetylated. Our study provides information regarding the interactions of mannan with other cell wall components and help to progress towards the understanding of monocot cell wall architecture and the mannan synthesis in wheat endosperm.

Molecular mechanism of Aspergillus fumigatus biofilm disruption by fungal and bacterial glycoside hydrolases

During infection, the fungal pathogen Aspergillus fumigatus forms biofilms that enhance its resistance to antimicrobials and host defenses. An integral component of the biofilm matrix is galactosaminogalactan (GAG), a cationic polymer of α-1,4-linked galactose and partially deacetylated N-acetylgalactosamine (GalNAc). Recent studies have shown that recombinant hydrolase domains from Sph3, an A. fumigatus glycoside hydrolase involved in GAG synthesis, and PelA, a multifunctional protein from Pseudomonas aeruginosa involved in Pel polysaccharide biosynthesis, can degrade GAG, disrupt A. fumigatus biofilms, and attenuate fungal virulence in a mouse model of invasive aspergillosis. The molecular mechanisms by which these enzymes disrupt biofilms have not been defined. We hypothesized that the hydrolase domains of Sph3 and PelA (Sph3h and PelAh, respectively) share structural and functional similarities given their ability to degrade GAG and disrupt A. fumigatus biofilms. MALDI-TOF enzymatic fingerprinting and NMR experiments revealed that both proteins are retaining endo-α-1,4-N-acetylgalactosaminidases with a minimal substrate size of seven residues. The crystal structure of PelAh was solved to 1.54 Å and structure alignment to Sph3h revealed that the enzymes share similar catalytic site residues. However, differences in the substrate-binding clefts result in distinct enzyme-substrate interactions. PelAh hydrolyzed partially deacetylated substrates better than Sph3h, a finding that agrees well with PelAh's highly electronegative binding cleft versus the neutral surface present in Sph3h Our insight into PelAh's structure and function necessitate the creation of a new glycoside hydrolase family, GH166, whose structural and mechanistic features, along with those of GH135 (Sph3), are reported here.

Enzymatic fingerprinting of isomalto/malto-polysaccharides

In this study, we present an enzymatic fingerprinting method for the characterization of isomalto/malto-polysaccharides (IMMPs). IMMPs are produced by the modification of starch with the 4,6-α-glucanotransferase (GTFB) enzyme and consist of α-(1→4), α-(1→6) and α-(1→4,6) linked glucoses. Enzymes were used separately, simultaneously or in successive order to specifically degrade and/or reveal IMMP substructures. The enzymatic digests were subsequently analysed with HPSEC and HPAEC to reveal the chain length distribution (CLD) of different IMMP substructures. The presence of amylose in the substrate resulted in the formation of linear α-(1→6) linked glycosidic chains (13.5 kDa) in the former amylopectin fraction. The length of these chains indicates that GTFB transferase activity on amylopectin is more likely to elongate single amylopectin chains than to provide an even distribution. Enzymatic fingerprinting also revealed that the GTFB enzyme is capable of introducing large (20 kDa) linear α-(1→6) linked glycosidic chains in the α-glucan substrate.

Endochitinase 1 (Tv-ECH1) from Trichoderma virens has high subsite specificities for acetylated units when acting on chitosans

Chitosans with defined characteristics have been shown to possess reproducible bioactivities for numerous applications. A promising approach for producing chitosans with defined degrees of polymerization (DP), degrees of acetylation (DA), and patterns of acetylation (PA) involves using chitin-modifying enzymes. One such enzyme, the chitinase Tv-ECH1 belonging to the glycoside hydrolase (GH) family 18, seems to have an important role in the biocontrol properties of the fungus Trichoderma virens, suggesting its potential in generating novel chitosans for plant health applications. In this study, the Tv-ECH1 enzyme was overexpressed in the methylotrophic yeast Pichia pastoris, yielding large amounts (up to 2mgmL−1) of purified recombinant enzyme of high activity, high purity, and high stability, making the system promising for industrial production of Tv-ECH1. The purified Tv-ECH1 chitinase displayed a wide optimal pH range from 4.5 to 6 and an optimal temperature of 37°C. Detailed subsite specificity analyses revealed high preference for acetylated residues at all four subsites analyzed (−2, −1, +1, +2), making Tv-ECH1 a promising candidate for the biotechnological production of specific chitosan oligomers and for the characterization of chitosan polymers via enzymatic fingerprinting.

