Improvement of heterologous protein production in Aspergillus oryzae by RNA interference with alpha-amylase genes.

PubMed

Nemoto, Takashi; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

2009-11-01

Aspergillus oryzae RIB40 has three alpha-amylase genes (amyA, amyB, and amyC), and secretes alpha-amylase abundantly. However, large amounts of endogenous secretory proteins such as alpha-amylase can compete with heterologous protein in the secretory pathway and decrease its production yields. In this study, we examined the effects of suppression of alpha-amylase on heterologous protein production in A. oryzae, using the bovine chymosin (CHY) as a reporter heterologous protein. The three alpha-amylase genes in A. oryzae have nearly identical DNA sequences from those promoters to the coding regions. Hence we performed silencing of alpha-amylase genes by RNA interference (RNAi) in the A. oryzae CHY producing strain. The silenced strains exhibited a reduction in alpha-amylase activity and an increase in CHY production in the culture medium. This result suggests that suppression of alpha-amylase is effective in heterologous protein production in A. oryzae.

The hyperthermophilic α-amylase from Thermococcus sp. HJ21 does not require exogenous calcium for thermostability because of high-binding affinity to calcium.

PubMed

Cheng, Huaixu; Luo, Zhidan; Lu, Mingsheng; Gao, Song; Wang, Shujun

2017-05-01

The hyperthermophilic α-amylase from Thermococcus sp. HJ21 does not require exogenous calcium ions for thermostability, and is a promising alternative to commercially available α-amylases to increase the efficiency of industrial processes like the liquefaction of starch. We analyzed the amino acid sequence of this α-amylase by sequence alignments and structural modeling, and found that this α-amylase closely resembles the α-amylase from Pyrococcus woesei. The gene of this α-amylase was cloned in Escherichia coli and the recombinant α-amylase was overexpressed and purified with a combined renaturation-purification procedure. We confirmed thermostability and exogenous calcium ion independency of the recombinant α-amylase and further investigated the mechanism of the independency using biochemical approaches. The results suggested that the α-amylase has a high calcium ion binding affinity that traps a calcium ion that would not dissociate at high temperatures, providing a direct explanation as to why the addition of calcium ions is not required for thermostability. Understanding of the mechanism offers a strong base on which to further engineer properties of this α-amylase for better potential applications in industrial processes.

Activity and storage of commercial amylases in the 2013 Louisiana grinding season

USDA-ARS?s Scientific Manuscript database

A current problem in the application of amylases at sugarcane factories is the existence of a wide variation in the activities and activity per unit cost of commercial amylases. The efficiency of amylase action to break down starch in the factory is related to the activity of the amylase used. Until...

Amylase - urine

MedlinePlus

... page: //medlineplus.gov/ency/article/003607.htm Amylase - urine To use the sharing features on this page, ... test that measures the amount of amylase in urine. Amylase is an enzyme that helps digest carbohydrates. ...

Heterologous expression, biochemical characterization, and overproduction of alkaline α-amylase from Bacillus alcalophilus in Bacillus subtilis

PubMed Central

2011-01-01

Background Alkaline α-amylases have potential applications for hydrolyzing starch under high pH conditions in the starch and textile industries and as ingredients in detergents for automatic dishwashers and laundries. While the alkaline α-amylase gains increased industrial interest, the yield of alkaline α-amylases from wild-type microbes is low, and the combination of genetic engineering and process optimization is necessary to achieve the overproduction of alkaline α-amylase. Results The alkaline α-amylase gene from Bacillus alcalophilus JN21 (CCTCC NO. M 2011229) was cloned and expressed in Bacillus subtilis strain WB600 with vector pMA5. The recombinant alkaline α-amylase was stable at pH from 7.0 to 11.0 and temperature below 40°C. The optimum pH and temperature of alkaline α-amylase was 9.0 and 50°C, respectively. Using soluble starch as the substrate, the Km and Vmax of alkaline α-amylase were 9.64 g/L and 0.80 g/(L·min), respectively. The effects of medium compositions (starch, peptone, and soybean meal) and temperature on the recombinant production of alkaline α-amylase in B. subtilis were investigated. Under the optimal conditions (starch concentration 0.6% (w/v), peptone concentration 1.45% (w/v), soybean meal concentration 1.3% (w/v), and temperature 37°C), the highest yield of alkaline α-amylase reached 415 U/mL. The yield of alkaline α-amylase in a 3-L fermentor reached 441 U/mL, which was 79 times that of native alkaline α-amylase from B. alcalophilus JN21. Conclusions This is the first report concerning the heterologous expression of alkaline α-amylase in B. subtilis, and the obtained results make it feasible to achieve the industrial production of alkaline α-amylase with the recombinant B. subtilis. PMID:21978209

Production of Fungal Amylases Using Cheap, Readily Available Agriresidues, for Potential Application in Textile Industry

PubMed Central

Singh, Sanamdeep; Bali, Vrinda; Mangla, Jyoti

2014-01-01

The study aimed at isolation and screening of fungal amylase producer, optimization of solid state fermentation conditions for maximum amylase production by the best amylase producer, and characterization of the crude amylases, so produced. Aspergillus fumigatus NTCC1222 showed the highest amylase activity (164.1 U/mL) in secondary screening under SSF conditions and was selected for further studies. The test strain showed maximum amylase production (341.7 U/mL) and supernatant protein concentration (9.7 mg/mL) for incubation period (6 days), temperature (35°C), initial pH (6.0), nutrient salt solution as moistening agent, and beef extract as nitrogen source. Pomegranate peel produced maximum amylase activity, but wheat bran (only slightly lesser amylase activity as compared to that of pomegranate peel) was chosen for further studies, keeping in mind the seasonal availability of pomegranate peel. TLC confirmed the amylase produced to be α-type and 60 kDa was the molecular weight of the partially purified amylase. The enzyme showed maximum enzyme activity at pH 6.0, temperature of 55°C, and incubation time of 60 minutes. UV (616.0 U/mL) and chemical (814.2 U/mL) mutation enhanced amylase activity as compared to wild test strain. The study indicates that Aspergillus fumigatus NTCC1222 can be an important source of amylase and the crude enzyme, hence obtained, can be cost effectively applied in multiple sections of textile wet processing. PMID:24527439

Models for the action of barley alpha-amylase isozymes on linear substrates.

