Hans Goesaert

Triticum aestivum L. endoxylanase inhibitor (TAXI) consists of two inhibitors, TAXI I and TAXI II, with different specificities.

The Triticum aestivum L. endoxylanase inhibitor (TAXI) discovered by Debyser and Delcour [(1997) Eur. Pat. filed April 1997, published as WO 98/49278] and Debyser, Derdelinckx and Delcour [(1997) J. Am. Soc. Brew. Chem. 55, 153-156] seems to be a mixture of two different endoxylanase inhibitors, called TAXI I and TAXI II. By using Aspergillus niger as well as Bacillus subtilis endoxylanases for assaying inhibition activity, both inhibitors could be purified to homogeneity from wheat (Triticum aestivum L., var. Soissons). TAXI I and TAXI II have similar molecular structures. They both have a molecular mass of approx. 40.0 kDa, are not glycosylated and occur in two molecular forms, i.e. a non-proteolytically processed one and a proteolytically processed one. However, the pI of TAXI II (at least 9.3) is higher than that of TAXI I (8.8). TAXI I and TAXI II clearly show different inhibition activities towards different endoxylanases. The N-terminal amino acid sequences of both inhibitors show a high degree of identity, which might indicate that there is an evolutionary relationship between them.

The role of gluten in a pound cake system: A model approach based on gluten-starch blends.

In order to evaluate the role of gluten in cake-making, gluten-starch (GS) blends with different ratios of gluten to starch were tested in a research pound cake formula. The viscosities of batters made from commercial GS blends in the otherwise standardised formula increased with their gluten content. High viscosities during heating provide the batters with the capacity to retain expanding air nuclei, and thereby led to desired product volumes. In line with the above, increasing gluten levels in the cake recipes led to a more extended oven spring period. Cakes with a starch content exceeding 92.5% in the GS blend suffered from substantial collapse during cooling. They had a coarse crumb with a solid gummy layer at the bottom. Image analysis showed statistical differences in numbers of cells per cm(2), cell to total area ratio and mean cell area (p<0.05). Both density and mean cell area were related to gluten level. Moreover, mean cell area and cell to total area ratio were the highest for cakes with the lowest density and highest gluten levels. Relative sodium dodecyl sulfate (SDS, 2.0%) buffer (pH 6.8) extractabilities of protein from cakes baked with the different GS blends decreased with gluten content and were strongly correlated with the intensity of collapse. Taken together, the results teach that protein gives the cakes resistance to collapse, resulting in desirable volumes and an optimal grain structure with uniform cell distribution.

TLXI, a novel type of xylanase inhibitor from wheat (Triticum aestivum) belonging to the thaumatin family.

Wheat (Triticum aestivum) contains a previously unknown type of xylanase (EC 3.2.1.8) inhibitor, which is described in the present paper for the first time. Based on its >60% similarity to TLPs (thaumatin-like proteins) and the fact that it contains the Prosite PS00316 thaumatin family signature, it is referred to as TLXI (thaumatin-like xylanase inhibitor). TLXI is a basic (pI> or =9.3 in isoelectric focusing) protein with a molecular mass of approx. 18-kDa (determined by SDS/PAGE) and it occurs in wheat with varying extents of glycosylation. The TLXI gene sequence encodes a 26-amino-acid signal sequence followed by a 151-amino-acid mature protein with a calculated molecular mass of 15.6-kDa and pI of 8.38. The mature TLXI protein was expressed successfully in Pichia pastoris, resulting in a 21-kDa (determined by SDS/PAGE) recombinant protein (rTLXI). Polyclonal antibodies raised against TLXI purified from wheat react with epitopes of rTLXI as well as with those of thaumatin, demonstrating high structural similarity between these three proteins. TLXI has a unique inhibition specificity. It is a non-competitive inhibitor of a number of glycoside hydrolase family 11 xylanases, but it is inactive towards glycoside hydrolase family 10 xylanases. Progress curves show that TLXI is a slow tight-binding inhibitor, with a K(i) of approx. 60-nM. Except for zeamatin, an alpha-amylase/trypsin inhibitor from maize (Zea mays), no other enzyme inhibitor is currently known among the TLPs. TLXI thus represents a novel type of inhibitor within this group of proteins.

Antifirming effects of starch degrading enzymes in bread crumb.

