Protein Inhibitors ofAlpha-amylase in Mature and Germinating Grain of Rye (Secale cereale)

The activities of endogenous (R-type) and exogenous acting (D-type) protein inhibitors ofalpha-amylase and the activities ofalpha- and total amylase were determined in milling fractions of rye. High D-type amylase inhibitor activities were detected in the embryo (255 IU/g) and in the endosperm fraction (64·9 IU/g), low inhibitor activities were found in the aleurone layer fraction (25·9 IU/g). The highest R-typealpha-amylase inhibitor activity was found in the aleurone layer fraction (32·6 IU/g), and the lowest value in the epidermis containing fraction (5·0 IU/g). The D- and R-typealpha-amylase inhibitor activities varied with growing conditions. D-type amylase inhibitor activities were found to be high in those samples which grew under drought conditions and low in samples cultivated under wet and cool weather. Higher R-typealpha-amylase inhibitor activities were found in rye genotypes cultivated under wet conditions and lower values under dry weather. There were small variations inalpha-amylase inhibitor activities between sprout-stable and sprout-sensitive rye genotypes. The D- and R-typealpha-amylase inhibitor activities of all varieties were stable during 72 h of germination. Similar soil conditions will therefore lead to differentialalpha-amylase inhibitor activities depending on weather conditions during growth.     

Distribution of Enzymes in Wheat Flour Mill Streams

The distribution of alpha -amylase, protease, lipoxygenase, polyphenol oxidase and peroxidase in wheat roller flour mill streams was studied. Break flours had relatively less alpha -amylase and protease activity than reduction flours both on flour weight and a protein basis. Among the different flour streams, the 5^thand 6^threduction passage had the highest alpha -amylase activity, while the 4^threduction passage had the highest protease activity. The lipoxygenase activity was concentrated mostly in the last break and the reduction streams, whereas polyphenol oxidase activity was highest in break flour streams. Peroxidase activity was distributed unevenly among the different mill streams. The lipoxygenase, polyphenol oxidase and peroxidase were highly concentrated in different bran fractions. Except for protease, the other enzymes were concentrated in the «atta», a milling by-product comprising refined flour, bran and shorts; and are least active in semolina (farina).     

Effect of particle size on kinetics of starch digestion in milled barley and sorghum grains by porcine alpha-amylase

The influence of milled grain particle size on the kinetics of enzymatic starch digestion was examined. Two types of cereals (barley and sorghum) were ground, and the resulting grounds separated by size using sieving, with sizes ranging from 0.1 to 3 mm. In vitro enzymatic digestion was performed, using pancreatic alpha-amylase, amyloglucosidase and protease, to determine fractional-digestion rates over 24 h. The resulting glucose production rate data were well fitted by simple first-order kinetics. For each sieve screen size, the digestion rate of barley was always higher than that of sorghum. The rate coefficients for digestion showed a decrease with increasing size, and could be well fitted by an inverse square relationship. This is consistent with the supposition that starch digestion in these systems is controlled by diffusion of enzyme through the grain fragment. Apparent diffusion coefficients of alpha-amylase obtained by fitting the size dependence were 0.76 (sorghum) and 1.7 (barley) × 10⁻⁷ cm² s⁻¹, 9 (sorghum) and 4 (barley) times slower than predicted for a molecule of the size of alpha-amylase in water.     

Variation of Beta -Amylase Activity in Barley as Affected by Cultivar and Environment and its Relation to Protein Content and Grain Weight

The cultivar and environmental variation of beta -amylase activity was studied using two barley cultivars with contrasting growth properties. There was a significant difference in beta -amylase activity between the two cultivars used, 92-11 being significantly higher than Xiumai 3. A significant variation in beta -amylase activity was detected between grains at different positions within a spike. The two cultivars showed the same pattern, with top grains showing the highest and bottom ones the lowest activities. The relative difference within a spike varied between the cultivars, with 92-11 being larger than Xiumai 3. Both seeding rate and timing of N application dramatically affected the beta -amylase activity. With N application at the booting stage, beta -amylase activity increased, mainly due to the significantly increased beta-amylase activity in the topmost grains. The bottom grains showed a lower response to timing of N application. The variation in protein content and grain weight between cultivars and among the various treatments was also examined. The possible influence of these factors on beta -amylase activity are discussed.     