PubMed

MacGregor, E A; MacGregor, A W; Macri, L J; Morgan, J E

1994-05-05

The formation of maltodextrins, G1 to G12, during the hydrolysis of amylose by alpha-amylases 1 and 2 from barley malt was followed by HPLC. Similar, but not identical, distributions of products were obtained with the two alpha-amylase components. Maltose, G6, and G7 were major products, but G7 was degraded as hydrolysis proceeded. alpha-Amylase 1 produced more G1 and G3 than did alpha-amylase 2 at all stages of hydrolysis. Products formed during the hydrolysis of G9, G10, G11, and G12 by the two alpha-amylases were also determined. A different spectrum of products was observed with each substrate and small differences were observed in the action pattern of the two alpha-amylases, e.g., G3 and G7 were the major products formed during the hydrolysis of G10 by alpha-amylase 1, whereas G2 and G8 were the major products formed by alpha-amylase 2 on the same substrate. These results were used to develop a model of the active site of barley malt alpha-amylases. This site contains ten contiguous subsites with the catalytic site situated between subsites 7 and 8. The model can be used to predict hydrolysis patterns of amylose and maltodextrins by cereal alpha-amylases.

Sequence-structural features and evolutionary relationships of family GH57 α-amylases and their putative α-amylase-like homologues.

PubMed

Janeček, Stefan; Blesák, Karol

2011-08-01

The glycoside hydrolase family 57 (GH57) contains α-amylase and a few other amylolytic specificities. It counts ~400 members from Archaea (1/4) and Bacteria (3/4), mostly of extremophilic prokaryotes. Only 17 GH57 enzymes have been biochemically characterized. The main goal of the present bioinformatics study was to analyze sequences having the clear GH57 α-amylase features. Of the 107 GH57 sequences, 59 were evaluated as α-amylases (containing both GH57 catalytic residues), whereas 48 were assigned as GH57 α-amylase-like proteins (having a substitution in one or both catalytic residues). Forty-eight of 59 α-amylases were from Archaea, but 42 of 48 α-amylase-like proteins were of bacterial origin. The catalytic residues were substituted in most cases in Bacteroides and Prevotella by serine (instead of catalytic nucleophile glutamate) and glutamate (instead of proton donor aspartate). The GH57 α-amylase specificity has thus been evolved and kept enzymatically active mainly in Archaea.

Molecular cloning and characterization of an alpha-amylase from Pichia burtonii 15-1.

PubMed

Kato, Saemi; Shimizu-Ibuka, Akiko; Mura, Kiyoshi; Takeuchi, Akiko; Tokue, Chiyoko; Arai, Soichi

2007-12-01

An alpha-amylase secreted by Pichia burtonii 15-1 isolated from a traditional starter murcha of Nepal, named Pichia burtonii alpha-amylase (PBA), was studied. The gene was cloned and its nucleotide sequence was determined. PBA was deduced to consist of 494 amino acid residues. It shared certain degrees of amino acid sequence identity with other homologous proteins: 60% with Schwanniomyces occidentalis alpha-amylase, 58% with Saccharomycopsis sp. alpha-amylase, and 47% with Taka-amylase A from Aspergillus oryzae. A three-dimensional structural model of PBA generated using the known three-dimensional structure of Taka-amylase A as a template suggested high structural similarity between them. Kinetic analysis revealed that the K(m) values of PBA were lower than those of Taka-amylase A for the oligosaccharides. Although the k(cat) values of PBA were lower than those of Taka-amylase A for the oligosaccharide substrates, the k(cat)/K(m) values of PBA were higher.

Potency of Amylase-producing Bacteria and Optimization Amylase Activities

NASA Astrophysics Data System (ADS)

Indriati, G.; Megahati, R. R. P.; Rosba, E.

2018-04-01

Enzymes are capable to act as biocatalyst for a wide variety of chemical reactions. Amylase have potential biotechnological applications in a wide range of industrial processes and account for nearly 30% of the world’s enzyme market. Amylase are extracellular enzymes that catalyze the hydrolysis of internal α-1,4-glycosidic linkages in starch to dextrin, and other small carbohydrate molecules constituted of glucose units. Although enzymes are produced from animal and plant sources, the microbial sources are generally the most suitable for commercial applications. Bacteria from hot springs is widely used as a source of various enzymes, such as amylase. But the amount of amylase-producing bacteria is still very limited. Therefore it is necessary to search sources of amylase-producing bacteria new, such as from hot springs Pariangan. The purpose of this study was to isolation of amylase-producing bacteria from Pariangan hot spring, West Sumatera and amylase activity optimization. The results were obtained 12 isolates of thermophilic bacteria and 5 isolates of amyalse-producing bacteria with the largest amylolytic index of 3.38 mm. The highest amylase activity was obtained at 50°C and pH 7.5.

Carbodiimide for Covalent α-Amylase Immobilization onto Magnetic Nanoparticles

NASA Astrophysics Data System (ADS)

Milani, Zeinab Mortazavi; Jalal, Razieh; Goharshadi, Elaheh K.

Covalent cross-linking of enzymes to magnetite (Fe3O4) nanoparticles (MNPs) is one of the useful enzyme immobilization methods which provides repeated use of the catalyst, facilitates enzyme separation from the reaction mixture, and sometimes improves biocatalysts stability. The aim of this study was to immobilize α-amylase onto MNPs via covalent attachment using carbodiimide (CDI) molecules. MNPs were synthesized by the co-precipitation method. The size and the structure of the particles were characterized by X-ray diffraction and transmission electron microscopy. The effects of different operational conditions of direct α-amylase binding on MNPs in the presence of CDI were investigated by using the shaking method. Fourier transform infrared spectroscopy was used to confirm the success of immobilization. The optimum conditions and catalytic properties of immobilized α-amylase were also evaluated. The efficiency of immobilization and the residual activity of the immobilized α-amylase were dependent on the mass ratio of MNPs: CDI: α-amylase and the immobilization temperature. The optimum pH for the free and immobilized amylase was 6. The free and immobilized α-amylase showed maximum activity at 20∘C and 35∘C, respectively. The immobilized α-amylase was more thermostable than the free one. The retained activity for free α-amylase after 19 storage days was 57.7% whereas it was 100% for the immobilized α-amylase. In repeated batch experiments, the immobilized α-amylase retained a residual activity of 45% after 11 repeated uses. The Km and Vmax values for the immobilized enzyme were larger than those of the free enzyme. The immobilization of α-amylase on MNPs using CDI improves its stability and reusability.

Molecular, Biochemical, and Dietary Regulation Features of α-Amylase in a Carnivorous Crustacean, the Spiny Lobster Panulirus argus.