Antifirming properties of amylases in bread crumb were evaluated in straight dough breadmaking and related to the amylolytically modified starch structure. Amylase properties and action mechanisms determine starch structure in the breads and, hence, how amylopectin recrystallization, starch network formation, water redistribution, and water mobility occur during breadmaking and storage. A bacterial endo-alpha-amylase mainly hydrolyzed the longer starch polymer chains internally. It thus reduced the number of connections between the crystallites in the starch networks, resulting in a softer bread crumb. However, because the enzyme had only little impact on the outer amylopectin chains, amylopectin recrystallization and the concomitant water immobilization presumably were not hindered. The loss of plasticizing water as a result of recrystallization presumably reduces the flexibility of the gluten network and results in poor crumb resilience. In contrast, in breadmaking, the Bacillus stearothermophilus maltogenic alpha-amylase acted as an exoacting amylase with more pronounced endoaction at higher temperatures. This enzyme caused extensive degradation of the crystallizable amylopectin side chains and thus limited amylopectin recrystallization and network formation during storage. As a result, it prevented the incorporation of water in the amylopectin crystallites. In this way, the different starch and gluten networks kept their flexibility, resulting in a softer crumb with good resilience.

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

riticum estivum endo ylanase nhibitors (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.

XIP-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.

Purification and characterization of a XIP-type endoxylanase inhibitor from Rice (Oryza sativa)

A rice XIP-type inhibitor was purified by affinity chromatography with an immobilized Aspergillus aculeatus family 10 endoxylanase. Rice XIP is a monomeric protein, with a molecular mass of ca. 32 kDa and a pI of ca. 5.6. Its N-terminal amino acid sequence was identical to that of a rice chitinase homologue, demonstrating the difficulty when using sequence information to differentiate between endoxylanase inhibitors and (putative) chitinases in rice. Rice XIP inhibited different endoxylanases to a varying degree. In particular, it most strongly inhibited family 10 endoxylanases from A. niger and A. oryzae, while several family 11 enzymes from Bacillus subtilis, A. niger and Trichoderma sp. were not sensitive to inhibition. The above mentioned A. aculeatus endoxylanase was not inhibited either, although gel permeation chromatography revealed that it complexed rice XIP in a 1:1 molar stoichiometric ratio.

Endoxylanase Inhibition Activity in Different European Wheat Cultivars and Milling Fractions

ABSTRACT Twenty-three wheat samples from 19 different European wheat cultivars (Triticum aestivum L.) were tested for their quantitative and qualitative variation in inhibition activity against family 11 endoxylanases of Aspergillus niger, Bacillus subtilis, and Trichoderma viride and a family 10 endoxylanase of A. aculeatus. Under the experimental conditions, the A. aculeatus enzyme was not inhibited by the wheat extracts, the A. niger and B. subtilis endoxylanases were affected to a similar extent, while the T. viride enzyme was much more inhibited. The inhibition activities in the different wheat samples against the A. niger, B. subtilis, and T. viride endoxylanases varied between 36.0 and 11.7, 34.0 and 12.9, and 86.2 and 46.6 IU/100 mg of dry whole meal, respectively. One IU (inhibition unit) corresponds to the amount of inhibitor resulting in 50% inhibition of endoxylanase activity under the conditions of the assay. The inhibitor activities were linearly related, indicating that the levels of differ...

Purification and partial characterization of an endoxylanase inhibitor from Barley

ABSTRACT Hordeum vulgare L. xylanase inhibitor (HVXI), an endoxylanase inhibitor with a protein structure, was purified to homogeneity from barley (Hordeum vulgare L.). HVXI is a nonglycosylated monomeric protein, with a molecular weight of ≈40,000 and a pI ≥ 9.3. Although it inhibits different endoxylanases to a varying degree, the activities of an α-L-arabinofuranosidase and a β-d-xylosidase were not inhibited. Apparently, HVXI occurs in two molecular forms. These characteristics and the N-terminal sequences of the composing polypeptides show that HVXI is homologous with Triticum aestivum L. xylanase inhibitor I, an endoxylanase inhibitor from wheat flour.

Hydrolysis of amylopectin by amylolytic enzymes: structural analysis of the residual amylopectin population.

Amylopectin fine structures were studied following limited hydrolysis of gelatinised waxy maize starch by amylases with a different level of inner chain attack (LICA). This was done by size exclusion chromatography as well as by debranching the (partially hydrolysed) amylopectin samples and studying the size distributions of the released chains. Alpha-amylases from Bacillus amyloliquefaciens and Aspergillus oryzae, with a relatively high LICA, drastically altered amylopectin chain length distribution and reduced the amylopectin molecular size (MS) significantly even at a low to moderate degree of hydrolysis (DH). Porcine pancreatic alpha-amylase (PPA), with a rather low LICA but a high multiple attack action on amylose, reduced the amylopectin MS much slower. Following hydrolysis by PPA to a DH of 10% and enzymic debranching of the amylopectin residue, several subpopulations of chains consisting of 2-12 glucose units were detected, indicating a multiple attack action on the amylopectin side chains. During the early stages of hydrolysis, the maltogenic Bacillus stearothermophilus alpha-amylase (BStA) preferentially hydrolysed the exterior chains of amylopectin. However, during the later phases, BStA also hydrolysed inner chains, presumably with a high multiple attack action. The present results clearly show that different enzymes can be used for (limited) conversion of amylopectin into structures differing in molecular weight and chain length distributions.