Biomedical subjects

Jan A Delcour

Publications and source records attributed to Jan A Delcour.

36 records · Page 2Linked to original sources

PubMed2004

Crystallization and preliminary X-ray diffraction study of two complexes of a TAXI-type xylanase inhibitor with glycoside hydrolase family 11 xylanases from Aspergillus niger and Bacillus subtilis.

Endo-beta-1,4-xylanases hydrolyze arabinoxylan, a major constituent of cereal cell walls, and are nowadays widely used in biotechnological processes. Purified complexes of family 11 xylanases from Aspergillus niger and Bacillus subtilis with TAXI I, a TAXI-type xylanase inhibitor from Triticum aestivum L., were prepared. In both cases the complex was crystallized using the hanging-drop vapour-diffusion method. The needle-like crystals of TAXI I in complex with A. niger xylanase belong to the trigonal space group P3(1) or P3(2), with unit-cell parameters a = b = 88.43, c = 128.99 A, and diffract to 1.8 A resolution. TAXI I in complex with B. subtilis xylanase crystallizes in the monoclinic space group C2, with a = 107.89, b = 95.33, c = 66.31 A, beta = 122.24 degrees. Complete data sets were collected for both crystal types using synchrotron radiation.

Aspergillus niger↗

PubMed2004

Properties of TAXI-type endoxylanase inhibitors.

Two types of proteinaceous endoxylanase inhibitors occur in different cereals, i.e. the TAXI [Triticum aestivum endoxylanase inhibitor]-type and XIP [endoxylanase inhibiting protein]-type inhibitors. The present paper focuses on the TAXI-type proteins and deals with their structural characteristics and the identification, characterisation and heterologous expression of a TAXI gene from wheat. In addition, to shed light on the mechanism by which TAXI-type endoxylanase inhibitors work, the enzyme specificity, the optimal conditions for maximal inhibition activity, the molar complexation ratio and the inhibition kinetics of the inhibitors are explained and the effect of mutations of an endoxylanase on the inhibition by TAXIs is discussed.

Amino Acid Sequence↗

PubMed2004

Occurrence of proteinaceous endoxylanase inhibitors in cereals.

Cereals contain proteinaceous inhibitors of endoxylanases, which affect the efficiency and functionality of these enzymes in cereal processing. This review relates their first discovery in wheat and the subsequent purification of two distinct classes of endoxylanase inhibitors, namely Triticum aestivum xylanase inhibitor (TAXI)-type and xylanase inhibitor protein (XIP)-type inhibitors in cereals. Both inhibitor classes occur in monocots as multi-isoform families. The reported data provide an overview of the relative quantitative and qualitative variation of these inhibitors in cereals. Wheat and rye are particularly rich in TAXI-type and XIP-type inhibitors with the latter inhibitors being more abundant. Lower inhibitor levels are present in durum wheat and barley, while maize contains solely XIP-type inhibitors. No inhibitors have been isolated from rice, oats and buckwheat.

Bacteria↗

PubMed2004

Potential physiological role of plant glycosidase inhibitors.

Carbohydrate-active enzymes including glycosidases, transglycosidases, glycosyltransferases, polysaccharide lyases and carbohydrate esterases are responsible for the enzymatic processing of carbohydrates in plants. A number of carbohydrate-active enzymes are produced by microbial pathogens and insects responsible of severe crop losses. Plants have evolved proteinaceous inhibitors to modulate the activity of several of these enzymes. The continuing discovery of new inhibitors indicates that this research area is still unexplored and may lead to new exciting developments. To date, the role of the inhibitors is not completely understood. Here we review recent results obtained on the best characterised inhibitors, pointing to their possible biological role in vivo. Results recently obtained with plant transformation technology indicate that this class of inhibitors has potential biotechnological applications.

Animals↗

PubMed2004

High-level expression, purification, and characterization of recombinant wheat xylanase inhibitor TAXI-I secreted by the yeast Pichia pastoris.

