Degradation of Native Starch Granules by Barley alpha-Glucosidases

The initial hydrolysis of native (unboiled) starch granules in germinating cereal kernels is considered to be due to α-amylases. We report that barley (Hordeum vulgare L.) seed α-glucosidases (EC 3.2.1.20) can hydrolyze native starch granules isolated from barley kernels and can do so at rates comparable to those of the predominant α-amylase isozymes. Two α-glucosidase charge isoforms were used individually and in combination with purified barley α-amylases to study in vitro starch digestion. Dramatic synergism, as much as 10.7-fold, of native starch granule hydrolysis, as determined by reducing sugar production, occurred when high pl α-glucosidase was combined with either high or low pl α-amylase. Synergism was also found when low pl α-glucosidase was combined with α-amylases. Scanning electron micrographs revealed that starch granule degradation by α-amylases alone occurred specifically at the equatorial grooves of lenticular granules. Granules hydrolyzed by combinations of α-glucosidases and α-amylases exhibited larger and more numerous holes on granule surfaces than did those granules attacked by α-amylase alone. As the presence of α-glucosidases resulted in more areas being susceptible to hydrolysis, we propose that this synergism is due, in part, to the ability of the α-glucosidases to hydrolyze glucosidic bonds other than α-1,4- and α-1,6- that are present at the granule surface, thereby eliminating bonds which were barriers to hydrolysis by α-amylases. Since both α-glucosidase and α-amylase are synthesized in aleurone cells during germination and secreted to the endosperm, the synergism documented here may function in vivo as well as in vitro.     

Production and Characteristics of Raw-Starch-Digesting α-Amylase from a Protease-Negative Aspergillus ficum Mutant

Mutational experiments were carried out to decrease the protease productivity of Aspergillus ficum IFO 4320 by using N-methyl-N′-nitro-N-nitrosoguanidine. A protease-negative mutant, M-33, exhibited higher α-amylaseactivity than the parent strain under submerged culture at 30°C for 24 h. About 70% of the total α-amylase activity in the M-33 culture filtrate was adsorbed onto starch granules. The electrophoretically homogeneous preparation of raw-starch-adsorbable α-amylase (molecular weight, 88,000), acid stable at pH 2, showed intensive raw-starch-digesting activity, dissolving corn starch granules completely. The preparation also exhibited a high synergistic effect with glucoamylase I. A mutant, M-72, with higher protease activity produced a raw cornstarch-unadsorbable α-amylase. The purified enzyme (molecular weight, 54,000), acid unstable, showed no digesting activity on raw corn starch and a lower synergistic effect with glucoamylase I in the hydrolysis of raw corn starch. The fungal α-amylase was therefore divided into two types, a novel type of raw-starch-digesting enzyme and a conventional type of raw-starch-nondigesting enzyme.     

Affinity chromatography of α-amylase from Bacillus licheniformis

An affinity chromatographic method with a novel eluant from Bacillus licheniformis is described. α-amylase was bound to starch, starch-celite, starch-Sepharose columns and the bound α-amylase was rapidly eluted with 2% (w/v) white dextrin. The binding capacity of α-amylase to starch column is 380 μmol/g of starch. The purified enzyme showed a single polypeptide on SDS-polyacrylamide gel electrophoresis with a molecular weight of 58 kD. The specificity of purified enzyme was confirmed by immunodiffusion, immunoelectrophoresis. Single radial immunodiffusion and western blotting studies analyzed the synthesis of enzyme at different time points.     

Rapid method for the affinity purification of thermostable α-amylase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Summary A rapid and efficient method the exploiting affinity of α-amylase for its substrate starch is described. α-amylase from Bacillus licheniformis was purified to homogeneity by ammonium sulphate precipitation and affinity chromatography with 230-fold purification. The α-amylase adsorption to various starches was examined in order to screen its ability for highest binding to starch. The α-amylase was bound to starch more tenaciously, hence various eluants like maltose, soluble starch and high salts could not elute the bound α-amylase. However, the bound α-amylase was instantly eluted using 2% (w/v) dextrin. The purified enzyme showed a single polypeptide on SDS-PAGE, with a molecular weight of 58 kD. Western blot analysis confirmed the specificity of antibody raised against purified α-amylase.     

