Elucidating structure of pectin in ramie fiber to customize enzyme cocktail for high-efficiency enzymatic degumming.

Pectin is one of the main components of bast fiber including ramie fiber, and must be removed before use. Enzymatic degumming is the preferred process as it is an environment-friendly, simple and controllable process for ramie degumming. However, an important problem limiting wide application of this process is the high cost due to the low efficiency of enzymatic degumming. In this study, pectin samples were extracted from raw ramie fiber and degummed ramie fiber, respectively, and their structures were characterized and compared to allow tailoring of an enzyme cocktail for degrading the pectin. It was elucidated that pectin from ramie fiber is composed of low esterified homogalacturonan (HG) and low branched rhamnogalacturonan I (RG-I), and the ratio of HG/RG-I is 1.72:1. Based on the pectin structure, potential enzymes to be used for enzymatic degumming of ramie fiber were proposed and an enzyme cocktail was customized. Degumming experiments confirmed that the customized enzyme cocktail can effectively remove pectin from ramie fiber. To our knowledge, this is the first time the structural characteristics of pectin in ramie fiber have been clarified, and it also provides an example of tailoring a specific enzyme system to achieve high-efficiency degumming for biomass containing pectin.

The specificity of pectate lyase VdPelB from Verticilium dahliae is highlighted by structural, dynamical and biochemical characterizations.

Pectins, complex polysaccharides and major components of the plant primary cell wall, can be degraded by pectate lyases (PLs). PLs cleave glycosidic bonds of homogalacturonans (HG), the main pectic domain, by ß-elimination, releasing unsaturated oligogalacturonides (OGs). To understand the catalytic mechanism and structure/function of these enzymes, we characterized VdPelB from Verticillium dahliae. We first solved the crystal structure of VdPelB at 1.2 Å resolution showing that it is a right-handed parallel ß-helix structure. Molecular dynamics (MD) simulations further highlighted the dynamics of the enzyme in complex with substrates that vary in their degree of methylesterification, identifying amino acids involved in substrate binding and cleavage of non-methylesterified pectins. We then biochemically characterized wild type and mutated forms of VdPelB. Pectate lyase VdPelB was most active on non-methylesterified pectins, at pH 8.0 in presence of Ca2+ ions. The VdPelB-G125R mutant was most active at pH 9.0 and showed higher relative activity compared to native enzyme. The OGs released by VdPelB differed to that of previously characterized PLs, showing its peculiar specificity in relation to its structure. OGs released from Verticillium-partially tolerant and sensitive flax cultivars differed which could facilitate the identification VdPelB-mediated elicitors of defence responses.

Antimicrobial high molecular weight pectin polysaccharides production from diverse citrus peels using a novel PL10 family pectate lyase.

The discovery of environmentally friendly enzymes that can convert inexpensive and abundant citrus peel pectin into high value-added product is a potential avenue for the citrus peel application. In this study, a novel PL10-family pectate lyase (pelA) was characterized from marine bacterium Echinicola pacifica. PelA was a Ca2+ dependent pectate lyase whose activity was highest at pH 8 and 40 °C. It was capable of degrading polygalacturonic acid (PGA) and citrus peel pectin (CPP), but not apple peel pectin. Notably, PelA hydrolyzed PGA to high molecular weight polysaccharide (average molecular weight 111.4 kDa). Moreover, PelA was also able to degrade CPP from nine distinct citrus species into polysaccharides (average molecular weight ranging from 84.7 to 539.2 kDa) that showed antimicrobial activity against Staphylococcus epidermidis (88.8 %), Bacillus subtilis (99.8 %), Staphylococcus aureus (92.1 %), Escherichia coli (100.0 %) and Klebsiella pneumoniae (86.4 %). Considering the high market value of pectin in the food industry, PelA's capacity to convert citrus pectin into high molecular weight polysaccharides lays a foundation for its applications.

Biochemical Characterization of a Pectate Lyase AnPL9 from Aspergillus nidulans.

