Alpha-amylase Isoenzymes Research Articles

Study of variability alpha-amylase isozymes in wheat varieties of Ukrainian breeding

Aim. To investigate the variability of alpha-amylase isoenzymes in soft wheat varieties of Ukrainian selection, created by breeding institutions located in different climatic zones (Forest Steppe and Steppe). Methods. Isoenzymes of alpha-amylase were detected by electrophoretic protein separation in a polyacrylamide gel. The fall number on the «Falling Number 1700» was determined. Results. For a representative assessment of the distribution of alpha-amylase isoenzymes, we studied the zoned varieties of common wheat from two breeding centers located in different climatic zones: the Institute of Plant Physiology and Genetics of the National Academy of Sciences of Ukraine, Kyiv (Forest-steppe) and the Breeding and Genetics Institute of the National Academy of Sciences, Odessa (Steppe). The variability of wheat grain alpha-amylase was revealed. A search for associations between alpha-amylase variants and resistance to pre-harvest grain germination was conducted. Conclusions. The prevalence of alpha-amylase isoenzyme variants in Ukrainian common wheat varieties was estimated. Among Odessa varieties, the AbCd alpha-amylase variant predominates with a frequency of 50.0 %. AbCd and AbCD phenotypes with a frequency of 38.8 % predominate in the selection varieties of the IFRG of the National Academy of Sciences of Ukraine. It was shown that the genotypes containing the AbCD and ABсD isoenzyme variant are more resistant to pre-harvest germination, and the ABCd and AbcD variants are less resistant to pre-harvest grain germination.

Design and synthesis of novel 1,2,3-triazole linked hybrids: Molecular docking, MD simulation, and their antidiabetic efficacy as α-Amylase inhibitors

Novel 1,2,3-triazole linked coumarin and cinnamic acid analogs were designed, synthesized, characterized, and evaluated for their ability to inhibit the α-amylase enzyme in order to treat diabetes. Porcine pancreatic α-amylase isoenzyme II was used in the investigation and analysis of in silico molecular docking and molecular dynamic simulations. The server PreADME/toxicity was used to predict pharmacokinetics and pharmacodynamics properties. In vitro and in silico results revealed that compounds 7d, 7e and 7f showed excellent antidiabetic activity with IC50 in the range of 0.133–0.192 μM and percentage inhibitions in the range of 88.22±1.43 to 98.87±2.63%, respectively, indicating their better potency than the standard acarbose. Furthermore, synthesized compounds were docked into the active sites of the porcine pancreatic alpha-amylase isoenzyme II to evaluate binding affinity and hybrid most potent 7f was lodged in the active site via many strong hydrogen and hydrophobic interactions. Through a 100-ns dynamic simulation research, stability and binding interactions between the promising hybrid 7f and the active residues of the investigated α-amylase isoenzyme II were confirmed. The biological assessments, ADMET, molecular docking, and MD simulations of synthesized analogs allude to the possibility of using hybrids 7d, 7e, and 7f in the development of new antidiabetic therapeutics.

Izozyme polymorphism of alpha-amylase in soft wheat

Aim. Study of alpha amylase polymorphism in winter wheat varieties. Methods. Isoenzymes of alpha-amylase were detected by electrophoretic protein separation in a polyacrylamide gel. The fall number on the Falling Number 1700 was determined. Results. Strains from various collections were explored: Institute of Plant Physiology and Genetics of the National Academy of Sciences of Ukraine, Kiev, The Plant Breeding and Genetics Institute – National Center of Seed and Cultivar Investigation of the National Academy of Agricultural Sciences of Ukraine, Odesa and german strains. The polymorphism of wheat grain alpha-amylase was detected by the method of electrophoretic protein separation. We searched associations between variants of alpha-amylase and resistance to pre-harvest germination, the falling number in the examined samples was determined. Conclusions. The frequency of the prevalence of variants for isoenzymes of alpha-amylase in soft wheat varieties was estimated. It was shown that genotypes containing the variant of the isoenzymes АbC are more resistant to pre-harvest germination. Among the studied varieties of PBGI NAAS and IPPG NAS of Ukraine, the variant of alpha-amylase AbC.
 Keywords: α-amylase, isoenzymes, soft wheat, electrophoresis, falling number.

