Endosperm α 1,4—α 1,6 Glucopolysaccharides Utilization During Germination of Sweet Corn and Other Maize Genotypes

The quantity variations and the structural changes of the α 1,4 —α 1,6 glucopolysaccharides contained in the endosperm of sweet corn, waxy and amylose extender maize genotypes, were studied during early germination. The α 1,4 —α 1,6 glucopolysaccharides were fractionated according to their branching degree. The results obtained showed that: the degradation pattern of the α 1,4 —α 1,6 glucopolysaccharides was similar for the different corn genotypes studied. In addition, soluble dextrins or partial degradation products were not detected, but reducing sugars were accumulated. Furthermore, the structure of the remaining polysaccharides was similar to those present in the non germinating seed. These results led us to propose that once a polysaccharide molecule was used as the substrate by degradative enzymes during germination, that molecule was totally degraded.     

Purification and Characterization of a Maltotriogenic α-Amylase I and a Maltogenic α-Amylase II Capable of Cleaving α-1,6-Bonds in Amylopectin

Extracellular α-amylases I and II, produced by a facultative thermophile Bacillus thermoamyloliquefaciens KP 1071 capable of growing at 30–66°C, were purified to homogeneity. α-Amylase I consisted of a single polypeptide with methionine residue at the NH₂-terminus. α-Amylase II consisted of two equivalent polypeptides each comprising a methionine at the NH₂-terminus. α-Amylase I hydrolyzed endotypically α-1,4-bonds in glycogen, amylopectin and β-limit dextrin, but not their α-1,6-bonds. α-Amylase II degraded amylopectin and β-limit dextrin in exo-fashion by cleaving preferentially α-maltose units from the non-reducing ends and hydrolyzing their α-1,6-branch points. α-Amylase II hydrolyzed maltotriose, phenyl-α-maltoside, α- and β-cyclodextrins and pullulan, whereas α-amylase I had no activity for all these sugars. α-Amylases I and II hydrolyzed maltotetraose, maltopentaose, α-limit dextrin and amylose, but they were inactive for maltose, isomaltose and panose. It was suggested that α-amylase I is the most thermostable type of hitherto known maltotriogenic endo-acting α-amylases, and α-amylase II is the first maltogenic exo-acting α-amylase able to split α-1,6-bonds in amylopectin.     

Characterization of an Exo-Maltotetraose-forming Amylase of Pseudomonas stutzeri and Application in p-Nitrophenyl β-D-Galactosyl-α-Maltooligosaccharides Production

Some kinetic characteristics and hydrolytic action patterns on various β-D -galactosyl-maltooligosaccharides (Gal-Gn), ranging in size from D.P. (degree of polymerization) 5 to 8, of an exo-maltotetraose-forming amylase of Pseudomonas stutzeri (G4-amylase) were examined to produce a few p-nitrophenyl β-D -galactosyl-α-maltooligosaccharides (Gal-GnP, n = 4,5). The relative hydrolytic reaction rates for larger Gal-Gn by the enzyme were larger than those for smaller saccharides tough the values for unmodified linear maltooligosachharides were almost same. Michaelis constants (Km) for hydrolysis of Gal-G4, Gal-G5, Gal-G6 and Gal-G7 by the enzyme were 1.3, 1.9, 1.3 and 1.3mM, and apparent molecular activities (ko) for these saccharides were 5.9, 38, 91 and 126s⁻¹, respectively. The values of ko/Km for them were remarkably smaller than those for unmodified linear maltooligosaccharides. The G4-amylase cleaved 2 points of the α-1,4-glucosidic linkage in β-1,4-Gal-G4 to give β-1,4-Gal-G2 and -Gal-G3 in the molar ratio of 3:1, whereas the enzyme attacked 3 points of the linkage in β-1,4-Gal-G5, -Gal-G6 and -Gal-G7 to form β-1,4-Gal-G2,-Gal-G3 and Gal-G4 in the molar ratios of 2:5:1, 1:3:6 and 1:3:6, in the early stage of the reaction, respectively. On the other hand, the enzyme showed no action on β-1,6-Gal-G4 and formed β-1,6-Gal-G4 solely from β-1,6-Gal-G5, and β-1,6-Gal-G4 and -Gal-G5 were from β-1,6-Gal-G6 and -Gal-G7 in the ratios of 8:1 and 2:1, respectively. The enzyme also catalyzed the transfer action to produce Gal-G3P, Gal-G4P and Gal-G5P, of which the formation ratio was coincided well with the hydrolytic action pattern on each Gal-Gn, from Gal-Gn tested as a donor and p-nitrophenyl α-glucoside (GP) as an acceptor in an aqueous solution containing 40% (v/v) methanol. By using this novel reaction, Gal-G5P is now producing on an industrial scale to apply as a substrate for the assay of human α-amylase.     