PubMed

Rodríguez-Viera, Leandro; Perera, Erick; Martos-Sitcha, Juan Antonio; Perdomo-Morales, Rolando; Casuso, Antonio; Montero-Alejo, Vivian; García-Galano, Tsai; Martínez-Rodríguez, Gonzalo; Mancera, Juan Miguel

2016-01-01

Alpha-amylases are ubiquitously distributed throughout microbials, plants and animals. It is widely accepted that omnivorous crustaceans have higher α-amylase activity and number of isoforms than carnivorous, but contradictory results have been obtained in some species, and carnivorous crustaceans have been less studied. In addition, the physiological meaning of α-amylase polymorphism in crustaceans is not well understood. In this work we studied α-amylase in a carnivorous lobster at the gene, transcript, and protein levels. It was showed that α-amylase isoenzyme composition (i.e., phenotype) in lobster determines carbohydrate digestion efficiency. Most frequent α-amylase phenotype has the lowest digestion efficiency, suggesting this is a favoured trait. We revealed that gene and intron loss have occurred in lobster α-amylase, thus lobsters express a single 1830 bp cDNA encoding a highly conserved protein with 513 amino acids. This protein gives rise to two isoenzymes in some individuals by glycosylation but not by limited proteolysis. Only the glycosylated isoenzyme could be purified by chromatography, with biochemical features similar to other animal amylases. High carbohydrate content in diet down-regulates α-amylase gene expression in lobster. However, high α-amylase activity occurs in lobster gastric juice irrespective of diet and was proposed to function as an early sensor of the carbohydrate content of diet to regulate further gene expression. We concluded that gene/isoenzyme simplicity, post-translational modifications and low Km, coupled with a tight regulation of gene expression, have arose during evolution of α-amylase in the carnivorous lobster to control excessive carbohydrate digestion in the presence of an active α-amylase.

Molecular, Biochemical, and Dietary Regulation Features of α-Amylase in a Carnivorous Crustacean, the Spiny Lobster Panulirus argus

PubMed Central

Martos-Sitcha, Juan Antonio; Perdomo-Morales, Rolando; Casuso, Antonio; Montero-Alejo, Vivian; García-Galano, Tsai; Martínez-Rodríguez, Gonzalo; Mancera, Juan Miguel

2016-01-01

Alpha-amylases are ubiquitously distributed throughout microbials, plants and animals. It is widely accepted that omnivorous crustaceans have higher α-amylase activity and number of isoforms than carnivorous, but contradictory results have been obtained in some species, and carnivorous crustaceans have been less studied. In addition, the physiological meaning of α-amylase polymorphism in crustaceans is not well understood. In this work we studied α-amylase in a carnivorous lobster at the gene, transcript, and protein levels. It was showed that α-amylase isoenzyme composition (i.e., phenotype) in lobster determines carbohydrate digestion efficiency. Most frequent α-amylase phenotype has the lowest digestion efficiency, suggesting this is a favoured trait. We revealed that gene and intron loss have occurred in lobster α-amylase, thus lobsters express a single 1830 bp cDNA encoding a highly conserved protein with 513 amino acids. This protein gives rise to two isoenzymes in some individuals by glycosylation but not by limited proteolysis. Only the glycosylated isoenzyme could be purified by chromatography, with biochemical features similar to other animal amylases. High carbohydrate content in diet down-regulates α-amylase gene expression in lobster. However, high α-amylase activity occurs in lobster gastric juice irrespective of diet and was proposed to function as an early sensor of the carbohydrate content of diet to regulate further gene expression. We concluded that gene/isoenzyme simplicity, post-translational modifications and low Km, coupled with a tight regulation of gene expression, have arose during evolution of α-amylase in the carnivorous lobster to control excessive carbohydrate digestion in the presence of an active α-amylase. PMID:27391425

Response of Fatty Acid Synthesis Genes to the Binding of Human Salivary Amylase by Streptococcus gordonii

PubMed Central

Nikitkova, Anna E.; Haase, Elaine M.; Vickerman, M. Margaret; Gill, Steven R.

2012-01-01

Streptococcus gordonii, an important primary colonizer of dental plaque biofilm, specifically binds to salivary amylase via the surface-associated amylase-binding protein A (AbpA). We hypothesized that a function of amylase binding to S. gordonii may be to modulate the expression of chromosomal genes, which could influence bacterial survival and persistence in the oral cavity. Gene expression profiling by microarray analysis was performed to detect genes in S. gordonii strain CH1 that were differentially expressed in response to the binding of purified human salivary amylase versus exposure to purified heat-denatured amylase. Selected genes found to be differentially expressed were validated by quantitative reverse transcription-PCR (qRT-PCR). Five genes from the fatty acid synthesis (FAS) cluster were highly (10- to 35-fold) upregulated in S. gordonii CH1 cells treated with native amylase relative to those treated with denatured amylase. An abpA-deficient strain of S. gordonii exposed to amylase failed to show a response in FAS gene expression similar to that observed in the parental strain. Predicted phenotypic effects of amylase binding to S. gordonii strain CH1 (associated with increased expression of FAS genes, leading to changes in fatty acid synthesis) were noted; these included increased bacterial growth, survival at low pH, and resistance to triclosan. These changes were not observed in the amylase-exposed abpA-deficient strain, suggesting a role for AbpA in the amylase-induced phenotype. These results provide evidence that the binding of salivary amylase elicits a differential gene response in S. gordonii, resulting in a phenotypic adjustment that is potentially advantageous for bacterial survival in the oral environment. PMID:22247133

Parallel beta/alpha-barrels of alpha-amylase, cyclodextrin glycosyltransferase and oligo-1,6-glucosidase versus the barrel of beta-amylase: evolutionary distance is a reflection of unrelated sequences.

PubMed

Janecek, S

1994-10-17

The structures of functionally related beta/alpha-barrel starch hydrolases, alpha-amylase, beta-amylase, cyclodextrin glycosyltransferase and oligo-1,6-glucosidase, are discussed, their mutual sequence similarities being emphasized. Since these enzymes (except for beta-amylase) along with the predicted set of more than ten beta/alpha-barrels from the alpha-amylase enzyme superfamily fulfil the criteria characteristic of the products of divergent evolution, their unrooted distance tree is presented.

Novel assay procedures for the measurement of α-amylase in weather-damaged wheat.

