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.

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 RV(80) 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 polysaccharides 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 RV(80) value. Bacillus amyloliquefaciens alpha-amylase and Aspergillus oryzae alpha-amylase, with the lowest RV(80) 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 RV(80) 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.

Flour sodium dodecyl sulfate (SDS)-extractable protein level as a cookie flour quality indicator.

Flour characteristics of laboratory-milled flour fractions of two wheat cultivars were related to their cookie-baking performance. Cultivar (cv.) Albatros wheat milling yielded fractions with lower damaged starch (DS) and arabinoxylan levels and higher sodium dodecyl sulfate-extractable protein (SDSEP) levels than did cv. Meunier wheat milling. During baking, cv. Albatros flour doughs spread faster and set later than their cv. Meunier counterparts and, hence, resulted in larger cookie diameters. DS levels negatively affected spread rate during both cv. Albatros (R2=0.68) and cv. Meunier (R2=0.51) cookie baking. SDSEP levels also influenced cookie quality. The use of flour heat-treated to reduce its SDSEP levels to different degrees led to reduction of the set time (R2=0.90). It was deduced that larger gluten polymer sizes limit dough spread time during baking and that, apart from DS level, the SDSEP level is an indicator for cookie flour quality.

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.

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.

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.

Temperature impacts the multiple attack action of amylases.

The action pattern of several amylases was studied at 35, 50, and 70 degrees C using potato amylose, a soluble (Red Starch) and insoluble (cross-linked amylose) chromophoric substrate. With potato amylose as substrate, Bacillus stearothermophilus alpha-amylase (BStA) and porcine pancreatic alpha-amylase displayed a high degree of multiple attack (DMA, i.e., the number of bonds broken during the lifetime of an enzyme-substrate complex minus one), the fungal alpha-amylase from Aspergillus oryzae a low DMA, and the alpha-amylases from B. licheniformis, Thermoactinomyces vulgaris, B. amyloliquifaciens, and B. subtilis an intermediate DMA. These data are discussed in relation to structural properties of the enzymes. The level of multiple attack (LMA), based on the relation between the drop in iodine binding of amylose and the increase in total reducing value, proved to be a good alternative for DMA measurements. The LMA of the endo-amylases increased with temperature to a degree depending on the amylase. In contrast, BStA showed a decreased LMA when temperature was raised. Furthermore, different enzymes had different activities on Red Starch and cross-linked amylose. Hence, next to the temperature, the action pattern of alpha-amylases is influenced by structural parameters of the starch substrate.

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.

Amylose-lipid complexes as controlled lipid release agents during starch gelatinization and pasting.

The effect of amylose-lipid (AM-L) complexes consisting of amylose populations with different peak degrees of polymerization (DP) and complexed with glyceryl monostearate (GMS) or docosanoic acid (C22) on the pasting properties of wheat and rice starches was evaluated with a rapid visco analyzer (RVA). AM-L complexes were formed by both (i) addition of lipids to amylose fractions with peak DP 20, 60, 400, or 950 at 60 degrees C or (ii) potato phosphorylase-catalyzed amylose synthesis in the presence of lipids. All AM-L complexes affected pasting properties in line with their dissociation characteristics. AM-L complexes therefore have potential as "controlled lipid release agents" with effects markedly different from those observable with emulsifier addition in starch pasting. More in particular, short chain AM-L complexes resulted in a starch pasting behavior comparable to that of cross-linked starch, as evidenced by reduced granule swelling, good viscosity stability in conditions of high temperature and shear, and a stable cold paste viscosity.

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.

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.

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.

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.

Potential role of glycosidase inhibitors in industrial biotechnological applications.

The nutrient content of food and animal feed may be improved through new knowledge about enzymatic changes in complex carbohydrates. Enzymatic hydrolysis of complex carbohydrates containing alpha or beta glycosidic bonds is very important in nutrition and in several technological processes. These enzymes are called glycosidases (Enzyme Class 3.2.1) and include amylases, pectinases and xylanases. They are present in many foods such as cereals, but their microbial analogues are often produced and added in many food processes, for instance to improve the shelf-life of bakery products, clear beer, produce glucose, fructose or dextrins, hydrolyse lactose, modify food pectins, or improve processes. However, many plant foods also contain endogenous inhibitors, which reduce the activity of glycosidases, in particular, proteins, peptides, complexing agents and phenolic compounds. The plant proteinaceous inhibitors of glycosidases are in focus in this review whose objective is to report the effect and implications of these inhibitors in industrial processes and applications. These studies will contribute to the optimisation of industrial processes by using modified enzymes not influenced by the natural inhibitors. They will also allow careful selection of raw material and reaction conditions, and future development of new genetic varieties low in inhibitors. These are all new and very promising concepts for the food and feed sector.

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.

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.

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.