Starch degradation in the cotyledons of germinating lentils

Starch, total amylolytic and phosphorylase activities were determined in lentil cotyledons during the first days of germination. Several independent criteria show that the amylolytic activity is due mainly to an amylase of the α type. Starch is degraded slowly in the first days; during this time, α- and β-amylase activity are very low, while phosphorylase increases and reach a peak on the 3rd day. On the 4th day, there is a more rapid depletion of starch which coincides with an increase in α-amylase activity. By polyacrylamide gel electrophoresis of the crude starch-degrading enzyme, five bands were obtained: one phosphorylase, three α-amylases, and one β-amylase. Based on their heat lability or heat stability, two sets of α-amylase seem to exist in lentil cotyledons.     

Specific Determination of alpha-Amylase Activity in Crude Plant Extracts Containing beta-Amylase

The specific measurement of α-amylase activity in crude plant extracts is difficult because of the presence of β-amylases which directly interfere with most assay methods. Methods compared in this study include heat treatment at 70°C for 20 min, HgCl₂ treatment, and the use of the α-amylase specific substrate starch azure. In comparing alfalfa (Medicago sativa L.), soybeans (Glycine max [L.] Merr.), and malted barley (Hordeum vulgare L.), the starch azure assay was the only satisfactory method for all tissues. While β-amylase can liberate no color alone, over 10 International units per milliliter β-amylase activity has a stimulatory effect on the rate of color release. This stimulation becomes constant (about 4-fold) at β-amylase activities over 1,000 International units per milliliter. Two starch azure procedures were developed to eliminate β-amylase interference: (a) the dilution procedure, the serial dilution of samples until β-amylase levels are below levels that interfere; (b) the β-amylase saturation procedure, addition of exogenous β-amylase to increase endogenous β-amylase activity to saturating levels. Both procedures yield linear calibrations up to 0.3 International units per milliliter. These two procedures produced statistically identical results with most tissues, but not for all tissues. Differences between the two methods with some plant tissues was attributed to inaccuracy with the dilution procedure in tissues high in β-amylase activity or inhibitory effects of the commercial β-amylase. The β-amylase saturation procedure was found to be preferable with most species. The heat treatment was satisfactory only for malted barley, as α-amylases in alfalfa and soybeans are heat labile. Whereas HgCl₂ proved to be a potent inhibitor of β-amylase activity at concentrations of 10 to 100 micromolar, these concentrations also partially inhibited α-amylase in barley malt. The reported α-amylase activities in crude enzyme extracts from a number of plant species are apparently the first specific measurements reported for any plant tissues other than germinating cereals.     

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.     

Amylases in Pea Tissues with Reduced Chloroplast Density and/or Function

Pea (Pisum sativum L.) tissues with reduced chloroplast density (e.g. petals and stems) or function (i.e. senescent leaves and leaves darkened for prolonged periods) were surveyed to determine whether tissues with genetically or environmentally reduced chloroplast density and/or function also have significantly different amylolytic enzyme activities and/or isoform patterns than leaf tissues with totally competent chloroplasts. Native PAGE followed by electrophoretically blotting through a starch or β-limit dextrin containing gel and KI/I₂ staining revealed that the primary amylases in leaves, stems, petals, and roots were the primarily vacuolar β-amylase (EC 3.2.1.2) and the primarily apoplastic α-amylase (EC 3.2.1.1). Among tissues of light grown pea plants, petals contained the highest levels of total amylolytic (primarily β-amylase) activity and considerably higher ratios of β- to α-amylase. In aerial tissues there was an inverse relationship between chlorophyll and starch concentration, and β-amylase activity. In sections of petals and stems there was a pronounced inverse relationship between chlorophyll concentration and the activity of α-amylase. Senescing leaves of pea, as determined by age, and protein and chlorophyll content, contained 3.8-fold (fresh weight basis) and 32-fold (protein basis) higher α-amylase activity than fully mature leaves. Leaves maintained in darkness for 12 days displayed a 14-fold (fresh weight basis) increase in α-amylase activity over those grown under continuous light. In senescence and prolonged darkness studies, the α-amylase that was greatly increased in activity was the primarily apoplastic α-amylase. These studies indicate that there is a pronounced inverse relationship between chloroplast function and levels of apoplastic α-amylase activity and in some cases an inverse relationship between chloroplast density and/or function and vacuolar β-amylase activity.     