PubMed

Cornaggia, Claudio; Ivory, Ruth; Mangan, David; McCleary, Barry V

2016-01-30

The measurement of α-amylase (EC 3.2.1.1) in sprout-damaged grains is a crucial analysis yet a problematic one owing to the typically low α-amylase levels in ground wheat samples. A number of standardised methods such as the Falling Number method and the Ceralpha method exist which are routinely used for the assay of α-amylase. These methods, however, are either highly substrate-dependent or lack the required sensitivity to assess sprout damage. Novel colorimetric and fluorometric reagents have been prepared (Amylase HR, Amylase SD, BzCNPG7 reagent and BzMUG7 reagent) for the direct and specific assay of α-amylase activity in sprout-damaged wheat. Assays employing these reagents have been developed and optimised to include a decolourisation step using activated charcoal. When used in a convenient assay format, Amylase SD--containing EtNPG7 (II) as the colorimetric substrate and α-glucosidase as the ancillary enzyme--was found to be an excellent reagent for the assessment of sprout damage in wheat with incubation times as short as 5 min. The assay using Amylase SD is completely specific for α-amylase. The use of the Amylase SD assay represents a sensitive and valid alternative to the traditionally used Falling Number values for the assessment of sprout damage in wheat samples. © 2015 Society of Chemical Industry.

Structural features, substrate specificity, kinetic properties of insect α-amylase and specificity of plant α-amylase inhibitors.

PubMed

Kaur, Rimaljeet; Kaur, Narinder; Gupta, Anil Kumar

2014-11-01

α-Amylase is an important digestive enzyme required for the optimal growth and development of insects. Several insect α-amylases had been purified and their physical and chemical properties were characterized. Insect α-amylases of different orders display variability in structure, properties and substrate specificity. Such diverse properties of amylases could be due to different feeding habits and gut environment of insects. In this review, structural features and properties of several insect α-amylases were compared. This could be helpful in exploring the diversity in characteristics of α-amylase between the members of the same class (insecta). Properties like pH optima are reflected in enzyme structural features. In plants, α-amylase inhibitors (α-AIs) occur as part of natural defense mechanisms against pests by interfering in their digestion process and thus could also provide access to new pest management strategies. AIs are quite specific in their action; therefore, these could be employed according to their effectiveness against target amylases. Potential of transgenics with α-AIs has also been discussed for insect resistance and controlling infestation. The differences in structural features of insect α-amylases provided reasons for their efficient functioning at different pH and the specificity towards various substrates. Various proteinaceous and non-proteinaceous inhibitors discussed could be helpful in controlling pest infestation. In depth detailed studies are required on proteinaceous α-AI-α-amylase interaction at different pH's as well as the insect proteinase action on these inhibitors before selecting the α-AI for making transgenics resistant to particular insect. Copyright © 2014 Elsevier Inc. All rights reserved.

Purification and characterization of α-Amylase from Miswak Salvadora persica.

PubMed

Mohamed, Saleh A; Almulaiky, Yaaser Q; Ahmed, Youssri M; Al-Bar, Omar A M; Ibrahim, Ibrahim H

2014-04-01

The miswak (Salvadora persica) is a natural toothbrush. It is well known that very little information has been reported on enzymes in miswak as medicinal plant. Recently, we study peroxidase in miswak. In the present study, the main goal of this work is to purify and characterize α-amylase from miswak. The second goal is to study the storage stability of α-amylase in toothpaste. The purification method included chromatography of miswak α-amylase on DEAE-Sepharose column and Sephacryl S-200 column. Molecular weight was determined by gel filtration and SDS-PAGE. Five α-amylases A1, A4a, A4b, A5a and A5b from miswak were purified and they had molecular weights of 14, 74, 16, 30 and 20 kDa, respectively. α-Amylases had optimum pH from 6 to 8. Affinity of the substrates toward all enzymes was studied. Miswak α-amylases A1, A4a, A4b, A5a and A5b had Km values for starch and glycogen of 3.7, 3.7, 7.1, 0.52, 4.3 mg/ml and 5.95, 5.9 4.16, 6.3, 6.49 mg/ml, respectively. The optimum temperature for five enzymes ranged 40°C- 60°C. Miswak α-amylases were stable up to 40°C- 60°C after incubation for 30 min. Ca+2 activated all the miswak α-amylases, while Ni2+, Co+2 and Zn+2 activated or inhibited some of these enzymes. The metal chelators, EDTA, sodium citrate and sodium oxalate had inhibitory effects on miswak α-amylases. PMSF, p-HMB, DTNB and 1,10 phenanthroline caused inhibitory effect on α-amylases. The analysis of hydrolytic products after starch hydrolysis by miswak α-amylases on paper chromatography revealed that glucose, maltose, maltotriose and oligosaccharide were the major products. Crude miswak α-amylase in the toothpaste retained 55% of its original activity after 10 months of storage at room temperature. From these findings, α-amylases from miswak can be considered as beneficial enzymes for pharmaceuticals. Therefore, we study the storage stability of the crude α-amylase of miswak, which contained the five α-amylases, in toothpaste. The enzyme in the toothpaste retained 55% of its original activity after 10 months of storage at room temperature.

Purification and characterization of α-Amylase from Miswak Salvadora persica

PubMed Central

2014-01-01

Background The miswak (Salvadora persica) is a natural toothbrush. It is well known that very little information has been reported on enzymes in miswak as medicinal plant. Recently, we study peroxidase in miswak. In the present study, the main goal of this work is to purify and characterize α-amylase from miswak. The second goal is to study the storage stability of α-amylase in toothpaste. Method The purification method included chromatographaphy of miswak α-amylase on DEAE-Sepharose column and Sephacryl S-200 column. Molecular weight was determined by gel filtration and SDS-PAGE. Results Five α-amylases A1, A4a, A4b, A5a and A5b from miswak were purified and they had molecular weights of 14, 74, 16, 30 and 20 kDa, respectively. α-Amylases had optimum pH from 6 to 8. Affinity of the substrates toward all enzymes was studied. Miswak α-amylases A1, A4a, A4b, A5a and A5b had Km values for starch and glycogen of 3.7, 3.7, 7.1, 0.52, 4.3 mg/ml and 5.95, 5.9 4.16, 6.3, 6.49 mg/ml, respectively. The optimum temperature for five enzymes ranged 40°C- 60°C. Miswak α-amylases were stable up to 40°C- 60°C after incubation for 30 min. Ca+2 activated all the miswak α-amylases, while Ni2+, Co+2 and Zn+2 activated or inhibited some of these enzymes. The metal chelators, EDTA, sodium citrate and sodium oxalate had inhibitory effects on miswak α-amylases. PMSF, p-HMB, DTNB and 1,10 phenanthroline caused inhibitory effect on α-amylases. The analysis of hydrolytic products after starch hydrolysis by miswak α-amylases on paper chromatography revealed that glucose, maltose, maltotriose and oligosaccharide were the major products. Crude miswak α-amylase in the toothpaste retained 55% of its original activity after 10 months of storage at room temperature. Conclusions From these findings, α-amylases from miswak can be considered as beneficial enzymes for pharmaceuticals. Therefore, we study the storage stability of the crude α-amylase of miswak, which contained the five α-amylases, in toothpaste. The enzyme in the toothpaste retained 55% of its original activity after 10 months of storage at room temperature. PMID:24690287