A Cell Wall Proteome and Targeted Cell Wall Analyses Provide Novel Information on Hemicellulose Metabolism in Flax

Experimentally-generated (nanoLC-MS/MS) proteomic analyses of four different flax organs/tissues (inner-stem, outer-stem, leaves and roots) enriched in proteins from 3 different sub-compartments (soluble-, membrane-, and cell wall-proteins) was combined with publically available data on flax seed and whole-stem proteins to generate a flax protein database containing 2996 nonredundant total proteins. Subsequent multiple analyses (MapMan, CAZy, WallProtDB and expert curation) of this database were then used to identify a flax cell wall proteome consisting of 456 nonredundant proteins localized in the cell wall and/or associated with cell wall biosynthesis, remodeling and other cell wall related processes. Examination of the proteins present in different flax organs/tissues provided a detailed overview of cell wall metabolism and highlighted the importance of hemicellulose and pectin remodeling in stem tissues. Phylogenetic analyses of proteins in the cell wall proteome revealed an important paralogy in the class IIIA xyloglucan endo-transglycosylase/hydrolase (XTH) family associated with xyloglucan endo-hydrolase activity.Immunolocalisation, FT-IR microspectroscopy, and enzymatic fingerprinting indicated that flax fiber primary/S1 cell walls contained xyloglucans with typical substituted side chains as well as glucuronoxylans in much lower quantities. These results suggest a likely central role of xyloglucans and endotransglucosylase/hydrolase activity in flax fiber formation and cell wall remodeling processes.

Delignification outperforms alkaline extraction for xylan fingerprinting of oil palm empty fruit bunch

Enzyme hydrolysed (hemi-)celluloses from oil palm empty fruit bunches (EFBs) are a source for production of bio-fuels or chemicals. In this study, after either peracetic acid delignification or alkaline extraction, EFB hemicellulose structures were described, aided by xylanase hydrolysis. Delignification of EFB facilitated the hydrolysis of EFB-xylan by a pure endo-β-1,4-xylanase. Up to 91% (w/w) of the non-extracted xylan in the delignified EFB was hydrolysed compared to less than 4% (w/w) of that in untreated EFB. Alkaline extraction of EFB, without prior delignification, yielded only 50% of the xylan. The xylan obtained was hydrolysed only for 40% by the endo-xylanase used. Hence, delignification alone outperformed alkaline extraction as pretreatment for enzymatic fingerprinting of EFB xylans. From the analysis of the oligosaccharide-fingerprint of the delignified endo-xylanase hydrolysed EFB xylan, the structure was proposed as acetylated 4-O-methylglucuronoarabinoxylan.

Isolation and structure elucidation of pectic polysaccharide from rose hip fruits (Rosa canina L.)

A pectic polysaccharide from rose hip (RH) fruits has been obtained by extraction with 1% aqueous citric acid. It was found that the polysaccharide fraction mainly consisted of galacturonic acid (45.5%) next to galactose (5.5%) and arabinose (4.7%). RH pectin is having a relatively high degree of methylesterification (62%) and acetylation (10%) and consists of different molecular weight populations in the range of 10–100kDa. Enzymatic fingerprinting was performed using a combination of pectin lyase (PL) and endo-polygalacturonase. Detailed information about the structure and level of galacturonic acid oligomers released was obtained using LC-HILIC-MS/ELSD and HPAEC. Predominantly, unsaturated and methyl-esterified oligomers (DP 3–5) were released indicating that high proportions of methylesterified ‘PL degradable’ areas were present within the pectin. The data revealed that homogalacturonan is the main building block of the extracted pectin and consists of long methylesterified/acetylated GalA sequences interspersed with small blocks of non-methyl-esterified GalA units.

Structural Variation and Content of Arabinoxylans in Endosperm and Bran of Durum Wheat (Triticum turgidum L.).

Arabinoxylans are one group of dietary fiber components in cereal grains, and specific health benefits have been linked with their molecular fine structures and hence with physicochemical properties such as solubility in aqueous media. To characterize the fiber quality for functional foods, starchy endosperm and bran fractions from 11 durum wheat lines were analyzed for total and water-soluble arabinoxylans, (1,3;1,4)-β-glucan, and bound ferulic acid. The arabinoxylan contents ranged from 11 to 16.4% (w/w) in bran and from 1.5 to 1.8% in the starchy endosperm. Of the starchy endosperm arabinoxylans, 37% was soluble in water. No correlation was found between arabinoxylan content and bound ferulic acid in bran, although a relatively high level of this antioxidant was found in endosperm (38.3 μg/g endosperm flour). Enzymatic fingerprinting was performed to define the major fine structural features of arabinoxylans from both regions of the grain. Five major oligosaccharides released by xylanase hydrolysis were identified and characterized in the 11 durum lines. In addition, DP5, DP6, and DP7 oligosaccharides containing five, six, and seven pentosyl residues, respectively, were purified.