Polymorphism of alpha-amylase of soft wheat

Aim. Study of polymorphism of alpha amylase in winter wheat varieties. Methods. Isoenzymes of alpha-amylase were detected by electrophoretic protein separation in a polyacrylamide gel. The germination index (GI) was calculated by Walker-Simmons: GI = (7xn1 + 6xn2 + ... + 1xn7) / 7 x N, where n1, n2, ..., n7 is the number of seeds sprouted on the first, second, and further days to the seventh day, respectively, N – the total number of grains. Results. Varieties were explored: Institute of Plant Physiology and Genetics of the National Academy of Sciences of Ukraine, Kiev, The Plant Breeding and Genetics Institute – National Center of Seed and Cultivar Investigation of the National Academy of Agricultural Sciences of Ukraine, Odesa and german varieties.The polymorphism of wheat grain alpha-amylase was detected by the method of electrophoretic protein separation. We searched associations between variants of alpha-amylase and resistance to pre-harvest germination, the germination index in the studied samples was determined. Conclusions. The frequency of the prevalence of variants for isoenzymes of alpha-amylase in soft wheat varieties was estimated. It was shown that genotypes containing the variant of the isoenzymes АbC are more resistant to pre-harvest germination. Among the studied varieties of PBGI NAAS and IPPG NAS of Ukraine, the variant of alpha-amylase AbC.
 Keywords: α-amylase, soft wheat, electrophoresis, varieties.

Distribution of alpha-amylase isozymes among the varieties of winter wheat in Russia and Ukraine

A catalog of winter wheat varieties bred in Russia and Ukraine for alpha-amylase isozymes is presented. Among them, 11 phenotypic classes were found. It was established that the observed differences in the frequency of specific phenotypes for alpha-amylase in the culture of winter wheat depended on the geographical origin. The percentage of the most widespread cultural phenotype, designated as AbCde, decreased from the south (45°–46° N) to the north (49°–50° N) by 31.0%. At the same time, the frequency of the abCde variant increased by 25.0%. The distribution of the Abcde phenotype changed significantly from 20.7% in the west (30o36′ E) to 0% in the east (40o18′ E). The observed territorial dynamics for alpha-amylase isoenzymes may indicate the adaptive value of the identified phenotypes of common winter wheat for the weather and climatic conditions of the region of their origin.

Polymorphism of alpha-amylase and conjugation in common wheat enzyme types with quantitative traits of plants

Using polymorphism of alpha-amylase in the winter common wheat studied inheritance isoenzymes and its conjugation enzyme types with germinating grain on the "vine", grain productivity, plant height and time of ear formation. It is shown that the polymorphism isoenzyme of alpha-amylase wheat is limited by the presence of different loci whose products are similar in electrophoretic parameters. In this regard, one component of the enzyme can be controlling at one or two or three genes. Identification of a locus controlling alpha-amylase isoenzyme in the fast moving part of the electrophoretogram, designated as α-Amy-B7. Determine the distance of the locus to factor α-Amy-B6.

Effect of Seed Soaking with Exogenous Proline on Seed Germination of Rice Under Salt Stress

To explore the germination mechanism of salt-stressed rice improved by exogenous proline, and provide a theoretical basis to rice direct sowing technology for salinized soil, the effects of soaking with proline on germination status, amylase activity and isoenzyme were studied in this paper. The results showed that germination status including germination energy (GE), germination rate (GR), relative germination energy (RGE) and relative germination rate (RGR) significantly decreased as the same as the activities of alpha-amylase, beta-amylase and the total amylase under salt stress. Soaking with exogenous proline improved the germination status of rice under salt stress. Moreover, GE and RGE of salt-stressed rice were improved with increasing of proline concentration at the range of 5-45 mmol •L-1. Soaking with 15 mmol •L-1 and 30 mmol •L-1 proline significantly improved the amylase activities (e.g. alpha-amylase, beta-amylase and total amylase) of rice under salt stress. Salt stress inhibited the express of beta-amylase isoenzyme temporarily, but had few impacts on alpha-amylase isozyme. Soaking with 30 mmol •L-1 proline brightened District I and increased the width of ‘i’ brand in District II of alpha-amylase isoenzyme, but had few impacts on beta-amylase isoenzyme. In a word, soaking with proline could effectively alleviate the inhibitory effects of salt stress on seed germination.