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: 8. Immunohistochemical Localization of beta-Amylase

Rabbit antiserum against β-amylase isolated from germinating seeds of rice was produced, and its specific cross-reactivity with β-amylase was confirmed by means of Ouchterlony double immunodiffusion and immunoelectrophoresis procedures. The cellular localization of β-amylase was studied by indirect fluorescence microscopy of thin sectioned germinating rice seed specimens (1-day stage) which had been fixed and treated with purified rabbit anti-β-amylase immunoglobulin G followed by conjugation with fluorescein isothiocyanate-labeled goat antirabbit immunoglobulin G. It has been demonstrated that β-amylase is uniformly associated with the periphery of starch granules in the starchy endosperm cells. The finding is discussed in relation to the general notion concerning the presence of the latent form of β-amylase bound to protein bodies in cereal seeds.     

Effect of α-Amylase Degradation on Physicochemical Properties of Pre-High Hydrostatic Pressure-Treated Potato Starch

The effect of high hydrostatic pressure (HHP) on the susceptibility of potato starch (25%, w/v) suspended in water to degradation by exposure to bacterial α-amylase (0.02%, 0.04% and 0.06%, w/v) for 40 min at 25°C was investigated. Significant differences (p < 0.05) in the structure, morphology and physicochemical properties were observed. HHP-treated potato starch (PS) exposed to α-amylase (0.06%, w/v) showed a significantly greater degree of hydrolysis and amount of reducing sugar released compared to α-amylase at a concentration of 0.04% (w/v) or 0.02% (w/v). Native PS (NPS) granules have a spherical and elliptical form with a smooth surface, whereas the hydrolyzed NPS (hNPS) and hydrolyzed HHP-treated PS granules showed irregular and ruptured forms with several cracks and holes on the surface. Hydrolysis of HHP-treated PS by α-amylase could decrease the average granule size significantly (p <0.05) from 29.43 to 20.03 μm. Swelling power decreased and solubility increased with increasing enzyme concentration and increasing pressure from 200–600 MPa, with the exception of the solubility of HHP-treated PS at 600 MPa (HHP₆₀₀ PS). Fourier transform infrared spectroscopy (FTIR) showed extensive degradation of the starch in both the ordered and the amorphous structure, especially in hydrolyzed HHP₆₀₀ PS. The B-type of hydrolyzed HHP₆₀₀ PS with α-amylase at a concentration 0.06% (w/v) changed to a B+V type with an additional peak at 2θ = 19.36°. The HHP₆₀₀ starch with 0.06% (w/v) α-amylase displayed the lowest value of T _o (onset temperature), T _c (conclusion temperature) and ΔH _gel (enthalpies of gelatinization). These results indicate the pre-HHP treatment of NPS leads to increased susceptibility of the granules to enzymatic degradation and eventually changes of both the amorphous and the crystalline structures.     

Degradation of starchy substrates by a crude enzyme preparation and utilization of the hydrolysates for lactic fermentation

An enzyme preparation obtained from Aspergillus ustus, possessing cellulase, α-amylase, amyloglucosidase, proteinase and d-xylanase activities, was used along with commercial bacterial α-amylase and amyloglucosidase for the degradation of ragi (Eleusine coracana) flour and wheat (Triticum vulgare) bran. Lactic acid yield from ragi hydrolysate, adjusted to 5% reducing sugars (w/v), was 25% when fermented with Lactobacillus plantarum. The yields increased to 78% and 94% when the ragi hydrolysate was fortified with 20% and 60% (v/v) wheat bran hydrolysate, respectively. When commercial α-amylase and amyloglucosidase were used for the hydrolysis of ragi and wheat bran and L. plantarum was employed to ferment the hydrolysates containing 5% reducing sugars (w/v), lactic acid yields were 10% in ragi hydrolysate and 57% and 90% when the ragi hydrolysate was fortified with 20% and 60% (v/v) of wheat bran hydrolysate, respectively. α-Amylase and amyloglucosidase hydrolysed wheat bran added at 20% (v/v) as the sole source of nutrient to soluble starch hydrolysate (5% reducing sugars) gave 22% yield of lactic acid. The yield increased to 55% by the utilization of A. ustus enzyme preparation in addition to α-amylase and amyloglucosidase for wheat bran hydrolysis.     