Three α-gliadins of Cappelle-Desprez

The strong α-doublet running behind the fastest major α-gliadin of Cappelle-Desprez on gel electrophoresis has been separated into three proteins. They were named: slow, middle and fast (S, M and F) from their mobilities on alkaline gels. The amino acid compositions were typical of gliadins, with no free thiol groups. Molecular weights and % amidation were: S31000, 96; M32 000, 94; F29000, 91. No convincing evidence for a glycoprotein nature was found. At acid pH the order seems to be, in decreasing mobility, F, S, M.     

Maize Mutants. Part 1: Studies on their Starch Components

The present study was conducted in order to evaluate the starch components contained in several maize mutants. The α1,4-α1,6 glucopolysaccharides were fractionated according to their branching degree. The determination of the wavelenght of maximum absorption (γ max) in the presence of Krisman's reagent [12] as well as the glucose α1,6 linkages involved in the branching point were determined. The mutants analyzed possess characteristics that allow us to identify them according to the composition and structural properties of their polysaccharides (phytoglycogen, amylopectin, amylose).     

Characterization of Pullulanase and α-Amylase Activities of a Thermus sp. AMD33

Thermostable Thermus sp. AMD 33 pullulanases (I and II) capable of cleaving α-1,6-links in pullulan as well as α-1,4-glucosidic linkages in amylose were purified to electrophoretically homogeneous states. Relative molecular masses and pI values were determined as 135,000 (I and II) by SDS-PAGE and 4.2 (I) and 4.3 (II) by isoelectric focusing, respectively. The pullulanase and α-amylase activities of the purified enzyme II responded similarly to temperature and pH, with optima at 70°C and pH 5.5–6.0. Both activities were activated by Ca²⁺ and inhibited by Hg²⁺, Fe³⁺, NBS, DBS, SDS and urea to almost the same extent. Both activities were also inhibited competitively by CDs. Enzyme II catalyzed the hydrolysis of α-1,6-glucosidic linkages in maltosyl- and maltotriosyl-α-CD as well as that of α-1,4-bonds in amylose and related linear malto-oligosaccarides larger than maltotriose, but exhibited no action on panose, isopanose or glucosyl α-CD.     

The influence of α-amylase supplementation, γ-irradiation (60Co) as well as long time of storage of wheat grain on flour technological properties

The varieties of winter wheat, Aria and Beta, were studied. The Aria variety was stored for the period of four years. The part of wheat grain from Beta variety was irradiated with γ rays (⁶⁰Co). In extracts from wheat kernels and flour protein content, total α- and β-amylolytic activity as well as α-amylolytic activity were determined. α-amylases of native and fungal origin were added to the flour obtained from samples of stored wheat kernels (Aria), irradiated and non-irradiated (Beta). Consequently native α-amylase activity of flour increased by 25 % and 50% respectively. Extensive technological estimation of grain and flour with amylase supplements was carried out. The study included: sedimentation analysis, falling number test, milling experiment, farinogram and extensogram analyses, measurement of the degree of damaged starch and flour colour, as well as baking experiment. The obtained experimental loaves of bread were tested for their ability to remain fresh. It was found out that the stored grain flour was characterized by the highest α -amylolytic activity and the lowest falling number value, whereas the irradiated grain flour showed the highest degree of starch damage and water absorption. When α-amylase supplementation to doughs wasn't accompanied either by irradiation or storage of grain, it definitely changed their physical properties for the worse. The negative influence of native α-amylases appeared to be less significant than that of fungal α-amylases. The positive influence of α-amylase supplementations, especially of those increasing by 25 % the native α-amylolytic activity of flour on volume, and freshness of loaves of bread was observed.     