Triticum aestivum xylanase inhibitor I (TAXI-I) is a wheat protein that inhibits microbial xylanases belonging to glycoside hydrolase family 11. In the present study, recombinant TAXI-I (rTAXI-I) was successfully produced by the methylotrophic yeast Pichia pastoris at high expression levels (approximately 75 mg/L). The rTAXI-I protein was purified from the P. pastoris culture medium using cation exchange and gel filtration chromatographic steps. rTAXI-I has an iso-electric point of at least 9.3 and a mass spectrometry molecular mass of 42,013 Da indicative of one N-linked glycosylation. The recombinant protein fold was confirmed by circular dichroism spectroscopy. Xylanase inhibition by rTAXI-I was optimal at 20-30 degrees C and at pH 5.0. rTAXI-I still showed xylanase inhibition activity at 30 degrees C after a 40 min pre-incubation step at temperatures between 4 and 70 degrees C and after 2 h pre-incubation at room temperature at a pH ranging from 3.0 to 12.0, respectively. All tested glycoside hydrolase family 11 xylanases were inhibited by rTAXI-I whereas those belonging to family 10 were not. Specific inhibition activities against family 11 Aspergillus niger and Bacillus subtilis xylanases were 3570 and 2940IU/mg protein, respectively. The obtained biochemical characteristics of rTAXI-I produced by P. pastoris (no proteolytical cleft) were similar to those of natural TAXI-I (mixture of proteolytically processed and non-processed forms) and non-glycosylated rTAXI-I expressed in Escherichia coli. The present results show that xylanase inhibition activity of TAXI-I is only affected to a limited degree by its glycosylation or proteolytic processing.

Amino Acid Sequence↗

PubMed2003

Impact of xylanases with different substrate selectivity on gluten-starch separation of wheat flour.

The influence on wheat flour gluten-starch separation of a xylanase from Aspergillus aculeatus (XAA) with hydrolysis selectivity toward water extractable arabinoxylan (WE-AX) and that is not inhibited by wheat flour xylanase inhibitors was compared to that of a xylanase from Bacillus subtilis (XBS) with hydrolysis selectivity toward water unextractable arabinoxylan (WU-AX) and that is inhibited by such inhibitors. XAA improved gluten agglomeration through degradation of WE-AX and concomitant reduction in viscosity, which in the laboratory scale batter procedure with a set of vibrating sieves (400, 250, and 125 microm), increased protein recoveries on the 400 microm sieve. In contrast, XBS had a negative effect as it decreased gluten protein recovery on this sieve, probably as a result of the viscosity increase that accompanied WU-AX solubilization. Hence, it was active even if most likely a considerable part of its activity was prevented by xylanase inhibitors. A combination of XAA and XBS at a low dosage yielded a distribution of gluten proteins on the different sieves comparable to that of the control. At a high combined dosage, the gluten agglomeration was better than that with XAA alone, indicating that both WE-AX and WU-AX have a negative impact on gluten agglomeration. Finally, experiments with endoxylanase addition at different moments during the separation process suggest that the status of the arabinoxylan population during dough mixing is far less critical for its impact on gluten agglomeration than that during the batter phase.

Aspergillus↗

PubMed2003

A screening method for endo-beta-1,4-xylanase substrate selectivity.

Endoxylanase (EC 3.2.1.8) substrate selectivity, i.e., its relative activity toward water-unextractable arabinoxylan (WU-AX) and water-extractable arabinoxylan (WE-AX) substrates, is important for its functionality in biotechnological processes such as bread-making and gluten starch separation. A screening method for rapidly determining said substrate selectivity was developed. Endoxylanase activity toward WU-AX was estimated by incubation of insoluble chromogenic substrate with a range of enzyme concentrations in microtiter plates, followed by colorimetric measurement of the dye released in the supernatant. A similar approach using soluble substrate and ethanol precipitation of unhydrolysed AX fragments was used to estimate enzyme activity toward WE-AX. A substrate selectivity factor was defined as the ratio of enzyme activity toward insoluble substrate over enzyme activity toward soluble substrate. A Bacillus subtilis and an Aspergillus aculeatus endoxylanase, known to have widely varying relative rates of hydrolysis of WU-AX and WE-AX, varied most in their substrate selectivity, while the endoxylanases of Aspergillus niger, Trichoderma longibrachiatum, and Trichoderma viride displayed intermediate such relative activities.