σ-Amylase inactivation during corn starch hydrolysis process

The effects of operating conditions on the enzymatic hydrolysis of corn starch were investigated. A commercial α-amylase produced by Bacillus sp. was used for the hydrolysis experiments. The degree of starch hydrolysis (%) and residual α-amylase activity (%) was investigated versus process variables, including pH, temperature, viscosity, impeller speed, processing time and some materials added such as hydrolysate, maltose, glucose, ethanol and CaCl₂ using a stirred batch reactor. The mathematical models depending on the operating conditions were also derived using the experimental data of residual starch concentration. Some inactivation models were tested to determine the relationship between process variables and enzyme stability during the hydrolysis process.     

Degradation of Raw Starch by a Wild Amylolytic Strain of Lactobacillus plantarum

Lactobacillus plantarum A6, isolated from fermented cassava, can break down cassava raw starch that has not been subjected to preliminary physicochemical treatment. When the pH was kept at 6, the microorganism cultured in a bioreactor excreted a high α-amylase activity (60 U/ml). Synthesis of the enzyme occurred during the stationary phase and resulted in full hydrolysis of the cassava starch granules. This gave 41 g of lactic acid from 45 g of raw starch after 3 days of fermentation. Enzymatic attack was evident under scanning electron microscopy in the rougher appearance of the surface of starch granules and in the presence of large cavities in some of them. In contrast, when the pH was not regulated, only a small amount of α-amylase activity was produced (2 U/ml) and no decrease in the starch content of the medium was observed. However, under scanning electron microscopy, some granules displayed a rougher surface, which might have been the result of weak enzymatic attack.     

Starch Granule Biosynthesis in Arabidopsis Is Abolished by Removal of All Debranching Enzymes but Restored by the Subsequent Removal of an Endoamylase

Several studies have suggested that debranching enzymes (DBEs) are involved in the biosynthesis of amylopectin, the major constituent of starch granules. Our systematic analysis of all DBE mutants of Arabidopsis thaliana demonstrates that when any DBE activity remains, starch granules are still synthesized, albeit with altered amylopectin structure. Quadruple mutants lacking all four DBE proteins (Isoamylase1 [ISA1], ISA2, and ISA3, and Limit-Dextrinase) are devoid of starch granules and instead accumulate highly branched glucans, distinct from amylopectin and from previously described phytoglycogen. A fraction of these glucans are present as discrete, insoluble, nanometer-scale particles, but the structure and properties of this material are radically altered compared with wild-type amylopectin. Superficially, these data support the hypothesis that debranching is required for amylopectin synthesis. However, our analyses show that soluble glucans in the quadruple DBE mutant are degraded by α- and β-amylases during periods of net accumulation, giving rise to maltose and branched malto-oligosaccharides. The additional loss of the chloroplastic α-amylase AMY3 partially reverts the phenotype of the quadruple DBE mutant, restoring starch granule biosynthesis. We propose that DBEs function in normal amylopectin synthesis by promoting amylopectin crystallization but conclude that they are not mandatory for starch granule synthesis.     

Arabidopsis thaliana AMY3 Is a Unique Redox-regulated Chloroplastic α-Amylase

α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. In vascular plants, α-amylases can be classified into three subfamilies. Arabidopsis has one member of each subfamily. Among them, only AtAMY3 is localized in the chloroplast. We expressed and purified AtAMY3 from Escherichia coli and carried out a biochemical characterization of the protein to find factors that regulate its activity. Recombinant AtAMY3 was active toward both insoluble starch granules and soluble substrates, with a strong preference for β-limit dextrin over amylopectin. Activity was shown to be dependent on a conserved aspartic acid residue (Asp⁶⁶⁶), identified as the catalytic nucleophile in other plant α-amylases such as the barley AMY1. AtAMY3 released small linear and branched glucans from Arabidopsis starch granules, and the proportion of branched glucans increased after the predigestion of starch with a β-amylase. Optimal rates of starch digestion in vitro was achieved when both AtAMY3 and β-amylase activities were present, suggesting that the two enzymes work synergistically at the granule surface. We also found that AtAMY3 has unique properties among other characterized plant α-amylases, with a pH optimum of 7.5–8, appropriate for activity in the chloroplast stroma. AtAMY3 is also redox-regulated, and the inactive oxidized form of AtAMY3 could be reactivated by reduced thioredoxins. Site-directed mutagenesis combined with mass spectrometry analysis showed that a disulfide bridge between Cys⁴⁹⁹ and Cys⁵⁸⁷ is central to this regulation. This work provides new insights into how α-amylase activity may be regulated in the chloroplast.     