Characteristics of alpha-amylase isozymes in cytologenetically different wheat cultivars

The isoenzyme composition of alpha-amylase is studied by polyacrylamide gel electrophoresis in Tris-glycine (pH 8.3) system in wheat cultivars with different genome composition. We show that durum wheat (Triticum durum, 2n=4x=28, BBAA) lacks the isoenzymes encoded by 6D and 7D chromosomes that are present in common wheat zymograms (Triticum aestivum, 2n=6x=42, BBAADD). A similar pattern is observed in a synthetic allohexaploid carrying the BBAA genomes of wheat and the HchHch genome of barley (Hordeum chilense). Our method of electrophoresis fails to reveal additional variants of alpha-amylase encoded by the barley genome, although C-banding analysis confirms the genomic structure BBAAHChHCh of this allopolyploid. The electrophoretic spectrum of the spring common wheat cultivar Dobrynya with the wheat-Agropyron translocation 7DL-7AiL contains all of the alpha-amylase isoenzymes typical for common wheat (2n=6x=42, BBAADD) except for the zymotype encoded by the long arm of chromosome 7D. This observation confirms the results of cytogenetic analysis that identified a 7DL-7AiL translocation in this cultivar. No additional alpha-amylase isoenzymes encoded by Agropyron chromosome have been observed. Our data indicate that analysis of wheat-alien hybrids or introgressive forms should be carried out using a complex of different methods.

The contribution of different alpha-amylase isoenzymes of the commodity grain spring wheat in the formation of falling number values

The participation of various isoenzymes of alpha-amylase in the formation of falling number values of the commodity grain of wheat grown in the Republic of Kazakhstan was investigated. It was found that active isoenzymes alpha-AMY1 and alpha-AMY2 of the embryonic shield were present in the grain with an index over 200. A significant decrease in the falling number depended mainly on the synthesis of alpha-AMY1 and alpha-AMY2 isoenzymes in the aleurone layer. In the grain, isoenzymes with high isoelectric points (p1 > or = 7.3) were found; these isoenzymes belong to alpha-amylase or late maturing or alpha-amylase of practically mature grains. It was discovered that the exogenous hormone (gibberellic acid) induced synthesis of alpha-amylase isoenzymes of scutellum, whole caryopses, and aleurone. It was shown that the impact of exogenous gibberellic acid on the activity and structure of alpha-amylase is reduced in grain with a low falling number.

Composição química e mobilização de reservas em sementes de soja de alto e baixo vigor

O estudo dos efeitos do vigor das sementes sobre os estádios iniciais do desenvolvimento das plântulas de soja é importante, já que compreendem o período de estabelecimento da cultura. Sementes de baixo vigor podem provocar reduções na velocidade de emergência e na produção de biomassa seca de plântulas, podendo afetar o estabelecimento da cultura. Este estudo teve por objetivo quantificar os componentes químicos e avaliar a capacidade de mobilização de reservas, na germinação de sementes de soja de alto e baixo vigor. Foram utilizadas três cultivares: BRS 243RR, BRS 246RR e CD 219RR. A caracterização do vigor foi por meio do teste de envelhecimento acelerado. Para determinação da composição química das sementes, foram quantificados os teores de proteínas, amido e açúcar solúvel, além da atividade da isoenzima alfa amilase. A capacidade de mobilização das reservas na germinação foi avaliada por meio da massa seca e do comprimento das plântulas. De acordo com os resultados desse trabalho, em sementes mais vigorosas há maiores conteúdos de proteínas solúveis, amido e açúcares solúveis, bem como maior capacidade de mobilização de reservas na germinação, resultando em plântulas de soja com melhor desempenho inicial.