Aspergillus↗

PubMed2003

Structural features of arabinoxylans extracted with water at different temperatures from two rye flours of diverse breadmaking quality.

The water extractable (WE) arabinoxylans from two rye flours differing in baking quality were studied following sequential extraction with water at 4, 40, and 100 degrees C. Ammonium sulfate fractionation of the resulting WE fractions and subsequent analysis revealed substantial differences in the structure of the isolated materials. Furthermore, it allowed us to identify the factors contributing to arabinoxylan water extractability. Our results provide compelling evidence for the existence of separate polymers in rye WE arabinoxylans with different substitution degrees, ranging from quantitatively dominating, lowly substituted populations (arabinose to xylose ratio, Ara/Xyl approximately 0.5) to comparatively less abundant, highly substituted analogues (Ara/Xyl approximately 1.3). Generally, arabinoxylan water extractability was governed by the relative proportion of lowly and highly branched structures. A gradually increasing proportion of highly substituted populations was observed from cold to hot WE fractions. This was associated with the lower proportion of monosubstituted xylopyranosyl residues in the backbone, the higher proportion of disubstituted xylopyranosyl residues, and the higher level of substitution with feruloyl residues. Notable differences in the ratio of phenolic compounds to arabinose residues were observed between corresponding polymers isolated from rye flours of high and low baking quality, whereas the differences in their molecular weights were much less pronounced.

Arabinose↗

PubMed2003

Refrigerated dough syruping in relation to the arabinoxylan population.

Refrigerated doughs develop syruping upon prolonged storage. To assess the role of arabinoxylans (AX), in this phenomenon, the evolution of the AX population and syruping in refrigerated doughs during storage were studied. When doughs were kept at 6 degrees C for up to 34 days of storage, dough syruping increased from 0% (fresh dough) to 22% of dough weight, reaching a plateau after 16 days of storage. High-performance size exclusion chromatography and gas-liquid chromatography showed hydrolysis of water-unextractable AX in the refrigerated dough, resulting in increased levels of solubilized AX in the first 2 days of storage. Longer storage resulted in further degradation of solubilized and water-extractable AX. Increased syruping was accompanied by a decrease in farinograph dough consistency. The results support the hypothesis that loss of water-holding capacity due to degradation of AX by endogenous xylanases is responsible for dough syruping.

Bread↗

PubMed2003

TAXI type endoxylanase inhibitors in different cereals.

An affinity-based purification procedure with the immobilized family 11 Bacillus subtilis endoxylanase XynA allowed us to obtain high yields of highly pure endoxylanase inhibitor fractions from rye, barley, and durum wheat. In contrast, no inhibitors interacting with the B. subtilis endoxylanase affinity column are present in corn, buckwheat, rice, and oats. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and inhibitor specificity showed that the isolated inhibitors belonged to the TAXI endoxylanase inhibitor family, thus providing a view on the diversity of this cereal inhibitor family. The isolated inhibitors are basic proteins of ca. 40 kDa, occurring in two molecular forms, with pI values of ca. 8.5 (durum wheat) and ca. 9.0 (rye, barley). They are, in general, strong inhibitors of family 11 endoxylanases but not of family 10 endoxylanases. Because cereal endogenous endoxylanases belong to the latter family, this probably indicates that they do not influence cereal metabolic processes. For the first time, endoxylanase inhibitors, similar to TAXI I and TAXI II from wheat, were isolated from durum wheat and characterized. For each cereal, high-resolution cation exchange chromatography revealed the presence of multiple isoinhibitors, each of which occurs in two molecular forms. However, in durum wheat and barley, a single isoform is predominantly present.