Physicochemical Studies on Resistant StarchIn VitroandIn Vivo

Resistant starch (RS), producedin vitroby hydrolysis of retrograded pea starch gels and amylose gels by porcine pancreaticalpha-amylase, was characterised by X-ray diffraction, size exclusion chromatography and methylation analysis. These techniques showed that RSin vitroconsisted of semi-crystalline, mostly linear material that was present in two main molecular size subfractions (DP_n>100 andDP_n20–30) with a third, minor subfraction (DP_n≤5). The extent of retrogradation of amylose was found to be of primary importance in determining the RS content of starch. Analysis ofin vivoRS, recovered during an ileostomy study, produced results that were similar to those obtained from RSin vitro. Anin vitromodel for the structure of resistant starch is proposed.     

In VitroDigestion of Wheat Microstructure with Xylanase and Cellulase fromTrichoderma reesei

Resistant starch (RS), producedin vitroby hydrolysis of retrograded pea starch gels and amylose gels by porcine pancreaticalpha-amylase, was characterised by X-ray diffraction, size exclusion chromatography and methylation analysis. These techniques showed that RSin vitroconsisted of semi-crystalline, mostly linear material that was present in two main molecular size subfractions (DP_n>100 andDP_n20–30) with a third, minor subfraction (DP_n≤5). The extent of retrogradation of amylose was found to be of primary importance in determining the RS content of starch. Analysis ofin vivoRS, recovered during an ileostomy study, produced results that were similar to those obtained from RSin vitro. Anin vitromodel for the structure of resistant starch is proposed.     

Differences in conformational stability of barley alpha-amylase isozymes 1 and 2. Role of charged groups and isozyme 2 specific salt-bridges

Barley alpha-amylase isozymes 1 (AMY1) and 2 (AMY2) have 80% sequence identity but possess different physico-chemical properties. By incubation in the range 37–85 °C T₅₀ is 75.2 °C of AMY1 and 79.2 °C of AMY2. While AMY2 is also most stable in urea at pH 6.7, [urea]₅₀ being 8.2 M compared to 7.9 M for AMY1, AMY1 has highest stability in urea below pH 6 or in the presence of NaCl. Moreover AMY1 is most stable in guanidinium chloride. Charge screening thus destabilises AMY2 but stabilises AMY1. Isozyme sequence comparison suggests that AMY1 lacks four of the 20 salt-bridges identified in the crystal structure of AMY2. The four residues that differ comprise Lys67_AMY2 and Asp267_AMY2, forming salt-bridges on the surface of the catalytic (β/α)₈-barrel (domain A), and Glu96_AMY2 and His344_AMY2 that participate in charged networks between domain A and the small domain B and the C-terminal domain, respectively. Four corresponding AMY2 mimics A68K; D97E; Q269D; N346H were made in AMY1 by site-directed mutagenesis. While D97E and Q269D have slightly improved stability compared to AMY1 wild-type, N346H and, under certain conditions, A68K are destabilised. The four mutants show 22–176% activity (k_cat/K_m) toward 2-chloro-4-nitrophenol β- -maltoheptaoside and amylose DP17 and 43–117% activity for insoluble starch.     

The Role of Dextrins in the Stickiness of Bread Crumb made from Pre-Harvest Sprouted Wheat or Flour Containing ExogenousAlpha-Amylase

Dextrins were extracted in water from bread made from pre-harvest sprouted wheat or standard flour supplemented with exogenousalpha-amylases. The dextrins were separated by gel permeation chromatography and the dextrin content (% of crumb weight) determined for different degree of polymerisation (DP) size classes; DP 1–2, DP 3–10, DP 11–50, DP 51–200 and DP >200. There were significant correlations between the dextrin content in each size class and crumb stickiness (r=0·84–0·91, 22 df ). The most significant correlation (r=0·96) was between total dextrin content and crumb stickiness. Addition of dextrins of various DP ranges from various sources to standard flour produced bread with sticky crumb. Again, the degree of stickiness was generally related to the amount of total dextrin in the crumb and not to size distribution of dextrins. In this instance, extensive enzymic hydrolysis of starch was not necessary to produce sticky crumb; the dextrins caused crumb stickiness directly. Addition of dextrins to reconstituted gluten–starch flour produced bread with unexpectedly low dextrin levels and correspondingly low stickiness scores. It is concluded that, to produce sticky crumb, high levels of dextrin of any size are necessary in the crumb; a sticky mass is produced when dextrins dissolve in the excess «free» water that is normally «bound» to starch, gluten and other insoluble components of bread crumb.     