Digestive α‐amylases of the flour moth Ephestia kuehniella– adaptation to alkaline environment and plant inhibitors

The digestive tract of lepidopteran insects is extremely alkaline. In the present work, molecular adaptation of amylolytic enzymes to this environment was investigated in the flour moth Ephestia kuehniella, an important stored-product pest. Three digestive alpha-amylases [Ephestia kuehniella alpha-amylase isoenzymes 1-3 (EkAmy1-3)] with an alkaline pH optimum were purified from larvae and biochemically characterized. These isoenzymes differ significantly in their sensitivity to alpha-amylase inhibitors of plant origin that are directed against herbivores as antifeedants. Such functional variability renders the amylolytic system less vulnerable to suppression by plant defensive molecules. Moreover, we found that expression of alpha-amylases is upregulated in larvae feeding on a diet enriched with an alpha-amylase inhibitor. The alpha-amylases are secreted into the larval midgut by an exocytotic mechanism, as revealed by immunogold microscopy. The cDNA sequence of EkAmy3 was determined, and a homology model of EkAmy3 was built in order to analyze the structural features responsible for adaptation to alkaline pH. First, the overall fold was found to be stabilized by remodeling of ion pairs. Second, molecular simulations supported by activity measurements showed that EkAmy3 does not bind a Cl(-), owing to an Arg-to-Gln mutation in a conserved binding site. The Cl(-)-binding residues are in contact with the catalytic residues, and this change might help to fine-tune the catalytic pK(a) values to an alkaline pH optimum. We conclude that lepidopteran alpha-amylases are evolutionarily adapted in terms of structure and expression dynamics for effective functioning in the digestive system.

Accurate quantitation of salivary and pancreatic amylase activities in human plasma by microchip electrophoretic separation of the substrates and hydrolysates coupled with immunoinhibition

A high-performance determination system for alpha-amylase isoenzyme activities in human plasma involving microchip electrophoresis with a plastic chip was developed. The combination of microchip electrophoresis for substrate and hydrolysate separation and an immunoinhibition method for the differentiation of isoenzyme activities using antihuman salivary amylase (S-AMY) mAb allowed the highly selective determination of amylase isoenzyme (S-AMY and pancreatic amylase (P-AMY)) activities even in a complex matrix such as a crude plasma sample. We used 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled maltohexaose (G6) as a substrate. Amylase in a human plasma sample hydrolyzed APTS-G6 into APTS-maltotriose (G3) and G3, which was measured as the fluorescence intensity of APTS-G3 on microchip electrophoresis. A double logarithm plot revealed a linear relationship between amylase activity and fluorescence intensity in the range of 5-500 U/L of amylase activity (r2=0.9995, p<0.01), and the LOD was 4.38 U/L. Amylase activities in 13 subjects determined by the present method were compared with the results obtained by conventional methods with nitrophenylated oligosaccharides as substrates, respectively. Good correlations were observed for each method on simple linear regression analysis (both p<0.01). The reproducibilities of within-days for total amylase and P-AMY were 2.98-6.27 and 3.83-6.39%, respectively, and these between-days were 2.88-5.66 and 3.64-5.63%, respectively. This system enables us to determine amylase isoenzyme activities in human plasma with high sensitivity and accuracy, and thus will be applicable to clinical diagnosis.

The role of two isoenzymes of alpha-amylase of Araucaria araucana (Araucariaceae) on the digestion of starch granules during germination.

Starch is the principal reserve of Araucaria araucana seeds, and it is hydrolysed during germination mainly by alpha-amylase. There are several alpha-amylase isoenzymes whose patterns change in the embryo and in the megagametophyte from the one observed in quiescent seeds (T(0)) to a different one observed 90 h after imbibition (T(90)). The objective of this research was to study the roles of two purified alpha-amylase isoenzymes by in vitro digestion of starch granules extracted from the tissues at two times of imbibition: one is abundant in quiescent seeds and the other is abundant after 90 h of imbibition. The isoenzymes digested the starch granules of their own stage of germination better, since the isoenzyme T(0) digested starch granules mainly from quiescent seeds, while the isoenzyme T(90) digested starch mainly at 90 h of imbibition. The sizes of the starch granule and the tissue from which these granules originated make a difference to digestion by the isoenzymes. Embryonic isoenzyme T(0) digested large embryonic starch granules better than small and medium-sized granules, and better than those isolated from megagametophytes. Similarly isoenzyme T(90) digested small embryonic starch granules better than medium-sized and large granules, and better than those isolated from megagametophytes. However, a mixture of partially purified megagametophytic isoenzymes T(0) and T(90) digested the megagametophytic granules better than those isolated from embryos. Studies of in vitro sequential digestion of starch granules with these isoenzymes corroborated their specificity. The isoenzyme T(90) digested starch granules previously digested by the isoenzyme T(0). This suggests that in vivo these two isoenzymes may act sequentially in starch granule digestion.