Amylose is not Strictly Linear

Amyloses from selected corn starches were used to measure the glucoses involved in the branching points. The number of glucose α 1,6 glycosidically linked were determined by two different methods: 1) The enzymatic one measures directly quantiatively and specifically the glucoses α, 1,6 linked. 2) The other, a chemical one that measures the different n-methylated glucoses involved in the terminal, α 1,4 linked and α1,6 linked positions. The results obtained through both methodologies clearly indicate a good correlation. Amylose is not linear. It has very small proportion of branches and they are α1,6 glycosidically linked.     

Molecular Weights,Lengths, and Distribution of Side-chains in α-D-Polyglucanes

Freshly prepared starch (from potatoes P, from maize M) was separated quantitavely into amyloses and amylopectins under mild conditions. Furthermore, glycogen (G) was extracted from livers of rabbits under observation of physiological provisoes (feeding, rapid removal of the organ under anaesthetic and freezing-stop). The molecular weights, determined by analytical ultracentrifuge . The side-chain parts of the amylopectins and of the liver glycogen which were checked by partial enzymatic break-down into β-dextrin by β-amylase (EC 3.2.1.2) and into the basic structure by pullulanase (EC 3.2.1.41) is 64% for P, 62.7% for M, and 65.4% for G. The lengths of the side-chains, expressed in glucoside residues, are calculated at 12.0 for P and M, and 8.0 for G; the intervals between the side-chains are 7.0 for P and M, and 4.0 for G. Sources of error with respect to the lengths of and to the intervals between the side-chains are, firstly, free-ending, undecomposed side-chains inside the big molecules which cannot be reached by the two enzymes because fo steric obstruction, and, secondly, the degree of ramification of the basic structures and, consequently, the number of α-1,4 and α-1,6-bound glucoside residues which cannot be split off by pullulanase.     

Analysis of the Amylolytic Enzyme System of Clostridium thermosulfurogenes EM1: Purification and Synergistic Action of Pullulanases and Maltohexaose Forming α-Amylase

During growth of C. thermosulfurogenes EM1 on starch, seven forms of pullulanase and one form of α-amylase were detected after gel electrophoretic separation of proteins. By determining the isoelectric points and N-terminal sequences of various pullulanase species it was evident that no structural differences exist between these proteins. These pullulanases hydrolyzed α-1,4- and α-1,6-linkages in various soluble sugar polymers causing their breakdown to maltose and maltotriose. Unlike these enzymes, the α-amylase produced by C. thermosulfurogenes EM1 preferentially attacked non-soluble starch. The main product of hydrolysis was maltohexaose. The combined action of pullulanases and α-amylase on native starch showed synergistic effect. This synergistic effect was not observed if soluble starch was used as substrate.     

Nitrogen fertilizer and seed rate effects on Hagberg falling number of hybrid wheats and their parents are associated with α‐amylase activity,grain cavity size and dormancy

Field experiments were carried out to assess the effects of nitrogen fertilization and seed rate on the Hagberg falling number (HFN) of commercial wheat hybrids and their parents. Applying nitrogen (200 kg N ha^?1) increased HFN in two successive years. The HFN of the hybrid Hyno Esta was lower than either of its parents (Estica and Audace), particularly when nitrogen was not applied. Treatment effects on HFN were negatively associated with α‐amylase activity. Phadebas grain blotting suggested two populations of grains with different types of α‐amylase activity: Estica appeared to have a high proportion of grains with low levels of late maturity endosperm α‐amylase activity (LMEA); Audace had a few grains showing high levels of germination amylase; and the hybrid, Hyno Esta, combined the sources from both parents to show heterosis for α‐amylase activity. Applying nitrogen reduced both apparent LMEA and germination amylase. The effects on LMEA were associated with the size and disruption of the grain cavity, which was greater in Hyno Esta and Estica and in zero‐nitrogen treatments. External grain morphology failed to explain much of the variation in LMEA and cavity size, but there was a close negative correlation between cavity size and protein content. Applying nitrogen increased post‐harvest dormancy of the grain. Dormancy was greatest in Estica and least in Audace. It is proposed that effects of seed rate, genotype and nitrogen fertilizer on HFN are mediated through factors affecting the size and disruption of the grain cavity and therefore LMEA, and through factors affecting dormancy and therefore germination amylase. Copyright © 2004 Society of Chemical Industry     