Comparative Enzyme Kinetics of Two Allelic Forms of Barley (Hordeum vulgare L.) Beta -amylase

The barley (Hordeum vulgare L.) varieties, Franklin and Schooner, contain two different allelic forms of beta -amylase (EC 3.2.1.2) encoded on chromosome 4H by the Bmy 1-Sd1 and Bmy 1-Sd2L alleles, respectively. The corresponding enzymes, referred to as Sd1 and Sd2L, were purified from both mature barley grain and germinated barley (green malt), and their physical and kinetic properties studied. Approximately 4 kDa were cleaved from both Sd1 and Sd2Lbeta -amylases after germination. The K_mvalue for green malt beta -amylase was less than that of mature grain beta -amylase for both varieties when potato starch was used as a substrate, although V_maxwas similar. This indicated that proteolysis after germination increased the affinity of beta -amylase for potato starch. No significant kinetic differences were observed between beta -amylase from mature grain and green malt of the two barley varieties when amylose (degree of polymerisation 100 and 18) and maltopentaose were used as substrates. Kinetic differences were also observed between the two allelic forms of beta -amylase. Sd1 beta -amylase from green malt exhibited a lower K_mvalue for potato starch than Sd2L beta -amylase, demonstrating that at non-saturating starch concentrations Sd1 beta -amylase is better able to hydrolyse starch than Sd2L beta -amylase. As the degree of polymerisation of the substrates decreased from approximately 740 (potato starch) to 5 (maltopentaose), the K_mvalues for beta -amylase increased, whereas V_maxvalues decreased. Maltose, the hydrolytic product of beta -amylase, was found to be a weak competitive inhibitor of both Sd1 and Sd2L green malt beta -amylases with respect to potato starch and amylose. Taken together the kinetic observations for bet a-amylase suggest that the allelic differences and C-terminal proteolysis might be exploited to improve the efficiency of starch hydrolysis during the mashing stage of the brewing process.     

Developmental Expression of Amylases During Barley Malting

Amylase activity and qualitative changes in amylase isoenzymes as a function of barley seedling age were investigated in 10 Brazilian barley cultivars. All cultivars showed few isoenzymes in early germination. An increase in general activity ensued in the following days when new isoenzymes were detected and those already observed since early germination had their activity increased. All cultivars disclosed increase in amylase activity until the third or fourth day of germination. Some cultivars maintained this high activity until the last day analysed. Other cultivars presented a decrease in activity in the fifth or sixth day. No electrophoretic pattern or allelomorph responsible for a higher amylase activity were detected. Beta -amylase activity was always superior to alpha -amylase activity. High beta -amylase activity was already observed on the second day of germination while alpha -amylase activity began to increase only from the third day on. The results obtained suggest that, at least for the cultivars analysed, there is a high general amylase activity around the fourth day of germination, indicating that germination could stop at this moment, ensuring that hydrolitic enzyme activity required in the brewing process is met. Beta -amylase was lightly correlated with diastatic power (r=0·565) but no correlation was observed between alpha -amylase and diastatic power (r=-0·128), or neither betweenalpha - and beta -amylase with malting quality (r=0·153 andr =−0·348, respectively). These results indicate that beta -amylase activity in barley grains, more than alpha -amylase, can be a good predictor of diastatic power.     

Late-maturity α-amylase: Low falling number in wheat in the absence of preharvest sprouting

Late maturity α-amylase (LMA), or prematurity α-amylase (PMAA) as it has been termed in the UK, in wheat involves the untimely synthesis of high pI α-amylase during the middle to later stages of grain development and ripening. The enzyme activity is retained in the grain at harvest ripeness, resulting in low falling number and failure to meet receival standards and customer specifications. This phenomenon, which is restricted to specific genotypes, appears to be controlled by 1 or 2 recessive genes acting alone or in combination and in most cases appears to be triggered by a temperature shock. This shock is only effective if it occurs during a window of sensitivity around 25–30 days postanthesis. Expression of LMA is reduced in the presence of dwarfing genes such as Rht1, Rht2 and Rht3 that confer insensitivity to gibberellin. Screening technologies, including molecular markers and high pI-specific ELISA, have been developed to assist wheat breeders and will be required to meet new challenges posed by novel germplasm such as primary synthetic wheats.     