Pectinolytic enzymes have diverse industrial applications. Among these, pectate lyases act on the internal α-1,4-linkage of the pectate backbone, playing a critical role in pectin degradation. While most pectate lyases characterized thus far are of bacterial origin, fungi can also be excellent sources of pectinolytic enzymes. In this study, we performed biochemical characterization of the pectate lyase AnPL9 belonging to the polysaccharide lyase family 9 (PL9) from the filamentous fungus Aspergillus nidulans. Recombinant AnPL9 was produced using a Pichia pastoris expression system and purified. AnPL9 exhibited high activity on homogalacturonan (HG), pectin from citrus peel, pectin from apple, and the HG region in rhamnogalacturonan-I. Although digalacturonic acid and trigalacturonic acid were not degraded by AnPL9, tetragalacturonic acid was converted to 4,5-unsaturated digalacturonic acid and digalacturonic acid. These results indicate that AnPL9 degrades HG oligosaccharides with a degree of polymerization > 4. Furthermore, AnPL9 was stable within a neutral-to-alkaline pH range (pH 6.0-11.0). Our findings suggest that AnPL9 is a candidate pectate lyase for biotechnological applications in the food, paper, and textile industries. This is the first report on a fungal pectate lyase belonging to the PL9 family.

A Novel Alginate Lyase: Identification, Characterization, and Potential Application in Alginate Trisaccharide Preparation.

Alginate oligosaccharides (AOS) have many biological activities and significant applications in prebiotics, nutritional supplements, and plant growth development. Alginate lyases have unique advantages in the preparation of AOS. However, only a limited number of alginate lyases have been so far reported to have potentials in the preparation of AOS with specific degrees of polymerization. Here, an alginate-degrading strain Pseudoalteromonasarctica M9 was isolated from Sargassum, and five alginate lyases were predicted in its genome. These putative alginate lyases were expressed and their degradation products towards sodium alginate were analyzed. Among them, AlyM2 mainly generated trisaccharides, which accounted for 79.9% in the products. AlyM2 is a PL6 lyase with low sequence identity (≤28.3%) to the characterized alginate lyases and may adopt a distinct catalytic mechanism from the other PL6 alginate lyases based on sequence alignment. AlyM2 is a bifunctional endotype lyase, exhibiting the highest activity at 30 °C, pH 8.0, and 0.5 M NaCl. AlyM2 predominantly produces trisaccharides from homopolymeric M block (PM), homopolymeric G block (PG), or sodium alginate, with a trisaccharide production of 588.4 mg/g from sodium alginate, indicating its promising potential in preparing trisaccharides from these polysaccharides.

Biochemical characterization and immobilization of a novel pectate lyase ErPL2 for efficient preparation of pectin oligosaccharides.

Pectate lyase (ErPL2) from Echinicola rosea JL3085 showed maximal activity at 45 °C and pH 9.0 with 0.6 mM CaCl2. The Km and Vmax values of ErPL2 for polygalacturonic sodium were 2.098 mmol/L and 0.955 mmol/s, respectively. ErPL2 endolytically degraded pectic substances into oligosaccharides with degree of polymerization (DP) 1-5. To improve the thermostability and pH operation range, recombinant ErPL2 was immobilized onto mesoporous titanium oxide particles (MTOPs). MTOPs have abundant hydroxyl groups on the surface, which is a non-toxicity and good biocompatibility carrier. The residual enzyme activity of immobilized ErPL2 at 40 °C increased remarkably from 11% to 91% compared with free enzyme. The operable pH range was extended from 8-9 to 9-11. Surprisingly, the catalytic efficiency of immobilized ErPL2 was about 19 times higher than free enzyme. To our knowledge this is the first attempt of pectate lyase immobilized on MTOPs and it provides a new option for improving the catalytic performance.

Structure of an Alkaline Pectate Lyase and Rational Engineering with Improved Thermo-Alkaline Stability for Efficient Ramie Degumming.