Enzymatic Analysis of an Amylolytic Enzyme from the Hyperthermophilic Archaeon Pyrococcus furiosus Reveals Its Novel Catalytic Properties as both an α-Amylase and a Cyclodextrin-Hydrolyzing Enzyme

Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF1939) similar to the enzymes in glycoside hydrolase family 13. This amylolytic enzyme, designated PFTA (Pyrococcus furiosus thermostable amylase), was cloned and expressed in Escherichia coli. The recombinant PFTA was extremely thermostable, with an optimum temperature of 90°C. The substrate specificity of PFTA suggests that it possesses characteristics of both α-amylase and cyclodextrin-hydrolyzing enzyme. Like typical α-amylases, PFTA hydrolyzed maltooligosaccharides and starch to produce mainly maltotriose and maltotetraose. However, it could also attack and degrade pullulan and β-cyclodextrin, which are resistant to α-amylase, to primarily produce panose and maltoheptaose, respectively. Furthermore, acarbose, a potent α-amylase inhibitor, was drastically degraded by PFTA, as is typical of cyclodextrin-hydrolyzing enzymes. These results confirm that PFTA possesses novel catalytic properties characteristic of both α-amylase and cyclodextrin-hydrolyzing enzyme.     

Starch metabolism in the leaf sheaths and culm of rice

The levels of starch and dextrin, free sugars, soluble protein, and enzymes involved in starch metabolism—α-amylase, β-amylase, phosphorylase, Q-enzyme, R-enzyme, and ADP-glucose starch synthetases—were assayed in the leaf sheaths and culm of the rice plant (Oryza sativa L., variety IR8) during growth.     

An endogenous alpha-amylase inhibitor in barley kernels

Barley (Hordeum distichum cv Klages) kernels were shown to contain a factor that converted malted barley α-amylase II to the α-amylase III form. After purification by ammonium sulfate fractionation, ion exchange chromatography on DEAE-Sephacel, and gel-filtration on Bio Gel P60, the factor gave a single band of protein on isoelectric focusing. The purified factor inhibited hydrolysis of soluble starch by α-amylase II from malted barley and germinated wheat (Triticum aestivum cv Neepawa). However, α-amylase I from these cereals was not affected. The inhibitor was not dialyzable and was retained by a PM 10 ultrafiltration membrane suggesting a molecular weight greater than 10,000 daltons. Heat treatment of the inhibitor at 70°C for 15 minutes at pH 5.5 and 8.0 resulted in considerable loss of inhibitory activity.     

Thermostable Amylolytic Enzymes from a New Clostridium Isolate

A new Clostridium strain was isolated on starch at 60°C. Starch, pullulan, maltotriose, and maltose induced the synthesis of α-amylase and pullulanase, while glucose, ribose, fructose, and lactose did not. The formation of the amylolytic enzymes was dependent on growth and occurred predominantly in the exponential phase. The enzymes were largely cell bound during growth of the organism with 0.5% starch, but an increase of the starch concentration in the growth medium was accompanied by the excretion of α-amylase and pullulanase into the culture broth; but also by a decrease of total activity. α-Amylase, pullulanase, and α-glucosidase were active in a broad temperature range (40 to 85°C) and displayed temperature optima for activity at 60 to 70°C. During incubation with starch under aerobic conditions at 75°C for 2 h, the activity of both enzymes decreased to only 90 or 80%. The apparent K_m values of α-amylase, pullulanase, and α-glucosidase for their corresponding substrates, starch, pullulan, and maltose were 0.35 mg/ml, 0.63 mg/ml, and 25 mM, respectively.     

Purification and Characterization of Pea Epicotyl beta-Amylase

The most abundant β-amylase (EC 3.2.1.2) in pea (Pisum sativum L.) was purified greater than 880-fold from epicotyls of etiolated germinating seedlings by anion exchange and gel filtration chromatography, glycogen precipitation, and preparative electrophoresis. The electrophoretic mobility and relative abundance of this β-amylase are the same as that of an exoamylase previously reported to be primarily vacuolar. The enzyme was determined to be a β-amylase by end product analysis and by its inability to hydrolyze β-limit dextrin and to release dye from starch azure. Pea β-amylase is an approximate 55 to 57 kilodalton monomer with a pl of 4.35, a pH optimum of 6.0 (soluble starch substrate), an Arrhenius energy of activation of 6.28 kilocalories per mole, and a K_m of 1.67 milligrams per milliliter (soluble starch). The enzyme is strongly inhibited by heavy metals, p-chloromer-curiphenylsulfonic acid and N-ethylmaleimide, but much less strongly by iodoacetamide and iodoacetic acid, indicating cysteinyl sulfhydryls are not directly involved in catalysis. Pea β-amylase is competitively inhibited by its end product, maltose, with a K_i of 11.5 millimolar. The enzyme is partially inhibited by Schardinger maltodextrins, with α-cyclohexaamylose being a stronger inhibitor than β-cycloheptaamylose. Moderately branched glucans (e.g. amylopectin) were better substrates for pea β-amylase than less branched or non-branched (amyloses) or highly branched (glycogens) glucans. The enzyme failed to hydrolyze native starch grains from pea and glucans smaller than maltotetraose. The mechanism of pea β-amylase is the multichain type. Possible roles of pea β-amylase in cellular glucan metabolism are discussed.     