Purification of alpha-amylase isoenzymes from Scytalidium thermophilum on a fluidized bed of alginate beads followed by concanavalin A-agarose column chromatography.

An alpha-amylase has been purified from the thermophilic fungus Scytalidium thermophilum. A ninefold purification was achieved in a single step using fluidized bed chromatography wherein alginate was used as the affinity matrix. There are at least two isoenzymes as shown by concanavalin A (Con A)-agarose column chromatography. The isoenzyme binding to Con A is stable for at least 3 h at 80 degrees C in the presence of calcium ions. The isoenzymes have similar molecular weights of around 45,000 Da as shown by SDS-PAGE analysis. The isoenzymes differ only slightly in their pH optima and temperature optima but the isoenzyme binding to Con A-agarose has slightly higher thermal stability.

5494804 Differential determination process of alpha-amylase isoenzyme activities : Uchida Riichiro; Tokutake Shoichi; Yamaji Nobuyuki; Motoyama Yoshinor; Hosoi Kenji Noda, Japan assigned to Kikkoman Corporation; Daiichi Pure Chemicals Co Ltd

5494804 Differential determination process of alpha-amylase isoenzyme activities : Uchida Riichiro; Tokutake Shoichi; Yamaji Nobuyuki; Motoyama Yoshinor; Hosoi Kenji Noda, Japan assigned to Kikkoman Corporation; Daiichi Pure Chemicals Co Ltd

Crystal Structure of Pig Pancreatic α‐amylase Isoenzyme II, in Complex with the Carbohydrate Inhibitor Acarbose

Two different crystal forms of pig pancreatic alpha-amylase isoenzyme II (PPAII), free and complexed to a carbohydrate inhibitor (acarbose), have been compared together and to previously reported structures of PPAI. A crystal form obtained at 4 degrees C, containing nearly 72% solvent, made it possible to obtain a new complex with acarbose, different from a previous one obtained at 20 degrees C [Qian, M., Buisson, G., Duée, E., Haser, H. & Payan, F. (1994) Biochemistry 33, 6284-6294]. In the present form, six contiguous subsites of the enzyme active site are occupied by the carbohydrate ligand; the structural data indicate that the binding site is capable of holding more than the five glucose units of the scheme proposed through kinetic studies. A monosaccharide ring bridging two protein molecules related by the crystal packing is located on the surface, at a distance of 2.0 nm from the reducing end of the inhibitor ligand; the symmetry-related glucose ring in the crystal lattice is found 1.5 nm away from the non-reducing end of the inhibitor ligand.

Automated measurement of alpha-amylase isoenzymes with 6(3)-deoxymaltotriose as selective amylase inhibitor

We developed an automated method for measurement of alpha-amylase isoenzymes in serum by a single kinetic assay (SKA) and a double kinetic assay (DKA) with 2-chloro-4-nitrophenyl-6(5)-azido-6(5)-deoxy-beta-maltopentaoside as a substrate and 6(3)-deoxymaltotriose (DOG3) as a novel selective amylase inhibitor. DOG3 showed a large difference in inhibitory activity between human pancreatic alpha-amylase (HPA; 86.9% inhibition) and salivary alpha-amylase (32.1% inhibition) at 0.33 mmol/L. Constant inhibition was obtained immediately after addition of DOG3. The inhibitory effect did not change with variation in concentrations of amylase up to approximately 3000 U/L. The results obtained by SKA correlated well with those obtained by three methods: monoclonal antibody (r = 0.988), wheat germ inhibitor (r = 0.989), and DKA (r = 0.995). The within-run and between-run CVs for HPA were 0.63-2.32% on SKA, 0.69-1.81% on DKA. No significant interferences by endogenous serum compounds were observed with the proposed methods.