Alkaline pectate lyases have biotechnological applications in plant fiber processing, such as ramie degumming. Previously, we characterized an alkaline pectate lyase from Bacillus clausii S10, named BacPelA, which showed potential for enzymatic ramie degumming because of its high cleavage activity toward methylated pectins in alkaline conditions. However, BacPelA displayed poor thermo-alkaline stability. Here, we report the 1.78 Å resolution crystal structure of BacPelA in apo form. The enzyme has the characteristic right-handed ß-helix fold of members of the polysaccharide lyase 1 family and shows overall structural similarity to them, but it displays some differences in the details of the secondary structure and Ca2+-binding site. On the basis of the structure, 10 sites located in flexible regions and showing high B-factor and positive ΔTm values were selected for mutation, aiming to improve the thermo-alkaline stability of the enzyme. Following site-directed saturation mutagenesis and screening, mutants A238C, R150G, and R216H showed an increase in the T5015 value at pH 10.0 of 3.0 °C, 6.5 °C, and 7.0 °C, respectively, compared with the wild-type enzyme, interestingly accompanied by a 24.5%, 46.6%, and 61.9% increase in activity. The combined mutant R150G/R216H/A238C showed an 8.5 °C increase in the T5015 value at pH 10.0, and an 86.1% increase in the specific activity at 60 °C, with approximately doubled catalytic efficiency, compared with the wild-type enzyme. Moreover, this mutant retained 86.2% activity after incubation in ramie degumming conditions (4 h, 60 °C, pH 10.0), compared with only 3.4% for wild-type BacPelA. The combined mutant increased the weight loss of ramie fibers in degumming by 30.2% compared with wild-type BacPelA. This work provides a thermo-alkaline stable, highly active pectate lyase with great potential for application in the textile industry, and also illustrates an effective strategy for rational design and improvement of pectate lyases.

Carbon catabolite repression in pectin digestion by the phytopathogen Dickeya dadantii.

The catabolism of pectin from plant cell walls plays a crucial role in the virulence of the phytopathogen Dickeya dadantii. In particular, the timely expression of pel genes encoding major pectate lyases is essential to circumvent the plant defense systems and induce massive pectinolytic activity during the maceration phase. Previous studies identified the role of a positive feedback loop specific to the pectin-degradation pathway, whereas the precise signals controlling the dynamics of pectate lyase expression were unclear. Here, we show that the latter is controlled by a metabolic switch involving both glucose and pectin. We measured the HPLC concentration profiles of the key metabolites related to these two sources of carbon, cAMP and 2-keto-3-deoxygluconate, and developed a dynamic and quantitative model of the process integrating the associated regulators, cAMP receptor protein and KdgR. The model describes the regulatory events occurring at the promoters of two major pel genes, pelE and pelD. It highlights that their activity is controlled by a mechanism of carbon catabolite repression, which directly controls the virulence of D. dadantii. The model also shows that quantitative differences in the binding properties of common regulators at these two promoters resulted in a qualitatively different role of pelD and pelE in the metabolic switch, and also likely in conditions of infection, justifying their evolutionary conservation as separate genes in this species.

Optimization of preparation conditions and in vitro sustained-release evaluation of a novel nanoemulsion encapsulating unsaturated guluronate oligosaccharide.

Unsaturated guluronate oligosaccharide (GOS) was prepared from alginate-derived homopolymeric blocks of guluronic acid by alginate lyase-mediated depolymerization. In this study, a GOS-based water-in-oil-in-water (W1/O/W2) nanoemulsion was prepared, and different influencing factors were investigated. First, linseed oil was selected as the optimal carrier oil. Then, other optimal conditions of the GOS nanoemulsion were determined based on response surface methodology (RSM). Under the optimal conditions, the obtained GOS nanoemulsion showed a spherical structure with an average particle size of 273.93 ±â€¯8.91 nm, and its centrifugal stability was 91.37 ±â€¯0.45 %. Moreover, the GOS nanoemulsion could achieve the aim of sustained release in vitro and be stably stored at 4°C for at least 5 days. This work prepared a novel GOS-based W1/O/W2 nanoemulsion that may effectively address the storage difficulties of unsaturated GOS and provides a valuable contribution to the application of GOS in the food and medicine fields.