Recognition and Binding of the PF2 Lectin to α-Amylase From Zabrotes subfasciatus (Coleoptera:Bruchidae) Larval Midgut

PubMed Central

Lagarda-Diaz, I.; Geiser, D.; Guzman-Partida, A.M.; Winzerling, J.; Vazquez-Moreno, L.

2014-01-01

Abstract Amylases are an important family of enzymes involved in insect carbohydrate metabolism that are required for the survival of insect larvae. For this reason, enzymes from starch-dependent insects are targets for insecticidal control. PF2 ( Olneya tesota ) is a lectin that is toxic to Zabrotes subfasciatus (Coleoptera: Bruchidae) larvae. In this study, we evaluated recognition of the PF2 lectin to α-amylases from Z. subfasciatus midgut and the effect of PF2 on α-amylase activity. PF2 caused a decrease of total amylase activity in vitro. Subsequently, several α-amylase isoforms were isolated from insect midgut tissues using ion exchange chromatography. Three enzyme isoforms were verified by an in-gel assay for amylase activity; however, only one isoform was recognized by antiamylase serum and PF2. The identity of this Z. subfasciatus α-amylase was confirmed by liquid chromatography−tandem mass spectrometry. The findings strongly suggest that a glycosylated α-amylase isoform from larval Z. subfasciatus midgut interacts with PF2, which interferes with starch digestion. PMID:25528751

Identification and properties of an alpha-amylase from a strain of Eubacterium sp. isolated from the rat intestinal tract.

PubMed

Delahaye, E P; Foglietti, M J; Andrieux, C; Chardon-Loriaux, I; Szylit, O; Raibaud, P

1991-01-01

1. A bacterial amylase was isolated from the intestinal content of monoxenic rats inoculated with Eubacterium sp. B86. 2. Affinity chromatography on cross-linked starch allowed its separation from rat endogenous amylases. 3. The bacterial enzyme was characterized by its pI, molecular weight and action pattern. It behaves as a typical endo-amylase (alpha-amylase).

Properties of Thermosensitive Extracellular α-Amylases of Bacillus subtilis

PubMed Central

Yamane, Kunio; Maruo, Bunji

1974-01-01

Enzymological properties of four thermosensitive α-amylases (M3, M9, M18, and M20) brought by different mutation sites in α-amylase structural gene of Bacillus subtilis were compared with those of the parental α-amylase NA64. Two thermosensitive α-amylases (M9 and M20) were altered not only in their thermosensitivity but also in their immunological properties, catalytic properties, molecular weights determined by the gel filtration on a Bio-Gel P-100 column, and others. The other two thermosensitive α-amylases (M3 and M18) were altered only in their thermosensitivity. Images PMID:4218234

Paper-based α-amylase detector for point-of-care diagnostics.

PubMed

Dutta, Satarupa; Mandal, Nilanjan; Bandyopadhyay, Dipankar

2016-04-15

We report the fabrication of a paper-sensor for quantitative detection of α-amylase activity in human blood serum. Pieces of filter papers were coated with starch-iodine solution leading to an intense blue coloration on the surface. Dispensing α-amylase solution on the starch-iodine coated paper reduced the intensity of the color because of starch-hydrolysis catalyzed by amylase. The variation in the intensity of the color with the concentration of amylase was estimated in three stages: (i) initially, the paper-surface was illuminated with a light emitting diode, (ii) then, the transmitted (reflected) rays emitted through (from) the paper were collected on a photoresistor, and (iii) the variations in the electrical resistance of the photoresistor were correlated with the amylase concentration in analyte. The resistance of photoresistor decreased monotonically with an increase in amylase concentration because the intensity of the reflected (transmitted) rays collected from (through) the paper increased with reduction in the color intensity on the paper surface. Since a specific bio-reaction was employed to detect the activity of amylase, the sensor was found to be equally efficient in detecting unknown quantities of amylase in human blood serum. The reported sensor has shown the potential to graduate into a point-of-care detection tool for α-amylase. Copyright © 2015 Elsevier B.V. All rights reserved.

Posttranscriptional regulation of alpha-amylase II-4 expression by gibberellin in germinating rice seeds.

PubMed

Nanjo, Yohei; Asatsuma, Satoru; Itoh, Kimiko; Hori, Hidetaka; Mitsui, Toshiaki; Fujisawa, Yukiko

2004-06-01

Hormonal regulation of expression of alpha-amylase II-4 that lacks the gibberellin-response cis-element (GARE) in the promoter region of the gene was studied in germinating rice (Oryza sativa L.) seeds. Temporal and spatial expression of alpha-amylase II-4 in the aleurone layer were essentially identical to those of alpha-amylase I-1 whose gene contains GARE, although these were distinguishable in the embryo tissues at the early stage of germination. The gibberellin-responsible expression of alpha-amylase II-4 was also similar to that of alpha-amylase I-1. However, the level of alpha-amylase II-4 mRNA was not increased by gibberellin, indicating that the transcriptional enhancement of alpha-amylase II-4 expression did not occur in the aleurone. Gibberellin stimulated the accumulation of 45Ca2+ into the intracellular secretory membrane system. In addition, several inhibitors for Ca2+ signaling, such as EGTA, neomycin, ruthenium red (RuR), and W-7 prevented the gibberellin-induced expression of alpha-amylase II-4 effectively. While the gibberellin-induced expression of alpha-amylase II-4 occurred normally in the aleurone layer of a rice dwarf mutant d1 which is defective in the alpha subunit of the heterotrimeric G protein. Based on these results, it was concluded that the posttranscriptional regulation of alpha-amylase II-4 expression by gibberellin operates in the aleurone layer of germinating rice seed, which is mediated by Ca2+ but not the G protein.

Galanin inhibits caerulein-stimulated pancreatic amylase secretion via cholinergic nerves and insulin.