Bacillus subtilis↗

PubMed2003

Fractionation of starch hydrolysates into dextrins with narrow molecular mass distribution and their detection by high-performance anion-exchange chromatography with pulsed amperometric detection.

Low levels of high-molecular-mass dextrins in starch hydrolysates can be detected by high-performance anion-exchange chromatography with pulsed amperometric detection in spite of their low responses by dialysis of the starch hydrolysate and fractionation of the resulting adialysate with ethanol (final concentration 30-80% at 6 degrees C). In doing so, dextrin fractions with a relatively narrow molecular mass distribution were obtained.

Anion Exchange Resins↗

PubMed2003

Molecular identification of wheat endoxylanase inhibitor TAXI-I1, member of a new class of plant proteins.

Triticum aestivum endoxylanase inhibitors (TAXIs) are wheat proteins that inhibit family 11 endoxylanases commonly used in different (bio)technological processes. Here, we report on the identification of the TAXI-I gene which encodes a mature protein of 381 amino acids with a calculated molecular mass of 38.8 kDa. When expressed in Escherichia coli, the recombinant protein had the specificity and inhibitory activity of natural TAXI-I, providing conclusive evidence that the isolated gene encodes an endoxylanase inhibitor. Bioinformatical analysis indicated that no conserved domains nor motifs common to other known proteins are present. Sequence analysis revealed similarity with a glycoprotein of carrot and with gene families in Arabidopsis thaliana and rice, all with unknown functions. Our data indicate that TAXI-I belongs to a newly identified class of plant proteins for which a molecular function as glycoside hydrolase inhibitor can now be suggested.

Amino Acid Sequence↗

PubMed2003

Crystallization and preliminary X-ray diffraction study of a wheat (Triticum aestivum L.) TAXI-type endoxylanase inhibitor.

A TAXI-type endoxylanase inhibitor from T. aestivum L. wheat flour has been crystallized using the hanging-drop vapour-diffusion method. The needle-like crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 49.92, b = 66.45, c = 106.42 A. From these crystals, a native data set and a gold-derivative data set were collected to 2.25 and 1.75 A resolution, respectively. The heavy-atom derivative of this crystal form was obtained by the soaking method and allowed determination of the initial phases.

Crystallization↗

PubMed2002

Wheat protein composition and properties of wheat glutenin in relation to breadmaking functionality.

The unique breadmaking properties of wheat are generally ascribed to the visco-elastic properties of its gluten proteins. While monomeric gluten proteins (gliadin) show viscous behavior, polymeric gluten proteins (glutenin) are elastic. The unique elasticity of glutenin results to a large extent from its polymeric nature. Glutenin is a highly heterogeneous mixture of polymers consisting of a number of different high- and low-molecular-weight glutenin subunits linked by disulfide bonds. Although glutenin obviously is the major polymeric protein in wheat, other polymeric proteins occur as well. Their importance in breadmaking may be underestimated. Nevertheless, variations in both quantity and quality of glutenin strongly determine variations in breadmaking performance. Structural features of different classes of glutenin subunits are described. Variations in glutenin quality may result from variations in its (1) structure, (2) size distribution, and (3) subunit composition. Some hypotheses on glutenin structure and current insights into the role of glutenin size distribution are evaluated. Finally, different ways in which variation in glutenin composition may directly or indirectly (by affecting glutenin structure and/or size distribution) influence glutenin quality are discussed.

Bread↗

PubMed2002

Purification of TAXI-like endoxylanase inhibitors from wheat (Triticum aestivum L.) whole meal reveals a family of iso-forms.