Characterization and enhanced extracellular overexpression of a new salt-activated alginate lyase.

BACKGROUND: Alginate lyases (EC 4.4.2.3/4.4.2.11) have been applied to produce alginate oligosaccharides, which have physiological advantages such as prebiotic and antidiabetic effects, and are of benefit in the food and pharmaceutical industries. Extracellular production of recombinant proteins in Escherichia coli presents advantages including simplified downstream processing and high productivity; however, the presence of certain signal peptides does not always ensure successful secretion, which make the extracellular production of alginate lyase in E. coli rarely reported but of great significance. RESULTS: A PL7 family alginate lyase, Aly01, with its native signal peptide from Vibrio natriegens SK42.001, was identified, characterized, and extracellularly expressed in E. coli. The enzyme specifically released trisaccharide from alginate and was strictly NaCl activated. Green fluorescent protein (GFP) was fused with the Aly01 signal peptide and successfully secreted in E. coli to expand the feasibility of using this signal peptide to produce other heterologous proteins extracellularly. Through a synergistic strategy of utilizing Terrific Broth (TB) medium supplemented with 120 mmol L-1 glycine and 10 mmol L-1 calcium, the lag phase of protein secretion was reduced to 3 h from 12 h; meanwhile calcium remedied glycine-related cell growth impairment, leading to further enhancement of overall enzyme productivity, reaching a maximum of 4.55 U mL-1 . CONCLUSION: A new salt-activated alginate lyase, Aly01, was identified and characterized. E. coli employed its signal peptide and extracellularly expressed both Aly01 and a GFP, which indicated the signal peptide of Aly01 could be a powerful tool for extracellular production of other heterologous proteins in E. coli. © 2021 Society of Chemical Industry.

Purification and biochemical characterization of Hel a 6, a cross-reactive pectate lyase allergen from Sunflower (Helianthus annuus L.) pollen.

Sunflower pollen was reported to contain respiratory allergens responsible for occupational allergy and pollinosis. The present study describes the comprehensive characterization of a major sunflower allergen Hel a 6. Natural Hel a 6 was purified from sunflower pollen by anion exchange and gel filtration chromatography. Hel a 6 reacted with IgE-antibodies from 57% of 39 sunflower-sensitized patient sera suggesting it to be a major allergen. The patients were of Indian origin and suffering from pollinosis and allergic rhinitis. Hel a 6 exhibited allergenic activity by stimulating mediator release from basophils. Monomeric Hel a 6 displayed pectate lyase activity. The effect of various physicochemical parameters such as temperature, pH, and calcium ion on the functional activity of Hel a 6 revealed a stable nature of the protein. Hel a 6 was folded, and its melting curve showed reversible denaturation in which it refolded back to its native conformation from a denatured state. Hel a 6 displayed a high degree of sequence conservation with the pectate lyase allergens from related taxonomic families such as Amb a 1 (67%) and Art v 6 (57%). The IgE-cross reactivity was observed between Hel a 6 and its ragweed and mugwort homologs. The cross-reactivity was further substantiated by the mediator release when Hel a 6-sensitized effector cells were cross-stimulated with Art v 6 and Amb a 1. Several putative B cell epitopes were predicted and mapped on these 3 allergens. Two antigenic regions were found to be commonly shared by these 3 allergens, which could be crucial for cross-reactivity. In conclusion, Hel a 6 serves as a candidate molecule for diagnosis and immunotherapy for weed allergy.

Thermostable and alkalistable exopolygalacturonase of Bacillus pumilus dcsr1: Characteristics and applicability.