PubMed

Barreto, Savio G; Woods, Charmaine M; Carati, Colin J; Schloithe, Ann C; Jaya, Surendra R; Toouli, James; Saccone, Gino T P

2009-08-01

Pancreatic exocrine secretion is affected by galanin, but the mechanisms involved are unclear. We aimed to determine the effect and elucidate the mechanism of action of exogenous galanin on basal and stimulated pancreatic amylase secretion in vitro. The effect of galanin on basal-, carbachol-, and caerulein-stimulated amylase secretion from isolated murine pancreatic lobules was measured. Carbachol and caerulein concentration-response relationships were established. Lobules were coincubated with galanin (10(-12) M to 10(-7) M), carbachol (10(-6) M), or caerulein (10(-10) M). Lobules were preincubated with atropine (10(-5) M), tetrodotoxin (10(-5) M), hexamethonium (10(-5) M), or diazoxide (10(-7) M and 10(-4) M) for 30 min followed by incubation with caerulein (10(-10) M) alone or combined with galanin (10(-12) M). Amylase secretion was expressed as percent of total lobular amylase. Immunohistochemical studies used the antigen retrieval technique and antisera for galanin receptor (GALR) 1, 2, and 3. Carbachol and caerulein stimulated amylase secretion in a concentration-dependent manner with maximal responses of two- and 1.7-fold over control evoked at 10(-6) M and 10(-10) M, respectively. Galanin (10(-12) M) completely inhibited caerulein-stimulated amylase secretion but had no effect on carbachol-stimulated or basal secretion. Atropine and tetrodotoxin pretreatment abolished the caerulein-stimulated amylase secretion, whereas hexamethonium had no significant effect. Diazoxide significantly reduced caerulein-stimulated amylase secretion by approximately 80%. Galanin did not affect caerulein-stimulated amylase secretion in the presence of hexamethonium or diazoxide. Glucose-stimulated amylase secretion was also inhibited by galanin. Immunohistochemistry revealed islet cells labeled for GALR2. These data suggest that galanin may modulate caerulein-stimulated amylase secretion by acting on cholinergic nerves and/or islet cells possibly via GALR2 to regulate insulin release.

Individual Differences in AMY1 Gene Copy Number, Salivary α-Amylase Levels, and the Perception of Oral Starch

PubMed Central

Mandel, Abigail L.; Peyrot des Gachons, Catherine; Plank, Kimberly L.; Alarcon, Suzanne; Breslin, Paul A. S.

2010-01-01

Background The digestion of dietary starch in humans is initiated by salivary α-amylase, an endo-enzyme that hydrolyzes starch into maltose, maltotriose and larger oligosaccharides. Salivary amylase accounts for 40 to 50% of protein in human saliva and rapidly alters the physical properties of starch. Importantly, the quantity and enzymatic activity of salivary amylase show significant individual variation. However, linking variation in salivary amylase levels with the oral perception of starch has proven difficult. Furthermore, the relationship between copy number variations (CNVs) in the AMY1 gene, which influence salivary amylase levels, and starch viscosity perception has not been explored. Principal Findings Here we demonstrate that saliva containing high levels of amylase has sufficient activity to rapidly hydrolyze a viscous starch solution in vitro. Furthermore, we show with time-intensity ratings, which track the digestion of starch during oral manipulation, that individuals with high amylase levels report faster and more significant decreases in perceived starch viscosity than people with low salivary amylase levels. Finally, we demonstrate that AMY1 CNVs predict an individual's amount and activity of salivary amylase and thereby, ultimately determine their perceived rate of oral starch viscosity thinning. Conclusions By linking genetic variation and its consequent salivary enzymatic differences to the perceptual sequellae of these variations, we show that AMY1 copy number relates to salivary amylase concentration and enzymatic activity level, which, in turn, account for individual variation in the oral perception of starch viscosity. The profound individual differences in salivary amylase levels and salivary activity may contribute significantly to individual differences in dietary starch intake and, consequently, to overall nutritional status. PMID:20967220

OPTIMIZATION OF ALKALINE Α-AMYLASE PRODUCTION BY THERMOPHILIC BACILL US SUBTILIS.

PubMed

Al-Johani, Nuha Bakeet; Al-Seeni, Madeha N; Ahmed, Youssri Mohamed

2017-01-01

Starch-degrading amylase enzyme is important in biotechnological applications as food, fermentation, textile, paper and pharmaceutical purposes. The aim of current study to isolate alkaline thermostable α-amylase bacteria and then study the composition of medium and culture conditions to optimize cells growth and a-amylase production. Thermophilic amylase producing bacterium was isolated from local hot water-springs in Gazan city Saudi Arabia. Phylogenetic analysis of 16 S rRNA sequence for the strain revealed that the strain have the same sequence of Bacillus subtilis . Maximum amylase production was observed, when B. subtilis cultured in medium containing starch at concentration 0.5%, and 10 g/L peptones as nitrogen source at pH 8.5 in when it was incubated for 48 h at 45°C. An amylase-producing bacterium were isolated from hot-spring water and was identified as B. subtilis . Amylase produced from B.subtilis had optimum temperature 45°C and pH 8.5 in shaking media.

Amylase Synthesis and Stability in Crested Wheatgrass Seeds at Low Water Potentials 1

PubMed Central

Wilson, A. M.

1971-01-01

Drying of seeds of Agropyron desertorum (Fisch. ex Link) Schult. did not result in breakdown of α-amylase nor impair the ability of seeds to resume its synthesis when moistened again. β-Amylase activity did not change during 5 days of germination at a water potential of 0 atmosphere nor during 40 days of incubation at −40 atmospheres. Seeds synthesized α-amylase at 0, −20, and −40 atmospheres, but not at −60 atmospheres. At 0 and −20 atmospheres, the log of α-amylase activity was linearly related to hastening of germination. But at −40 atmospheres, seeds synthesized α-amylase during a time when there was little hastening of germination. Thus, it appears that other biochemical reactions are less drought-tolerant than synthesis of α-amylase. It is concluded that inhibition of α-amylase synthesis is not a controlling factor in the germination of these seeds at low water potentials. PMID:16657835

Application of α-amylase as a novel biodemulsifier for destabilizing amphiphilic polymer-flooding produced liquid treatment.