Further Methods for the Characterization of Natural Amyloses as Potential Substrates for the Assay of α-Amylase

Improved methods have been devised for the assessment of amylose samples and their suitability for conversion by dyeing with Cibacron Blue 3G-A to give insoluble coloured substrates for the assay of α-amylase. The new methods reported herein are based on: the determination of oligosaccharide component composition of water soluble fractions and enzymatic hydrolysates using gel permeation chromatography with Bio-Gel P-2 media; gel permeation chromatography to assess molecular weight distributions; and enzymic degradation using α-amylase (1,4-α-D -glucan glucanohydrolase, EC 3.2.1.1) and β-amylase (1,4-α-D -glucan maltohydrolase, EC 3.2.1.2).     

α‐(1,4)‐Amylase,but not α‐ and β‐(1,3)‐glucanases,may be responsible for the impaired growth and morphogenesis of Paracoccidioides brasiliensis induced by N‐glycosylation inhibition

The cell wall of Paracoccidioides brasiliensis, which consists of a network of polysaccharides and glycoproteins, is essential for fungal pathogenesis. We have previously reported that N‐glycosylation of proteins such as N‐acetyl‐β‐d ‐glucosaminidase is required for the growth and morphogenesis of P. brasiliensis. In the present study, we investigated the influence of tunycamicin (TM)‐mediated inhibition of N‐linked glycosylation on α‐ and β‐(1,3)‐glucanases and on α‐(1,4)‐amylase in P. brasiliensis yeast and mycelium cells. The addition of 15 µg/ml TM to the fungal cultures did not interfere with either α‐ or β‐(1,3)‐glucanase production and secretion. Moreover, incubation with TM did not alter α‐ and β‐(1,3)‐glucanase activity in yeast and mycelium cell extracts. In contrast, α‐(1,4)‐amylase activity was significantly reduced in underglycosylated yeast and mycelium extracts after exposure to TM. In spite of its importance for fungal growth and morphogenesis, N‐glycosylation was not required for glucanase activities. This is surprising because these activities are directed to wall components that are crucial for fungal morphogenesis. On the other hand, N‐glycans were essential for α‐(1,4)‐amylase activity involved in the production of malto‐oligosaccharides that act as primer molecules for the biosynthesis of α‐(1,3)‐glucan. Our results suggest that reduced fungal α‐(1,4)‐amylase activity affects cell wall composition and may account for the impaired growth of underglycosylated yeast and mycelium cells. © 2013 The Authors. Yeast published by John Wiley & Sons Ltd.     

Characterisation of Dextrins Solubilised by α‐Amylase from Barley Starch Granules

Large granules from barley starch were packed into a column and hydrolysed with α‐amylase by pumping a diluted enzyme solution through the starch bed. The enzyme was then trapped onto an ion‐exchanger and the dextrins that solubilised from the granules were collected and characterised. The size‐distribution of the solubilished dextrins ranged from degree of polymerisation (DP) 2—500. The linear and branched products originated from both the amylose and the amylopectin components. The rate of solubilisation and the composition of the solubilised dextrins from barley starch were very similar to those found for large wheat starch granules.     

Die Wirkung von α-Amylasen auf β-Cyclodextrin und der Nachweis von α-Amylasefremdaktivitäten in ausgewählten Enzympräparaten