STARCH-EXCESS4 Is a Laforin-Like Phosphoglucan Phosphatase Required for Starch Degradation in Arabidopsis thaliana

Starch is the major storage carbohydrate in plants. It is comprised of glucans that form semicrystalline granules. Glucan phosphorylation is a prerequisite for normal starch breakdown, but phosphoglucan metabolism is not understood. A putative protein phosphatase encoded at the Starch Excess 4 (SEX4) locus of Arabidopsis thaliana was recently shown to be required for normal starch breakdown. Here, we show that SEX4 is a phosphoglucan phosphatase in vivo and define its role within the starch degradation pathway. SEX4 dephosphorylates both the starch granule surface and soluble phosphoglucans in vitro, and sex4 null mutants accumulate phosphorylated intermediates of starch breakdown. These compounds are linear α-1,4-glucans esterified with one or two phosphate groups. They are released from starch granules by the glucan hydrolases α-amylase and isoamylase. In vitro experiments show that the rate of starch granule degradation is increased upon simultaneous phosphorylation and dephosphorylation of starch. We propose that glucan phosphorylating enzymes and phosphoglucan phosphatases work in synergy with glucan hydrolases to mediate efficient starch catabolism.     

Enzymes of starch metabolism in the developing rice grain

The levels of starch, soluble sugars, protein, and enzymes involved in starch metabolism—α-amylase, β-amylase, phosphorylase, Q-enzyme, R-enzyme, and starch synthetase —were assayed in dehulled developing rice grains (Oryzasativa L., variety IR8). Phosphorylase, Q-enzyme, and R-enzyme had peak activities 10 days after flowering, whereas α- and β-amylases had maximal activities 14 days after flowering. Starch synthetase bound to the starch granule increased in activity up to 21 days after flowering. These enzymes (except the starch synthetases) were also detected by polyacrylamide gel electrophoresis. Their activity in grains at the midmilky stage (8-10 days after flowering) was determined in five pairs of lines with low and high amylose content from different crosses. The samples had similar levels of amylases, phosphorylase, R-enzyme, and Q-enzyme. The samples consistently differed in their levels of starch synthetase bound to the starch granule, which was proportional to amylose content. Granule-bound starch synthetase may be responsible for the integrity of amylose in the developing starch granule.     

Crosslinked potato starch as an affinity adsorbent for bacterial α-amylase

Crosslinked potato starch was prepared as an affinity adsorbent for bacterial α-amylase. To this end, reaction parameters for crosslinking in an ethanol/water solvent were investigated. The degree of crosslinking, and consequently the suitability of crosslinked starch as an adsorbent for α-amylase, changed by altering these parameters. An increase in the degree of crosslinking of the adsorbent caused lower affinity for bacterial α-amylase which resulted in an unfavourable decrease in adsorption capacity and a favourable decrease in the degradation of the adsorbent by the enzyme. 1 g of a suitable adsorbent for bacterial α-amylase, prepared with an epichlorohydrin/glucose monomer ratio of 0·65 (starch concentration 150 mg/ml, ethanol/water ratio 2·0, sodium hydroxide/epichlorohydrin ratio 1·0), can adsorb 9·8 mg of an α-amylase from B. licheniformis at 4°C in 20 h.The equilibrium constant between bound and unbound α-amylase is dependent on the temperature. An effective desorption was possible by a shift to higher temperatures. Degradation values smaller than 0·1% were measured after an incubation of 1 h at 70°C in a desorption buffer with 20% glycerol.It was concluded that coulombic interactions and hydrogen bonds are of no or little importance in enzyme adsorption. Van der Waals forces, which are responsible for the large temperature effect, are the main forces in the interaction between α-amylase and crosslinked starch.     