The bioactive form of thermostable and alkali stable pectinase of Bacillus pumilus dcsr1 is a homodimer of the molecular mass of 60 kDa with a pI of 4.6. The enzyme is optimally active at 50 °C and pH 10.5, and its Michaelis constant (Km), maximum rate of reaction (Vmax), activation energy (Ea), and temperature quotient (Q10) values (for citrus pectin) are 0.29 mg mL-1, 116 µmole mg-1 min-1, 74.73 KJmol-1 and 1.57, respectively. The enzyme has a shelf life of one and a half years at room temperature as well as 4 °C. The activity of the enzyme is stimulated by Mn2+ and Ca2+ and inhibited by Hg+, Cd2+, Co2+, Zn2+, Fe2+, Pb2+, EDTA and urea to a varied extent. The conformational studies of the enzyme revealed a high ß-sheet content in the bioactive dimer, and high α-helix in the inactive monomer. The Circular Dichroism (CD) spectra of the dimer in the presence of inhibitors suggested a marked decrease in ß-sheet, and a significant increase in α-helix, suggesting a key role of ß-sheets in the enzyme catalysis. Based on the end product analysis, the enzyme is an exopolygalacturonase with a unique ability of transglycosylation. When ramie fibers were treated with the enzyme, removal of gummy material (pectin) was visible, confirming its applicability in the degumming process.

Production pectin oligosaccharides using Humicola insolens Y1-derived unusual pectate lyase.

The economical production of pectin oligosaccharides with a specific degree of polymerization and structure from agro-food waste is an industrially important process. This study identified a novel pectate lyase gene (plhy1) from the thermophilic cellulolytic fungus H. insolens Y1 and tested its ability to produce pectin oligosaccharides. The recombinant PLHY1 produced in Pichia pastoris was superior to other similar enzymes due to its high thermal and pH stability. PLHY1 demonstrated optimal enzymatic activity at 55°C and pH 10.0 in the presence of 0.4 mM Ca2+, and preferred methyl esterified substrates for digestion. High performance anion exchange chromatography-pulsed amperometric detector and ultra high performance liquid chromatography in combination with electrospray ionization tandem mass spectrometry analysis showed that galacturonic acid-oligosaccharides with a small degree of polymerization (4-6) were the major hydrolysates produced by the degradation of apple peel pectin by PLHY1. The properties of PLHY1 make it valuable for application in the agro-food industry for the production of pectin oligosaccharides.

A surrogate structural platform informed by ancestral reconstruction and resurrection of a putative carbohydrate binding module hybrid illuminates the neofunctionalization of a pectate lyase.

Yersinia enterocolitica is a pectinolytic zoonotic foodborne pathogen, the genome of which contains pectin-binding proteins and several different classes of pectinases, including polysaccharide lyases (PLs) and an exopolygalacturonase. These proteins operate within a coordinated pathway to completely saccharify homogalacturonan (HG). Polysaccharide lyase family 2 (PL2) is divided into two major subfamilies that are broadly-associated with contrasting 'endolytic' (PL2A) or 'exolytic' (PL2B) activities on HG. In the Y. enterocolitica genome, the PL2A gene is adjacent to an independent carbohydrate binding module from family 32 (YeCBM32), which possesses a N-terminal secretion tag and is known to specifically bind HG. Independent CBMs are rare in nature and, most commonly, are fused to enzymes in order to potentiate catalysis. The unconventional gene architecture of YePL2A and YeCBM32, therefore, may represent an ancestral relic of a fission event that decoupled PL2A from its cognate CBM. To provide further insight into the evolution of this pectinolytic locus and the molecular basis of HG depolymerisation within Y. enterocolitica, we have resurrected a YePL2A-YeCBM32 chimera and demonstrated that the extant PL2A digests HG more efficiently. In addition, we have engineered a tryptophan from the active site of the exolytic YePL2B into YePL2A (YePL2A-K291W) and demonstrated, using X-ray crystallography of substrate complexes, that it is a structural determinant of exo-activity within the PL2 family. In this manner, surrogate structural platforms may assist in the study of phylogenetic relationships informed by extant and resurrected sequences, and can be used to overcome challenging structural problems within carbohydrate active enzyme families.

A novel and cost-effective methodology for enhanced production of industrially valuable alkaline xylano-pectinolytic enzymes cocktail in short solid-state fermentation cycle.