Alpha-amylase/subtilisin Inhibitor Levels in Australian Barleys

An enzyme-linked immunosorbent assay (ELISA) was used to determine the bifunctional alpha-amylase/subtilisin inhibitor (BASI) content of barley grain from 11 cultivars grown in six diverse locations in Australia. The inhibitor ranged from 119 to 254 μg/g in 57 barley samples. Genotype had a significant (P<0·05) effect on BASI content but there was no effect due to environment. Total protein varied independently of BASI and was influenced by environment and genotype. BASI content was higher (P<0·05) in malting barley than in feed barley and was correlated positively (r=0·29;P<0·05) with alpha-amylase activity in corresponding malts. The ELISA used monoclonal and polyclonal antibodies raised against purified BASI. In immunoblot analysis the monoclonal antibody showed high specificity for the inhibitor in barley and also detected the inhibitor in wheat. Low levels of inhibitor (mean 3·2 μg/g) were found in 12 Australian wheat cultivars using the ELISA developed for barley. The assay had a linear working range of 5–50 ng/mL with a detection limit of 2 ng/mL. Reproducibility between assays was good (CV=4·9%) but mean recoveries were high, ranging from 116–129% when purified inhibitor was added to barley extracts. The ELISA may have useful applications in brewing research and barley breeding programmes.     

Pre-maturity α-amylase in wheat: The role of abscisic acid and gibberellins

The occurrence of pre-maturity α-amylase (PMA) is a major cause of poor bread-making quality (low Hagberg Falling Number) in wheat grain. In susceptible genotypes, it involves the excessive accumulation of high isoelectric point (pI) α-amylase in mature grain prior to germination and in the absence of pre-harvest sprouting. Several factors regulate PMA formation in developing grain, including genotype, agronomy, and environmental conditions. In particular, a cold period during mid-grain development has been found to be a major stimulus for PMA induction. Although the factors affecting the PMA occurrence are well known, little is known about the molecular mechanism governing its induction. The plant hormones abscisic acid (ABA) and gibberellins (GAs) influence various aspects of grain development, and it has been suggested that PMA involves changes in the amount of these hormones or the sensitivity of the grain to these hormones. This review summarizes recent studies investigating the role of ABA and GAs in PMA induction and PMA occurrence.     

Production of Proteases by Fusarium Species Grown on Barley Grains and in Media Containing Cereal Proteins

The production of proteases by the cereal plant pathogens Fusarium culmorum, F. graminearum and F. poae was followed through seven days of cultivation. The fungi were grown in mineral and in gluten culture media, and on autoclaved barley grains. The proteolytic activities of each sample were analysed at pH 2·2, 5·0 and 8·0 and the pH optima of the most active proteases were determined. All of the fungi grown in the gluten medium produced proteases that were active at pH levels between 6 and 10 and were most active at about pH 9·0. Fusarium poae also produced acid protease(s) with pH optima between 3.0 and 3.5 when grown in the gluten medium. No protease activity was detected in the cultures that were grown in the mineral medium, except that a small amount was formed after the glucose substrate was depleted. When grown on the barley grain medium the Fusarium species produced protease activities that were similar to the neutral and alkaline ones present in the gluten cultures, but no pH 2·2 protease activity was detected. The alkaline proteases had some characteristics that were similar to those of chymotrypsin.     

A rapid,automated method for measuring α-amylase in pre-harvest sprouted (sprout damaged) wheat

The quality of wheat for baking is critically dependent on the level of α-amylase (1,4-α-D-glucan glucanohydrolase, EC 3.2.1.1), which can be present as “late maturity α-amylase” (LMA), or due to pre-harvest sprouting due to high rainfall and humidity at the time of harvesting. The most commonly used method to measure α-amylase in wheat grain is the Hagberg Falling Number method, but values are also influenced by rheological properties of starch in the grain. In this study we describe a simple, rapid, automated method (Amylase SD) for measurement of α-amylase in pre-harvest sprouted (sprout damaged) wheat grain. The method (Amylase SD) measures the release of p-nitrophenol from 4,6-O-ethylidene-α-4-nitrophenyl-maltoheptaoside by α-amylase in the presence of α-glucosidase. The absorbance of p-nitrophenolate measured at 405 nm in a ChemWell^®-T auto-analyser is directly related to the level of α-amylase activity present in the milled wheat grain extract. The Amylase SD method generated <6%CV and correlation to the Falling Number method was represented by an inflection point at ∼160 s. The precision, sensitivity and speed of this method provides an ideal alternative to the Falling Number method for measurement of α-amylase (sprout damage) in wheat grain in wheat breeding programmes or at grain receival points.     