Mammalian Mucosal α-Glucosidases Coordinate with α-Amylase in the Initial Starch Hydrolysis Stage to Have a Role in Starch Digestion beyond Glucogenesis

Starch digestion in the human body is typically viewed in a sequential manner beginning with α-amylase and followed by α-glucosidase to produce glucose. This report indicates that the two enzyme types can act synergistically to digest granular starch structure. The aim of this study was to investigate how the mucosal α-glucosidases act with α-amylase to digest granular starch. Two types of enzyme extracts, pancreatic and intestinal extracts, were applied. The pancreatic extract containing predominantly α-amylase, and intestinal extract containing a combination of α-amylase and mucosal α-glucosidase activities, were applied to three granular maize starches with different amylose contents in an in vitro system. Relative glucogenesis, released maltooligosaccharide amounts, and structural changes of degraded residues were examined. Pancreatic extract-treated starches showed a hydrolysis limit over the 12 h incubation period with residues having a higher gelatinization temperature than the native starch. α-Amylase combined with the mucosal α-glucosidases in the intestinal extract showed higher glucogenesis as expected, but also higher maltooligosaccharide amounts indicating an overall greater degree of granular starch breakdown. Starch residues after intestinal extract digestion showed more starch fragmentation, higher gelatinization temperature, higher crystallinity (without any change in polymorph), and an increase of intermediate-sized or small-sized fractions of starch molecules, but did not show preferential hydrolysis of either amylose or amylopectin. Direct digestion of granular starch by mammalian recombinant mucosal α-glucosidases was observed which shows that these enzymes may work either independently or together with α-amylase to digest starch. Thus, mucosal α-glucosidases can have a synergistic effect with α-amylase on granular starch digestion, consistent with a role in overall starch digestion beyond their primary glucogenesis function.     

Sweet potato: A novel substrate for pullulan production by Aureobasidium pullulans

A strain Aureobasidium pullulans AP329, was used for the production of pullulan by employing hydrolysed sweet potato as cultivation media. Hydrolysis with α-amylase alone resulted in the lowest yields of pullulan. In contrast continuous hydrolysis with pullulanase and the β-amylase in sweet potato itself gave higher yields, but prolonged hydrolysis with amyloglucosidase decreased the yield. The maximum pullulan yield (29.43 g/l) was achieved at the dextrose equivalent value of 45 and pH of 5.5 for 96 h. As a substitute of sucrose, hydrolysed sweet potato was found to be hopeful and the yield of pullulan was higher than that of glucose and sucrose. The molecular weight of pullulan obtained from hydrolysed sweet potato media was much higher than that of sucrose and glucose media. Results of this work indicated that sweet potato was a promising substrate for the economical production of pullulan.     

Characterization of alpha-Amylase from Shoots and Cotyledons of Pea (Pisum sativum L.) Seedlings

The most abundant α-amylase (EC 3.2.1.1) in shoots and cotyledons from pea (Pisum sativum L.) seedlings was purified 6700-and 850-fold, respectively, utilizing affinity (amylose and cycloheptaamylose) and gel filtration chromatography and ultrafiltration. This α-amylase contributed at least 79 and 15% of the total amylolytic activity in seedling cotyledons and shoots, respectively. The enzyme was identified as an α-amylase by polarimetry, substrate specificity, and end product analyses. The purified α-amylases from shoots and cotyledons appear identical. Both are 43.5 kilodalton monomers with pls of 4.5, broad pH activity optima from 5.5 to 6.5, and nearly identical substrate specificities. They produce identical one-dimensional peptide fingerprints following partial proteolysis in the presence of SDS. Calcium is required for activity and thermal stability of this amylase. The enzyme cannot attack maltodextrins with degrees of polymerization below that of maltotetraose, and hydrolysis of intact starch granules was detected only after prolonged incubation. It best utilizes soluble starch as substrate. Glucose and maltose are the major end products of the enzyme with amylose as substrate. This α-amylase appears to be secreted, in that it is at least partially localized in the apoplast of shoots. The native enzyme exhibits a high degree of resistance to degradation by proteinase K, trypsin/chymostrypsin, thermolysin, and Staphylococcus aureus V8 protease. It does not appear to be a high-mannose-type glycoprotein. Common cell wall constituents (e.g. β-glucan) are not substrates of the enzyme. A very low amount of this α-amylase appears to be associated with chloroplasts; however, it is unclear whether this activity is contamination or α-amylase which is integrally associated with the chloroplast.     