The aim of this study was to enhance the production of xylano-pectinolytic enzymes concurrently and also to reduce the fermentation period. In this study, the effect of agro-residues extract-based inoculum on yield and fermentation time of xylano-pectinolytic enzymes was studied. Microbial inoculum and fermentation media were supplemented with xylan and pectin polysaccharides derived from agro-based residues. Enzymes production parameters were optimized through two-stage statistical design approach. Under optimized conditions (temperature 37°C, pH 7.2, K2 HPO4 0.22%, MgSO4 0.1%, gram flour 5.6%, substrate: moisture ratio 1:2, inoculum size 20%, agro-based crude xylan in production media 0.45%, and agro-based crude xylan-pectin in inoculum 0.13%), nearly 28,255 ± 565 and 9,202 ± 193 IU of xylanase and pectinase, respectively, were obtained per gram of substrate in a time interval of 6 days only. The yield of both xylano-pectinolytic enzymes was enhanced along with a reduction of nearly 24 h in fermentation time in comparison with control, using polysaccharides extracted from agro-residues. The activity of different types of pectinase enzymes such as exo-polymethylgalacturonase (exo-PMG), endo-PMG, exo-polygalacturonase (exo-PG), endo-PG, pectin lyase, pectate lyase, and pectin esterase was obtained as 1,601, 12.13, 5637, 24.86, 118.62, 124.32, and 12.56 IU/g, respectively, and was nearly twofold higher than obtained for all seven types in control samples. This is the first report mentioning the methodology for enhanced production of xylano-pectinolytic enzymes in short solid-state fermentation cycle using agro-residues extract-based inoculum and production media.

Ginseng Extract Modified by Pectin Lyase Inhibits Retinal Vascular Injury and Blood-Retinal Barrier Breakage in a Rat Model of Diabetes.

GS-E3D is an enzymatically modified ginseng extract by pectin lyase. In this study, we evaluated the preventive effects of GS-E3D on blood-retinal barrier (BRB) leakage in a rat model of diabetes. To produce diabetes, rats were injected with streptozotocin. GS-E3D was orally gavaged at 25, 50, and 100 mg/kg body weight for 6 weeks. We then compared the effect of GS-E3D with that of an unmodified ginseng extract (UGE) on retinal vascular leakage. The administration of GS-E3D significantly blocked diabetes-induced BRB breakdown. Immunofluorescence staining showed that GS-E3D reduced the loss of occludin in diabetic rats. In TUNEL staining, the number of apoptotic retinal microvascular cells was dose dependently decreased by GS-E3D treatment. GS-E3D decreased the accumulations of advanced glycation end products in the retinal vessels. In addition, the inhibition potential of GS-E3D on BRB breakage was stronger compared with UGE. These results indicate that GS-E3D could be a beneficial treatment option for preventing diabetes-induced retinal vascular injury.

Flax rhamnogalacturonan lyases: phylogeny, differential expression and modeling of protein structure.

Rhamnogalacturonan lyases (RGLs; EC 4.2.2.23) degrade the rhamnogalacturonan I (RG-I) backbone of pectins present in the plant cell wall. These enzymes belong to polysaccharide lyase family 4, members of which are mainly from plants and plant pathogens. RGLs are investigated, as a rule, as pathogen 'weapons' for plant cell wall degradation and subsequent infection. Despite the presence of genes annotated as RGLs in plant genomes and the presence of substrates for enzyme activity in plant cells, evidence supporting the involvement of this enzyme in certain processes is limited. The differential expression of some RGL genes in flax (Linum usitatissimum L.) tissues, revealed in our previous work, prompted us to carry out a total revision (phylogenetic analysis, analysis of expression and protein structure modeling) of all the sequences of flax predicted as coding for RGLs. Comparison of the expressions of LusRGL in various tissues of flax stem revealed that LusRGLs belong to distinct phylogenetic clades, which correspond to two co-expression groups. One of these groups comprised LusRGL6-A and LusRGL6-B genes and was specifically upregulated in flax fibers during deposition of the tertiary cell wall, which has complex RG-I as a key noncellulosic component. The results of homology modeling and docking demonstrated that the topology of the LusRGL6-A catalytic site allowed binding to the RG-I ligand. These findings lead us to suggest the presence of RGL activity in planta and the involvement of special isoforms of RGLs in the modification of RG-I of the tertiary cell wall in plant fibers.