PubMed

Jiang, Jiatong; Wu, Hairong; Lu, Yao; Ma, Tao; Li, Zhe; Xu, Derong; Kang, Wanli; Bai, Baojun

2018-07-01

The performance and de-emulsification mechanism of α-amylase, a novel environmental friendly biodemulsifier in petroleum industry, was investigated at room temperature. The effects of α-amylase on the viscosity of amphiphilic polymer solution and de-emulsification rate were studied by changing the concentration of α-amylase, temperature and salinity. Polymer molecular weight, Zeta potential, interfacial film strength and interfacial tension were measured to investigate the de-emulsification mechanism of α-amylase. The results show that α-amylase is an efficient biodemulsifier to increase the de-emulsification rate of amphiphilic polymer emulsions. Hydrolysis of α-amylase to amphiphilic polymers destroys the structure of the amphiphilic polymer, thereby reduces the viscosity and the interfacial film strength of the system. Once de-emulsification is completed, the lower layer, i.e. the emulsified layer, will be clear. Thus, α-amylase can be applied as an effective de-emulsifier for amphiphilic polymer-stabilized O/W emulsion. Copyright © 2018 Elsevier Ltd. All rights reserved.

Recognition and binding of the PF2 lectin to α-amylase from Zabrotes subfasciatus (Coleoptera:Bruchidae) larval midgut.

PubMed

Lagarda-Diaz, I; Geiser, D; Guzman-Partida, A M; Winzerling, J; Vazquez-Moreno, L

2014-01-01

Amylases are an important family of enzymes involved in insect carbohydrate metabolism that are required for the survival of insect larvae. For this reason, enzymes from starch-dependent insects are targets for insecticidal control. PF2 (Olneya tesota) is a lectin that is toxic to Zabrotes subfasciatus (Coleoptera: Bruchidae) larvae. In this study, we evaluated recognition of the PF2 lectin to α-amylases from Z. subfasciatus midgut and the effect of PF2 on α-amylase activity. PF2 caused a decrease of total amylase activity in vitro. Subsequently, several α-amylase isoforms were isolated from insect midgut tissues using ion exchange chromatography. Three enzyme isoforms were verified by an in-gel assay for amylase activity; however, only one isoform was recognized by antiamylase serum and PF2. The identity of this Z. subfasciatus α-amylase was confirmed by liquid chromatography-tandem mass spectrometry. The findings strongly suggest that a glycosylated α-amylase isoform from larval Z. subfasciatus midgut interacts with PF2, which interferes with starch digestion. © The Author 2014. Published by Oxford University Press on behalf of the Entomological Society of America.

Bottleneck in secretion of α-amylase in Bacillus subtilis.

PubMed

Yan, Shaomin; Wu, Guang

2017-07-19

Amylase plays an important role in biotechnology industries, and Gram-positive bacterium Bacillus subtilis is a major host to produce heterogeneous α-amylases. However, the secretion stress limits the high yield of α-amylase in B. subtilis although huge efforts have been made to address this secretion bottleneck. In this question-oriented review, every effort is made to answer the following questions, which look simple but are long-standing, through reviewing of literature: (1) Does α-amylase need a specific and dedicated chaperone? (2) What signal sequence does CsaA recognize? (3) Does CsaA require ATP for its operation? (4) Does an unfolded α-amylase is less soluble than a folded one? (5) Does α-amylase aggregate before transporting through Sec secretion system? (6) Is α-amylase sufficient stable to prevent itself from misfolding? (7) Does α-amylase need more disulfide bonds to be stabilized? (8) Which secretion system does PrsA pass through? (9) Is PrsA ATP-dependent? (10) Is PrsA reused after folding of α-amylase? (11) What is the fate of PrsA? (12) Is trigger factor (TF) ATP-dependent? The literature review suggests that not only the most of those questions are still open to answers but also it is necessary to calculate ATP budget in order to better understand how B. subtilis uses its energy for production and secretion.

Phenolic group on A-ring is key for dracoflavan B as a selective noncompetitive inhibitor of α-amylase.

PubMed

Toh, Zhi Siang; Wang, Hongyu; Yip, Yew Mun; Lu, Yuyun; Lim, Benedict Jeffrey Ang; Zhang, Daiwei; Huang, Dejian

2015-12-15

A high throughput assay was applied to guide the isolation of a new pancreatic α-amylase inhibitor, dracoflavan B, from the dragon's blood resin from Daemonorops draco. Applying C18 column, we successfully isolated both diastereomers and their structures verified by (1)H NMR spectra in comparison with the literature values. Their activity in inhibition of pancreatic α-amylase with comparable IC50 values of 23μM (A type) and 27μM (B type) that are similar to that of acarbose. Dracoflavan B shows much weaker activity in inhibiting bacterial α-amylase and no activity towards fungal α-amylase. Moreover, both isomers show no inhibitory activity towards mammalian α-glucosidase. Kinetic analysis revealed that using starch as the substrate, dracoflavan B was a non-competitive α-amylase inhibitor with a Ki value of 11.7μM. Lack of α-amylase inhibition for proanthocyanidin A2 dimer demonstrated that dracoflavan B hydrophobic nature of the B, A', C' and B' rings are important for its α-amylase inhibition. In addition, selective chemical modification studies revealed that the phenolic group is also vital to dracoflavan B for its pancreatic α-amylase inhibition activity. Without the A ring phenolic hydrogen bond donor, the derivatives of dracoflavan B showed detrimental α-amylase inhibition. On the contrary, galloylation on the A ring phenolic OH group enhanced the activity as shown by the low IC50 (12μM) against α-amylase which is 56% more potent as compared to dracoflavan B. Copyright © 2015 Elsevier Ltd. All rights reserved.

Low serum amylase and obesity, diabetes and metabolic syndrome: A novel interpretation

PubMed Central

Nakajima, Kei

2016-01-01

For the last decade, low serum amylase (hypoamylasemia) has been reported in certain common cardiometabolic conditions such as obesity, diabetes (regardless of type), and metabolic syndrome, all of which appear to have a common etiology of insufficient insulin action due to insulin resistance and/or diminished insulin secretion. Some clinical studies have shown that salivary amylase may be preferentially decreased in obese individuals, whereas others have revealed that pancreatic amylase may be preferentially decreased in diabetic subjects with insulin dependence. Despite this accumulated evidence, the clinical relevance of serum, salivary, and pancreatic amylase and the underlying mechanisms have not been fully elucidated. In recent years, copy number variations (CNVs) in the salivary amylase gene (AMY1), which range more broadly than the pancreatic amylase gene (AMY2A and AMY2B), have been shown to be well correlated with salivary and serum amylase levels. In addition, low CNV of AMY1, indicating low salivary amylase, was associated with insulin resistance, obesity, low taste perception/satiety, and postprandial hyperglycemia through impaired insulin secretion at early cephalic phase. In most populations, insulin-dependent diabetes is less prevalent (minor contribution) compared with insulin-independent diabetes, and obesity is highly prevalent compared with low body weight. Therefore, obesity as a condition that elicits cardiometabolic diseases relating to insulin resistance (major contribution) may be a common determinant for low serum amylase in a general population. In this review, the novel interpretation of low serum, salivary, and pancreas amylase is discussed in terms of major contributions of obesity, diabetes, and metabolic syndrome. PMID:27022442

Liver alpha-amylase gene expression as an early obesity biomarker.