The molecular deposition of transgenically modified starch in the starch granule as imaged by functional microscopy

The molecular deposition of starch extracted from normal plants and transgenically modified potato lines was investigated using a combination of light microscopy, environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy (CLSM). ESEM permitted the detailed (10 nm) topographical analysis of starch granules in their hydrated state. CLSM could reveal internal molar deposition patterns of starch molecules. This was achieved by equimolar labelling of each starch molecule using the aminofluorophore 8-amino-1,3,6-pyrenetrisulfonic acid (APTS). Starch extracted from tubers with low amylose contents (suppressed granule bound starch synthase, GBSS) showed very little APTS fluorescence and starch granules with low molecular weight amylopectin and/or high amylose contents showed high fluorescence. Growth ring structures were sharper in granules with normal or high amylose contents. High amylose granules showed a relatively even distribution in fluorescence while normal and low amylose granules had an intense fluorescence in the hilum indicating a high concentration of amylose in the centre of the granule. Antisense of the starch phosphorylating enzyme (GWD) resulted in low molecular weight amylopectin and small fissures in the granules. Starch granules with suppressed starch branching enzyme (SBE) had severe cracks and rough surfaces. Relationships between starch molecular structure, nano-scale crystalline arrangements and topographical-morphological features were estimated and discussed.     

Chemical and physical properties of ginkgo (Ginkgo biloba) starch

Starch isolated from mature Ginkgo biloba seeds and commercial normal maize starches were subjected to α-amylolysis and acid hydrolysis. Ginkgo starch was more resistant to pancreatic α-amylase hydrolysis than the normal maize starch. The chain length distribution of debranched amylopectin of the starches was analyzed by using high performance anion-exchange chromatography equipped with an amyloglucosidase reactor and a pulsed amperometric detector. The chain length distribution of ginkgo amylopectin showed higher amounts of both short and long chains compared to maize starch. Naegeli dextrins of the starches prepared by extensive acid hydrolysis over 12 days demonstrated that ginkgo starch was more susceptible than normal maize to acid hydrolysis. Ginkgo dextrins also demonstrate a lower concentration of singly branched chains than maize dextrins, and unlike maize dextrin, debranched ginkgo shows no multiple branched chains. The ginkgo starch displayed a C-type X-ray diffraction pattern, compared to an A-type pattern for maize. Ginkgo starch and maize starch contained 24.0 and 17.6% absolute amylose contents, respectively.     

Systematic Analysis of Pericarp Starch Accumulation and Degradation during Wheat Caryopsis Development

Although wheat (Triticum aestivum L.) pericarp starch granule (PSG) has been well-studied, our knowledge of its features and mechanism of accumulation and degradation during pericarp growth is poor. In the present study, developing wheat caryopses were collected and starch granules were extracted from their pericarp to investigate the morphological and structural characteristics of PSGs using microscopy, X-ray diffraction and Fourier transform infrared spectroscopy techniques. Relative gene expression levels of ADP-glucose pyrophosphorylase (APGase), granule-bound starch synthase II (GBSS II), and α-amylase (AMY) were quantified by quantitative real-time polymerase chain reaction. PSGs presented as single or multiple starch granules and were synthesized both in the amyloplast and chloroplast in the pericarp. PSG degradation occurred in the mesocarp, beginning at 6 days after anthesis. Amylose contents in PSGs were lower and relative degrees of crystallinity were higher at later stages of development than at earlier stages. Short-range ordered structures in the external regions of PSGs showed no differences in the developing pericarp. When hydrolyzed by α-amylase, PSGs at various developmental stages showed high degrees of enzymolysis. Expression levels of AGPase, GBSS II, and AMY were closely related to starch synthesis and degradation. These results help elucidate the mechanisms of accumulation and degradation as well as the functions of PSG during wheat caryopsis development.