Molecular identification of wheat endoxylanase inhibitor TAXI-I11The nucleotide sequence reported in this paper is available at the EMBL/GenBank/DDBJ databases (accession number AJ438880)., member of a new class of plant proteins

AbstractTriticum 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

Residual amylopectin structures of amylase-treated wheat starch slurries reflect amylase mode of action

Some amylases can delay bread staling and/or starch (amylopectin) retrogradation, but the molecular basis of this effect remains little understood. In order to increase our insight in these aspects of amylase functionality, several amylases were added in a pure wheat-starch-containing model system and subjected to a heating step corresponding to that in the baking phase in bread making. Next, the effects of the limited amylolytic degradation on the rapid visco analyser (RVA) rheological properties of starch were studied and the accompanying changes in the amylopectin molecular properties (such as chain length distribution) investigated. The different amylases clearly affected the molecular structure of amylopectin to a different extent, which could be related to their mode of action and the enzyme activity levels added. Bacillus subtilis and Aspergillus oryzae alpha-amylases had only a limited impact on the side chain distribution of the amylopectin molecules, presumably due to their preferential hydrolysis of internal chain segments and the low enzyme activity added in the RVA. In contrast, porcine pancreatic alpha-amylase and Bacillus stearothermophilus maltogenic alpha-amylase, both with higher degree of multiple attack and used at higher enzyme activity levels, had a marked influence on the amylopectin molecular structure. More in particular, under the test conditions, the maltogenic a-amylase reduced the average chain length of the outer chains by 50%. Presumably, this will affect amylopectin retrogradation to a large extent. The results contribute to a better understanding of amylase functionality in starchy foods. (C) 2007 Elsevier Ltd. All rights reserved.status: publishe

Model approach to starch functionality in bread making

We used modified wheat starches in gluten-starch flour models to study the role of starch in bread making. Incorporation of hydroxypropylated starch in the recipe reduced loaf volume and initial crumb firmness and increased crumb gas cell size. Firming rate and firmness after storage increased for loaves containing the least hydroxypropylated starch. Inclusion of cross-linked starch had little effect on loaf volume or crumb structure but increased crumb firmness. The firming rate was mostly similar to that of control samples. Presumably, the moment and extent of starch gelatinization and the concomitant water migration influence the structure formation during baking. Initial bread firmness seems determined by the rigidity of the gelatinized granules and leached amylose. Amylopectin retrogradation and strengthening of a long-range network by intensifying the inter- and intramolecular starch-starch and possibly also starch-gluten interactions (presumably because of water incorporation in retrograded amylopectin crystallites) play an important role in firming.status: publishe

Representations of facial identity information in the ventral visual stream investigated with multivoxel pattern analyses

The neural basis of face recognition has been investigated extensively. Using fMRI, several regions have been identified in the human ventral visual stream that seem to be involved in processing and identifying faces, but the nature of the face representations in these regions is not well known. In particular, multivoxel pattern analyses have revealed distributed maps within these regions, but did not reveal the organizing principles of these maps. Here we isolated different types of perceptual and conceptual face properties to determine which properties are mapped in which regions. A set of faces was created with systematic manipulations of featural and configural visual characteristics. In a second part of the study, personal and spatial context information was added to all faces except one. The perceptual properties of faces were represented in face regions and in other regions of interest such as early visual and object-selective cortex. Only representations in early visual cortex were correlated with pixel-based similarities between the stimuli. The representation of nonperceptual properties was less distributed. In particular, the spatial location associated with a face was only represented in the parahippocampal place area. These findings demonstrate a relatively distributed representation of perceptual and conceptual face properties that involves both face-selective/sensitive and non-face-selective cortical regions.status: publishe

Amylase action pattern on starch polymers

Several decades ago, the first reports on differences in action pattern between amylases from different sources indicated that the starch polymers are not degraded in a completely random manner. We here give an overview of different action patterns of amylases on amylose and amylopectin, focusing on the so-called multiple attack action of the enzymes. Nowadays, the multiple attack action is generally an accepted concept to explain the differences in amylase action pattern. However, the pancreatic alpha-amylase remains one of the few enzymes known with a considerable level of multiple attack action. Despite some recent studies, the molecular mechanism of the multiple attack action is still largely unclear. Probably, the degree to which the active site architecture and binding properties allow both the reorganization (sliding) of the substrate in the active site and the stabilisation of the productive enzyme/substrate complex mainly determine the multiple attack action of amylases.status: publishe

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. (C) 2009 Elsevier Ltd. All rights reserved.status: publishe

Hydrolysis of amylopectin by amylolytic enzymes: level of inner chain attack as an important analytical differentiation criterion