Germination of Wheat Grains at Various Temperatures in Relation to the Activities of α-Amylase and Endoprotease

Abstract

Germination percentages of wheat grains sampled at 3 grain-filling stages : yellow-ripe stage (water content 45-50%), dough-ripe stage (35-40%), and full-ripe stage (25-30%), and imbibed in water at 12°C and 20°C were examined in relation to the activities of α-amylase and endoprotease. Wheat varieties studied were Chihoku-komugi, which is susceptible to pre-harvest sprouting, and Satanta, which is resistant. Germination percentage was higher at 12°C than at 20°C in all grains sampled at all stages in both varieties, and was higher in Chihoku-komugi than in Satanta at 20°C. The activity of α-amylase in the grains at the yellow-ripe stage was higher at 12°C than at 20°C in both varieties, but that at the other 2 stages was higher only in Satanta. Endoprotease increased rapidly from 7 to 10 days after the start of imbibition, and exceeded 12 units only at 12°C in Chihoku-komugi grains at the dough and full-ripe stages. The results showed that α-amylase activity was lower than the value equivalent to 300 brabender unit (BU) in amylography when the germination percentage was 0%. Endoprotease activity exceeded 6 units when the germination percentage exceeded 90%.     

CerealBeta-Amylases

Cerealbeta-amylases are perhaps best known in terms of the vital role they play in releasing easily fermentable sugars from cereal grain starch to fuel the production of alcohol by yeast in brewing. The extent to which they have been investigated is indeed largely due to their significance in this economically important industry. However, cerealbeta-amylases are also, or could be, employed in many other aspects of the food industry and the analysis of starch, and they constitute valuable markers in cereal assessment and breeding studies. Quite apart from their practical significance, they are rewarding objects of biochemical and physiological research. They are interesting models for the study of enzyme polymorphism, post-translational modification and the differential expression of isoenzymes. In spite of their often high activitiesin situand all that is known about their generation, they are an enigma in that their physiological function, or even necessity, remains unclear. It has been recently recognised that there are two different categories of cerealbeta-amylases which exhibit different tissue and taxonomic specificities and physiological developmental patterns. The «classical»beta-amylases present at high activities in cereal seeds appear to be limited to the endosperm of the species of the Triticeae tribe of the Festucoideae subfamily of the Gramineae (wheat, barley and rye), whereas all cereals exhibit a different, tissue-«ubiquitous» form of the enzyme which is present at much lower activity levels. The physiological phenomenology and the usage of cerealbeta-amylases are discussed in relation to these two categories of enzyme.     

Associations between caryopsis dormancy, α-amylase activity,and pre-harvest sprouting in barley

Pre-harvest sprouting (PHS) is a concern for barley (Hordeum vulgare L.) producers, grain processors, and researchers worldwide. Pre-harvest sprouting has been mainly attributed to low dormancy, which is determined by genotype, stage of plant maturation, and environmental conditions during caryopsis development. Fourteen barley genotypes were sown in field experiments at two sites in North Dakota in 2004 and 2005. Spikes were harvested at four different stages: ≈500 g kg⁻¹ moisture content, physiological maturity, harvest maturity, and post-harvest maturity. Results indicated that barley genotypes were released from dormancy at different rates. The 14 barley genotypes were divided into three classes based on their dormancy loss rate during caryopsis development. C93-3230-24 was highly dormant, and ‘Stander’ and ‘Legacy’ were highly susceptible to PHS due to lack of dormancy from as early as 20 d after heading date. All other genotypes fell into the third group that had intermediate dormancy loss rate. No significant correlation was detected between barley α-amylase activity and germination percentage. A moderate association between malt α-amylase activity and caryopsis dormancy suggested that cultivars with increased malt α-amylase activity tend to have low dormancy and may be more prone to PHS.     

Amylolytic activity in stored potato tubers. 1. Estimation usingp-nitrophenyloligosaccharides

Summary Methods for the estimation of amylolytic activity are reviewed. A procedure for the routine extraction of amylolytic activity from freeze-dried powder prepared from potato tubers is described. The extraction medium is buffered at pH 7.0 and contains glycerol, dithiothreitol, calcium chloride and the non-ionic detergent, nonidet P-40. α-Amylase activity and exoamylolytic activity were estimated in crude extracts of potato tubers using the substrates, blockedp-nitrophenyl-maltoheptaoside andp-nitrophenyl-maltopentaoside respectively. These substrates are included in kits supplied by Biocon, (UK) Ltd to measure the α- and ?-amylase activity in cereals. The validity of using these kits for the determination of α- and ?-amylase activity in potato tuber tissue is discussed.