Screening of a Novel Polysaccharide Lyase Family 10 Pectate Lyase from Paenibacillus polymyxa KF-1: Cloning, Expression and Characterization.

Pectate lyase (EC 4.2.2.2) catalyzes the cleavage of α-1,4-glycosidic bonds of pectin polymers, and it has potential uses in the textile industry. In this study, a novel pectate lyase belonging to polysaccharide lyase family 10 was screened from the secreted enzyme extract of Paenibacillus polymyxa KF-1 and identified by liquid chromatography-MS/MS. The gene was cloned from P. polymyxa KF-1 genomic DNA and expressed in Escherichia coli. The recombinant enzyme PpPel10a had a predicted Mr of 45.2 kDa and pI of 9.41. Using polygalacturonic acid (PGA) as substrate, the optimal conditions for PpPel10a reaction were determined to be 50 °C and pH 9.0, respectively. The Km, vmax and kcat values of PpPel10a with PGA as substrate were 0.12 g/L, 289 µmol/min/mg, and 202.3 s-1, respectively. Recombinant PpPel10a degraded citrus pectin, producing unsaturated mono- and oligogalacturonic acids. PpPel10a reduced the viscosity of PGA, and weight loss of ramie (Boehmeria nivea) fibers was observed after treatment with the enzyme alone (22.5%) or the enzyme in combination with alkali (26.3%). This enzyme has potential for use in plant fiber processing.

Distinguishing allergens from non-allergenic homologues using Physical-Chemical Property (PCP) motifs.

Quantitative guidelines to distinguish allergenic proteins from related, but non-allergenic ones are urgently needed for regulatory agencies, biotech companies and physicians. In a previous study, we found that allergenic proteins populate a relatively small number of protein families, as characterized by the Pfam database. However, these families also contain non-allergenic proteins, meaning that allergenic determinants must lie within more discrete regions of the sequence. Thus, new methods are needed to discriminate allergenic proteins within those families. Physical-Chemical Properties (PCP)-motifs specific for allergens within a Pfam class were determined for 17 highly populated protein domains. A novel scoring method based on PCP-motifs that characterize known allergenic proteins within these families was developed, and validated for those domains. The motif scores distinguished sequences of allergens from a large selection of 80,000 randomly selected non-allergenic sequences. The motif scores for the birch pollen allergen (Bet v 1) family, which also contains related fruit and nut allergens, correlated better than global sequence similarities with clinically observed cross-reactivities among those allergens. Further, we demonstrated that the average scores of allergen specific motifs for allergenic profilins are significantly different from the scores of non-allergenic profilins. Several of the selective motifs coincide with experimentally determined IgE epitopes of allergenic profilins. The motifs also discriminated allergenic pectate lyases, including Jun a 1 from mountain cedar pollen, from similar proteins in the human microbiome, which can be assumed to be non-allergens. The latter lacked key motifs characteristic of the known allergens, some of which correlate with known IgE binding sites.

Crystal structure of exo-rhamnogalacturonan lyase from Penicillium chrysogenum as a member of polysaccharide lyase family 26.

Exo-rhamnogalacturonan lyase from Penicillium chrysogenum 31B (PcRGLX) was recently classified as a member of polysaccharide lyase (PL) family 26 along with hypothetical proteins derived from various organisms. In this study, we determined the crystal structure of PcRGLX as the first structure of a member of this family. Based on the substrate-binding orientation and substrate specificity, PcRGLX is an exo-type PL that cleaves rhamnogalacturonan from the reducing end. Analysis of PcRGLX-complex structures with reaction products indicate that the active site possesses an L-shaped cleft that can accommodate galactosyl side chains, suggesting side-chain-bypassing activity in PcRGLX. Furthermore, we determined the residues critical for catalysis by analyzing the enzyme activities of inactive variants.