The action of α-amylases on β-cyclodextrin and the evidence of foreign activity of α-amylase in selected preparations of enzymes The interaction between cyclodextrins and α-amylases taken from different sources is discribed contradictious in the literature. Some α-amylases e.g. isolated from Aspergillus oryzae, porcine pancreas and saliva hydrolized cyclodextrins to glucose. The hydrolysis of cyclodextrins catalysed by α-amylase from Bacillus species have been described conflicting. In this paper the action of hydrolysis of different preparations of α-amylases on β-cyclodextrin have been investigated. It has been shown that Rohalase M3 (α-amylase from Aspergillus niger) cleaves the ring of β-cyclodextrin. 2 α-amylases from Bacillus subtilis are not able to hydrolyse β-cyclodextrin. The reasons for the different actions of hydrolysis have been discussed with size and structure of the active centre of the enzymes. Moreover, different preparation of hydrolysis have been tested on secondary activity of α-amylase. 2 glucoamylases from Aspergillus oryzae have been shown secondary activity of α-amylase. With the hydrolases α-glucosidase from fungies, β-amylase from malt, saccharase from yeast, invertase from S. cerevisiae and pullulanase from Aerobacter aerogenes no cleavage of the ring of β-cyclodextrin could be detected.     

Electrophoretic studies of α-amylase in wheat. II

The lack of correlation between the degree of sprouting and the α-amylase activity in wheat and rye, as well as the apparent variation in the falling number during ripening, can be explained as the result of two amylase systems acting during different stages of the development of the grain. During the early stages of the development of the grain, α-amylase is continuously inactivated. This process is reversible, however, and when the evaporation of moisture is retarded the α-amylase activity increases as a consequence of the higher amount of dissolved enzyme. This results in an occasional decrease in the falling number, which might amount to more than 50 sec. During germination, a new kind of α-amylase develops. The synthesis of the new form of α-amylase is irreversible and causes a much greater and permanent reduction in the falling number. During the initial stages of germination, the amylase activity thus increases owing to the combined action of both the original amylase and the new form. The two kinds of amylases show different electrophoretic patterns. Drying the grain after harvesting reduces the activity of ‘green’ amylase (amylase in unripened grain), which might explain the frequent observations of increasing falling number during storage.     

ENZYMATIC DETERMINATION OF 1,3:1,4-β-GLUCANS IN BARLEY GRAIN AND OTHER CEREALS†

A direct and specific enzymatic method is described for the determination of 1,3:1,4-β-glucans in barley grain and other cereals. In the procedure, purified, amylase-free, bacterial 1,3:1,4-β-glucan hydrolase is used to depolymerize the 1,3:1,4-β-glucan in autoclaved and ethanol-extracted flour prepared from whole grain. The liberated oligoglucosides are extracted with 80% (vol/vol) ethanol and, following acid hydrolysis, measured by the glucose oxidase method. The method can be used to measure total β-glucan and β-glucan in 65°C water-soluble and water-insoluble fractions of cereal grains. The procedure has been applied to barley grains allowed to develop under controlled environmental conditions and to a series of barleys of different geographical origins. The results for Canadian cultivars are compared with estimates based on viscometric data for the same samples. The 1,3:1,4-β-glucan content of a number of other cereals has also been measured.     

The Significance of Iso‐α‐Acids for Beer Quality Cambridge Prize Paper

The iso‐α‐acids and their chemically‐modified variants play a disproportionately large role in the final quality of beer. Here, fundamental aspects of two of these quality issues — foam and bitterness — are discussed. A common feature of both issues is the dependence on the hydrophobic character of the hop compounds on both bitterness potency and ability to stabilise foams. Thus the isocohumulones appear significantly less bitter than the other, more hydrophobic hop compounds. Also apparent were the differences in bitterness between the cis‐ and the trans‐isomers, with the former being the more potent. Also described are the differences in the partitioning of the cis‐ and trans‐iso‐α‐acids into beer foam. The trans‐isomers are enriched in foam relative to their cis‐counterparts and may account for the observed enrichment of cis‐isomers in the final beer relative to the common ratios observed upstream.     

Corn Starch (α1,4-α1,6) Glucopolysaccharides - Correlation Between Amylose: Amylopectin Ratios and Physical Properties of the Grains

The starch components from corn inbreads and hybrids, mature seeds of commercial interest, were fractionated according to their branching degree. The amount and structural properties of them was determined, allowing a better definition of the polysaccharides present in each fraction. The methodology used allowed the visualization of the introduction of some traits if they result in a structural change of the starch components. It is, therefore, suggested that it is possible to predict the endosperm type related to hardness. A linear correspondence was obtained between amylose % and % of endosperm type floury or horny.