Effect of modifying histidine residues on the action of Bacillus amyloliquefaciens and barley-malt α-amylases

Modification of porcine pancreatic alpha-amylase (PPA) and Taka-amylase A(TAA) with diethyl pyrocarbonate (DEP) causes activation of the release of p-nitrophenol from p-nitrophenol alpha-maltoside (G2PNP), and a decrease in amylase activity (hydrolysis of alpha-1,4 glucosidic bonds in starch). Among the possible sites of modification, attention focuses on three histidine residues present around the active site of alpha-amylases of many different origins. In PPA these are His 101, His 201, and His 299, with His 101 and His 299 being very close to the site of catalysis and thus perhaps directly or indirectly involved in the catalysis. On the other hand, His 201 is located on the aglycon side of the catalytic site, and we have suggested that it is involved in the increase of PNP release after chemical modification. Investigations of site-directed mutagenesis of the histidine residues of human pancreatic alpha-amylase support this identification. Although the degree of sequence similarity among alpha-amylases of different origins is low, there are several conserved short regions. Most belong to the structural components of the active site in PPA and TAA. Furthermore, there is a close similarity in the three-dimensional structures of PPA and TAA. The conserved residues around the active site in all alpha-amylases suggest some universal structural similarities in these active sites. Therefore, we examined the effects of the chemical modification of histidine residues in Bacillus amyloliquefaciens alpha-amylase(BLA) with DEP and made a comparison with modification of barley alpha-amylase isoenzyme II(BAII), using identical substrate systems. These two alpha-amylases have more substrate binding subsites than PPA and TAA, and have similar action patterns with malto-oligosaccharides.

Expression of alpha-amylase isoenzymes and trypsin by the proliferating epithelium of large intrahepatic bile ducts and intrahepatic peribiliary glands in hepatolithiasis.

The expression of alpha-amylase isoenzymes (pancreatic and salivary) and trypsin by the epithelium of large intrahepatic bile ducts and peribiliary glands was examined immunohistochemically in hepatolithiasis (n = 22), extrahepatic biliary obstruction (n = 20) and normal liver (n = 22). Hepatolithiasis was associated with marked proliferation of bile duct cells and peribiliary glands. Expression of pancreatic and salivary amylase was observed in the proliferating bile duct cells and peribiliary glands of all livers, and trypsin was found in 68% of the livers. In extrahepatic biliary obstruction, proliferation of the biliary epithelium was less marked, but expression of amylase isoenzymes was observed in all livers and trypsin was found in 50%. All normal livers showed expression of amylase isoenzymes in large intrahepatic bile ducts, septal bile ducts and peribiliary glands, and trypsin was found in 73%. The density of enzyme-containing acini was highest in hepatolithiasis, intermediate in extrahepatic biliary obstruction and lowest in normal liver. These results show that the proliferating biliary epithelium in hepatolithiasis contains amylase isoenzymes and trypsin and that biliary epithelium retains the ability to produce these enzymes after proliferation, suggesting that a large amount of amylase isoenzyme and trypsin may be secreted into the bile ducts in hepatolithiasis. These enzymes may play an important role in the pathophysiology of hepatolithiasis.

Sensitive, simple staining method for use in electrophoretic determination of amylase isoenzymes in serum.

We have developed a sensitive and simple staining method for use in electrophoretic analysis of serum alpha-amylase (EC 3.2.1.1) isoenzymes. The principle of this method is as follows. alpha-Amylase hydrolyzes 4-nitrophenylmaltoheptaoside to generate oligosaccharide, which is then converted to gluconolactone in the presence of oligosaccharide dehydrogenase (no EC no. assigned), with concomitant reduction of 1-methoxy-5-methylphenazinium methylsulfate (1-m-PMS) to 1-m-PMSH. The hydrogen in 1-m-PMSH is then transferred to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to yield formazan. Each of the isoenzymes can then be measured densitometrically. The mean (and SD) values for total amylase, P1, S1, and S2 as determined by this method with sera from 25 healthy adults in fasting were 251 (64), 104 (35), 126 (40), and 22 (11) U/L, respectively. Between-assay CVs (n = 10) for determinations of P1, S1, and S2 were 3.54%, 4.03%, and 7.01%, respectively.