Diurnal oscillation of amylolytic activity in spinach chloroplasts

Chloroplasts isolated from spinach (Spinacia oleracea L., cv. vital^R) plants grown under controlled light/dark and temperature regimes, contained the phosphorolytic and amylolytic pathways for starch breakdown. The latter consists at least of α- and β-amylase and maltase. Only low amylolytic activity was observed in chloroplasts isolated during the light phase. In chloroplasts prepared during the dark phase, this activity was almost twice as high. These diurnal oscillations of the amylolytic activity were maintained when the plants were kept in prolonged darkness or continuous light. The amylolytic system exhibited a sharp pH optimum between 5.8 and 6.0. Phosphorylase activity, when assayed with saturating concentrations of inorganic phosphate, did not show diurnal fluctuations.     

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.     

Chloroplastic regulation of apoplastic alpha-amylase activity in pea seedlings

Photobleaching of pea (Pisum sativum L.) seedling leaves by treatment with norflurazon (San 9789) and 7 days of continuous white light caused a 76- to 85-fold increase in the activity of the primary α-amylase, a largely apoplastic enzyme, over normally greening seedlings. Levels of chlorophyll were near zero and levels of plastid marker enzyme activities were very low in norflurazon-treated seedlings, indicating severe photooxidative damage to plastids. As levels of norflurazon or fluence rates were lowered, decreasing photobleaching of tissues, α-amylase activity decreased. Levels of leaf β-amylase and starch debranching enzyme changed very little in norflurazon-treated seedlings. Infiltration extraction of leaves of norflurazon-treated and normally greening seedlings indicated that at least 57 and 62%, respectively, of α-amylase activity was in the apoplast. α-Amylase activity recovered from the apoplast of photobleached leaves of norflurazon-treated seedlings was 18-fold higher than that for green leaves. Inhibitors of photosynthesis (DCMU and atrazine) and an inhibitor of chlorophyll accumulation that does not cause photooxidation of plastid components (tentoxin) had little effect on levels of α-amylase activity, indicating norflurazon-caused loss of chlorophyll and lack of photosynthesis did not cause the large induction in α-amylase activity. An inhibitor of both abscisic acid and gibberellin synthesis (paclobutrazol [PP333]) and an analog of norflurazon which inhibits photosynthesis but not carotenoid synthesis (San 9785) caused only moderate (about five-fold) increases in α-amylase activity. Lincomycin and chloramphenicol increased α-amylase activity in light grown seedings to the same magnitude as norflurazon, indicating that the effect of norflurazon is probably through the destruction of plastid ribosomes. It is proposed that chloroplasts produce a negative signal for the regulation of the apoplastic α-amylase in pea.     

Localization of alpha-Amylase in the Apoplast of Pea (Pisum sativum L.) Stems

Most of the activity of an α-amylase present in crude pea (Pisum sativum L. cv Laxton's Progress No. 9) leaf preparations cannot be found in isolated pea leaf protoplasts. The same extrachloroplastic α-amylase is present in pea stems, representing approximately 6% of total stem amylolytic activity and virtually all of the α-amylase activity. By a simple infiltration-extraction procedure, the majority (87%) of this α-amylase activity was recovered from the pea stem apoplast without significantly disrupting the symplastic component of the tissue. Only 3% of the β-amylase activity and less than 2% of other cellular marker enzymes were removed during infiltration-extraction.     

The ram1 Mutant of Arabidopsis Exhibits Severely Decreased β-Amylase Activity

Despite extensive biochemical analyses, the biological function(s) of plant β-amylases remains unclear. The fact that β-amylases degrade starch in vitro suggests that they may play a role in starch metabolism in vivo. β-Amylases have also been suggested to prevent the accumulation of highly polymerized polysaccharides that might otherwise impede flux through phloem sieve pores. The identification and characterization of a mutant of Arabidopsis var. Columbia with greatly reduced levels of β-amylase activity is reported here. The reduced β-amylase 1 (ram1) mutation lies in the gene encoding the major form of β-amylase in Arabidopsis. Although the Arabidopsis genome contains nine known or putative β-amylase genes, the fact that the ram1 mutation results in almost complete loss of β-amylase activity in rosette leaves and inflorescences (stems) indicates that the gene affected by the ram1 mutation is responsible for most of the β-amylase activity present in these tissues. The leaves of ram1 plants accumulate wild-type levels of starch, soluble sugars, anthocyanin, and chlorophyll. Plants carrying the ram1 mutation also exhibit wild-type rates of phloem exudation and of overall growth. These results suggest that little to no β-amylase activity is required to maintain normal starch levels, rates of phloem exudation, and overall plant growth.     

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.     