Differences in amylase action pattern on amylopectin were demonstrated by the relation between the decrease in potassium iodide-iodine binding of waxy maize starch and the increase in reducing value during hydrolysis, as expressed by the RV80 value (i.e., the reducing value for a potassium iodide-iodine binding value of 80% of that of the starting material). In the initial stages of the hydrolysis, the ratio of the increase in the level of reducing polysaccharicles to the increase in the total level of reducing sugars formed during amylolysis of amylopectin can be considered as a measure of the level of inner chain attack (LICA) in the overall hydrolysis of the amylopectin structure and correlated with the respective RV80 value. Bacillus amyloliquefaciens alpha-amylase and Aspergillus oryzae alpha-amylase, with the lowest RV80 and the highest LICA values, hydrolysed the inner chains of amylopectin to a greater extent than did porcine pancreatic alpha-amylase. In the initial stages of hydrolysis, Bacillus stearothermophilus maltogenic amylase, like the Bacillus cereus beta-amylase, did not display any significant degree of internal hydrolysis of amylopectin, in line with the high RV80 and very low LICA values for these enzymes. However, at the later stages of hydrolysis, the maltogenic amylase probably exhibited a significant degree of internal hydrolysis of amylopectin, which itself seems to depend on temperature. The temperature dependence of the hydrolysis pattern of this enzyme is relevant for interpretation of its action as antifirming enzyme in bread-making applications. (C) 2009 Elsevier Ltd. All rights reserved.status: publishe

Amylases and bread firming - an integrated view

Despite much research, bread crumb firming during storage and amylase anti-staling properties are still ill understood. We present a coherent view on the topic based on literature, experimental data, and food polymer science-related concepts. During bread storage, the gelatinised starch (amylopectin) network, present in soft, fresh bread, is gradually transformed into an extensive, partially crystalline, permanent amylopectin network, with amylopectin crystallites acting as junction zones. This network increasingly accounts for the bulk theological behaviour of aging bread crumb. Furthermore, as amylopectin retrogradation proceeds, moisture migration within the crumb structure occurs, and more and more water is immobilised within amylopectin crystallites. The crystalline hydrate water can no longer plasticise the different networks, which goes hand in hand with increased crumb firmness and decreased crumb resilience, due to a less flexible gluten network. The efficiency of anti-staling amylases can be related to the extent they limit the formation and the strength of the permanent amylopectin network, and the water immobilisation. Conventional alpha-amylases weaken the amylopectin network by cutting the long polymer chains connecting the crystalline regions, but have little effect on amylopectin recrystallisation. In contrast, maltogenic alpha-amylase primarily shortens the amylopectin side chains, thus hindering amylopectin recrystallisation, and the concomitant network formation and water immobilisation. (C) 2009 Elsevier Ltd. All rights reserved.status: publishe

Amylose-lipid complexation: a new fractionation method

Amylose fractions of different peak Degree of Polymerisation (DP) (DP20, DP60, DP400, DP950) were complexed with docosanoic acid (C22) and glyceryl monostearate (GMS) at 60 and 90degreesC. Complexation yields, relative crystallinities, dissociation temperatures and enthalpies increased with amylose chain lengths (DP20-DP60-DP400). Relative crystallinities and thermal stabilities of the DP950-complexes were slightly lower than those of the other amylose fractions, probably due to increased conformational disorders, resulting in crystal defaults. Molecular weight distributions of the complexes revealed that, irrespective to the complexation temperature, the critical DP for complex formation and precipitation was 35 and 40 for complexes with GMS and C22, respectively, corresponding to the length needed to accommodate two GMS- or C22-molecules within an amylose helix. Complexation of dextrins with a well-chosen lipid, allows to separate starch derived dextrins with a predictable critical chain length as border. Dextrins, of sufficient DP will complex and precipitate, while the shorter dextrins will remain in solution. (C) 2004 Elsevier Ltd. All rights reserved.status: publishe

Enzyme and acid resistance of amylose-lipid complexes differing in amylose chain length, lipid and complexation temperature

To study parameters that influence enzyme and acid resistance of amylose-lipid complexes, complexes were formed between amylose of different average chain lengths [Degree of Polymerisation (DP); DP60, 400, 950] and docosanoic acid (C22) or glyceryl monostearate (GMS) at 60 or 90 °C. Complexes were hydrolysed with hog pancreatic alpha-amylase or acid (2.2 N HCl). DP400- and DP950-complexes were of type I when formed at 60 °C and of type II when formed at 90 °C. Enzyme and acid resistance increased with increasing amylose DP, lipid chain length and complexation temperature. DP60 yielded only type I complexes, independent of the complexation temperature. Enzymic and acid hydrolysis of all complexes gave rise to two or more dextrin subpopulations, which are interpreted to originate from a sequence of lamellar units (the smallest peak DP) with interconnecting, amorphous amylose chains. The peak DP of such lamellar unit increased with increasing amylose DP and complexation temperature, but remained constant when higher enzyme dosages were applied. Synthesis of amylose-lipid complexes from amylose and lipids of variable structure under variable temperature conditions followed by hydrolysis can yield dextrin populations of defined and relatively narrow molecular weight distributions.status: publishe