An affinity chromatography method has been developed for purification of endoxylanase inhibitors concentrated by cation exchange chromatography from wheat whole meal and is based on immobilisation of a Bacillus subtilis family 11 endoxylanase on N-hydroxysuccinimide activated Sepharose 4 Fast Flow. When followed by high-resolution cation exchange chromatography, the purification of seven TAXIs, Triticum aestivum L. endoxylanase inhibitors was achieved so extending the number of such proteins known to date (TAXI I and II). Based on their inhibition activities against a B. subtilis family 11 and an Aspergillus niger family 11 endoxylanase, six TAXI I- and only one TAXI II-like inhibitor could be distinguished. The first type of endoxylanase inhibitor is active against both endoxylanases and the second type only has significant activity against the B. subtilis endoxylanase.

Bacillus subtilis↗

PubMedDate not supplied

Designing new materials from wheat protein.

We recently discovered that wheat gluten could be formed into a tough, plasticlike substance when thiol-terminated, star-branched molecules are incorporated directly into the protein structure. This discovery offers the exciting possibility of developing biodegradable high-performance engineering plastics and composites from renewable resources that are competitive with their synthetic counterparts. Wheat gluten powder is available at a cost of less than dollars 0.5/lb, so if processing costs can be controlled, an inexpensive alternative to synthetic polymers may be possible. In the present work, we demonstrate the ability to toughen an otherwise brittle protein-based material by increasing the yield stress and strain-to-failure, without compromising stiffness. Water absorption results suggest that the cross-link density of the polymer is increased by the presence of the thiol-terminated, star-branched additive in the protein. Size-exclusion high performance liquid chromatography data of molded tri-thiol-modified gluten are consistent with that of a polymer that has been further cross-linked when compared directly with unmodified gluten, handled under identical conditions. Remarkably, the mechanical properties of our gluten formulations stored in ambient conditions were found to improve with time.

Absorption↗

PubMedDate not supplied

Amylopectin molecular structure reflected in macromolecular organization of granular starch.

For lintners with negligible amylose retrogradation, crystallinity related inversely to starch amylose content and, irrespective of starch source, incomplete removal of amorphous material was shown. The latter was more pronounced for B-type than for A-type starches. The two predominant lintner populations, with modal degrees of polymerization (DP) of 13-15 and 23-27, were best resolved for amylose-deficient and A-type starches. Results indicate a more specific hydrolysis of amorphous lamellae in such starches. Small-angle X-ray scattering showed a more intense 9-nm scattering peak for native amylose-deficient A-type starches than for their regular or B-type analogues. The experimental evidence indicates a lower contrasting density within the "crystalline" shells of the latter starches. A higher density in the amorphous lamellae, envisaged by the lamellar helical model, explains the relative acid resistance of linear amylopectin chains with DP > 20, observed in lintners of B-type starches. Because amylopectin chain length distributions were similar for regular and amylose-deficient starches of the same crystal type, we deduce that the more dense (and ordered) packing of double helices into lamellar structures in amylose-deficient starches is due to a different amylopectin branching pattern.

Amylopectin↗

PubMedDate not supplied

Potato phosphorylase catalyzed synthesis of amylose-lipid complexes.

On-line size-exclusion chromatography monitoring of potato phosphorylase catalyzed amylose synthesis--starting from alpha-D-glucose-1-P and maltohexaose--revealed rather monodisperse amylose populations. In the presence of lipids, amylose-lipid complexes spontaneously formed and precipitated. They were recovered by centrifugation, freeze-dried, and characterized by wide-angle X-ray diffraction and differential scanning calorimetry. The presence of lipids during amylose synthesis led to lower amylose degrees of polymerization (DP). Lipid chain length defined amylose DP, which increased in the order myristic acid (C14), glyceryl monostearate (GMS), stearic acid (C18), and docosanoic acid (C22). The thermal stability of the complexes increased in the same manner, with the C22 complexes having the highest dissociation temperature. In addition, we hypothesized that these results provide additional evidence for the fringed micellar organization of (semi-enzymically synthesized) amylose-lipid complexes.

Amylose↗