Bacillus stearothermophilus Neopullulanase Selective Hydrolysis of Amylose to Maltose in the Presence of Amylopectin

The specificity of Bacillus stearothermophilus TRS40 neopullulanase toward amylose and amylopectin was analyzed. Although this neopullulanase completely hydrolyzed amylose to produce maltose as the main product, it scarcely hydrolyzed amylopectin. The molecular mass of amylopectin was decreased by only one order of magnitude, from approximately 10⁸ to 10⁷ Da. Furthermore, this neopullulanase selectively hydrolyzed amylose when starch was used as a substrate. This phenomenon, efficient hydrolysis of amylose but not amylopectin, was also observed with cyclomaltodextrinase from alkaliphilic Bacillus sp. strain A2-5a and maltogenic amylase from Bacillus licheniformis ATCC 27811. These three enzymes hydrolyzed cyclomaltodextrins and amylose much faster than pullulan. Other amylolytic enzymes, such as bacterial saccharifying α-amylase, bacterial liquefying α-amylase, β-amylase, and neopullulanase from Bacillus megaterium, did not exhibit this distinct substrate specificity at all, i.e., the preference of amylose to amylopectin.     

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.     

Purification of a beta-Amylase that Accumulates in Arabidopsis thaliana Mutants Defective in Starch Metabolism

Amylase activity is elevated 5- to 10-fold in leaves of several different Arabidopsis thaliana mutants defective in starch metabolism when they are grown under a 12-hour photoperiod. Activity is also increased when plants are grown under higher light intensity. It was previously determined that the elevated activity was an extrachloroplastic β-(exo)amylase. Due to the location of this enzyme outside the chloroplast, its function is not known. The enzyme was purified to homogeneity from leaves of both a starchless mutant deficient in plastid phosphoglucomutase and from the wild type using polyethylene glycol fractionation and cyclohexaamylose affinity chromatography. The molecular mass of the β-amylase from both sources was 55,000 daltons as determined by denaturing gel electrophoresis. Gel filtration studies indicated that the enzyme was a monomer. The specific activities of the purified protein from mutant and wild-type sources, their substrate specificities, and K_m for amylopectin were identical. Based on these results it was concluded that the mutant contained an increased level of β-amylase protein. Enzyme neutralization studies using a polyclonal antiserum raised to purified β-amylase showed that in each of two starchless mutants, one starch deficient mutant and one starch overproducing mutant, the elevated amylase activity was due to elevated β-amylase protein.     

Washingtonia filifera seed extracts inhibit the islet amyloid polypeptide fibrils formations and α-amylase and α-glucosidase activity

Washingtonia filifera seeds have revealed to possess antioxidant properties, butyrylcholinesterase and xanthine oxidase inhibition activities. The literature has indicated a relationship between Alzheimer’s disease (AD) and type-2 diabetes (T2D). Keeping this in mind, we have now evaluated the inhibitory properties of W. filifera seed extracts on α-amylase, α-glucosidase enzyme activity and the Islet Amyloid Polypeptide (IAPP) fibrils formation.Three extracts from seeds of W. filifera were evaluated for their enzyme inhibitory effect and IC₅₀ values were calculated for all the extracts. The inhibition mode was investigated by Lineweaver-Burk plot analysis and the inhibition of IAPP aggregate formation was monitored.W. filifera methanol seed extract appears as the most potent inhibitor of α-amylase, α-glucosidase, and for the IAPP fibril formation.Current findings indicate new potential of this extract that could be used for the identification or development of novel potential agents for T2D and AD.     

Simultaneous and Enhanced Production of Thermostable Amylases and Ethanol from Starch by Cocultures of Clostridium thermosulfurogenes and Clostridium thermohydrosulfuricum

Clostridium thermohydrosulfuricum and Clostridium thermosulfurogenes produced ethanol and amylases with different components as primary metabolites of starch fermentation. Starch fermentation parameters were compared in mono- and cocultures of these two thermoanaerobes to show that the fermentation was dramatically improved as a consequence of coordinate action of amylolytic enzymes and synergistic metabolic interactions between the two species. Under given monoculture fermentation conditions, neither species completely degraded starch during the time course of the study, whereas in coculture, starch was completely degraded. In monoculture starch fermentation, C. thermohydrosulfuricum produced lower levels of pullulanase and glucoamylase, whereas C. thermosulfurogenes produced lower levels of β-amylase and glucoamylase. In coculture fermentation, improvement of starch metabolism by each species was noted in terms of increased amounts and rates of increased starch consumption, amylase production, and ethanol formation. The single-step coculture fermentation completely degraded 2.5% starch in 30 h at 60°C and produced 9 U of β-amylase per ml, 1.3 U of pullulanase per ml, 0.3 U of glucoamylase per ml, and >120 mM ethanol with a yield of 1.7 mol/mol of glucose in starch. The potential industrial applications of the coculture fermentation and the physiological basis for the interspecies metabolic interactions are discussed.