PubMed

Mojbafan, Marzieh; Afsartala, Zohreh; Amoli, Mahsa M; Mahmoudi, Mahdi; Yaghmaei, Parichehreh; Larijani, Bagher; Ebrahim-Habibi, Azadeh

2017-04-01

Obesity is a major health problem worldwide, for which preventive and therapeutic means are still needed. Alpha-amylase is a digestive enzyme whose inhibition has been targeted as a potential anti-obesity strategy. However, alpha-amylase gene expression has not been particularly attended to, and in contrast with pancreatic and salivary amylases, fewer studies have focused on liver alpha-amylase. The present study aimed at investigating the expression of alpha-amylase gene in obese and normal mice at RNA and protein level as well as acarbose effect on this gene expression in hepatocyte cell culture. Control and case groups were fed by normal mouse pellet and high-fat diet respectively, during 8 weeks. After this period, serum biochemical parameters including glucose, cholesterol, triglycerides, AST, ALT and alpha-amylase were assayed. Liver alpha-amylase gene was analyzed by real time PCR, and liver enzyme was assayed with Bernfeld and ELISA methods Hepatocyte cell culture derived from both group were also treated by acarbose and alpha-amylase activity and gene expression was analyzed by above mentioned methods. All biochemical factors showed an increase in obese mice, but the increase in ALT and AST were not statistically significant. Alpha-amylase levels were also increased in obese mice, both at RNA and protein level, while a decrease was seen in obese mice derived hepatocytes after acarbose treatment. Elevated liver alpha-amylase levels may be indicative of initial stages of obesity and the use of acarbose could be considered as a treatment of obesity which could be potentially effective at multiple levels. Copyright © 2016. Published by Elsevier Urban & Partner Sp. z o.o.

Stabilization of α-amylase by using anionic surfactant during the immobilization process

NASA Astrophysics Data System (ADS)

El-Batal, A. I.; Atia, K. S.; Eid, M.

2005-10-01

This work describes the entrapment of α-amylase into butylacrylate-acrylic acid copolymer (BuA/AAc) using γ irradiation. The effect of an anionic surfactant (AOT), the reuse efficiency, and kinetic behavior of immobilized α-amylase were studied. Covering of α-amylase with bis-(2-ethylhexyl)sulfosuccinate sodium salt (AOT) made the enzyme more stable than the uncovered form. The hydrolytic activity of the pre-coated immobilized α-amylase was increased below the critical micelle concentration (cmc) (10 mmol/L). The results showed an increase in the relative activity with increase in the degree of hydration. The pre-coated immobilized α-amylase showed a higher k/K and lower activation energy compared to the free and uncoated-immobilized preparation, respectively. The results suggest that the immobilization of α-amylase is a potentially useful approach for commercial starch hydrolysis in two-phase systems.

Evaluation of alpha- amylase inhibition by Urtica dioica and Juglans regia extracts.

PubMed

Rahimzadeh, Mahsa; Jahanshahi, Samaneh; Moein, Soheila; Moein, Mahmood Reza

2014-06-01

One strategy for the treatment of diabetes is inhibition of pancreatic α- amylase. Plants contains different chemical constituents with potential for inhibition of α-amylase and hence maybe used as therapeutic. Urtica dioica and Juglans regia Linn were tested for α-amylase inhibition. Different concentrations of leaf aqueous extracts were incubated with enzyme substrate solution and the activity of enzyme was measured. For determination of the type of inhibition, Dixon plot was depicted. Acarbose was used as the standard inhibitor. Both plant extracts showed time and concentration dependent inhibition of α-amylase. 60% inhibition was seen with 2 mg/ml of U. dioica and 0.4 mg/ml of J. regia aqueous extract. Dixon plots revealed the type of α-amylase inhibition by these two extracts as competitive inhibition. Determination of the type of α-amylase inhibition by these plant extracts could provide by successful use of plant chemicals as drug targets.

Encouragement of Enzyme Reaction Utilizing Heat Generation from Ferromagnetic Particles Subjected to an AC Magnetic Field.

PubMed

Suzuki, Masashi; Aki, Atsushi; Mizuki, Toru; Maekawa, Toru; Usami, Ron; Morimoto, Hisao

2015-01-01

We propose a method of activating an enzyme utilizing heat generation from ferromagnetic particles under an ac magnetic field. We immobilize α-amylase on the surface of ferromagnetic particles and analyze its activity. We find that when α-amylase/ferromagnetic particle hybrids, that is, ferromagnetic particles, on which α-amylase molecules are immobilized, are subjected to an ac magnetic field, the particles generate heat and as a result, α-amylase on the particles is heated up and activated. We next prepare a solution, in which α-amylase/ferromagnetic particle hybrids and free, nonimmobilized chitinase are dispersed, and analyze their activities. We find that when the solution is subjected to an ac magnetic field, the activity of α-amylase immobilized on the particles increases, whereas that of free chitinase hardly changes; in other words, only α-amylase immobilized on the particles is selectively activated due to heat generation from the particles.

21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false α-Amylase enzyme preparation from Bacillus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1012 α-Amylase enzyme preparation from Bacillus stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the culture...

21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false α-Amylase enzyme preparation from Bacillus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1012 α-Amylase enzyme preparation from Bacillus stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the culture...

21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false α-Amylase enzyme preparation from Bacillus... Specific Substances Affirmed as GRAS § 184.1012 α-Amylase enzyme preparation from Bacillus stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the culture filtrate that results from a pure...

21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true α-Amylase enzyme preparation from Bacillus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1012 α-Amylase enzyme preparation from Bacillus stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the culture...

21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false α-Amylase enzyme preparation from Bacillus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1012 α-Amylase enzyme preparation from Bacillus stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the culture...

Evaluation of commercial a-amylase enzyme-linked immunosorbent assy (ELISA) test kits for wheat

USDA-ARS?s Scientific Manuscript database

a-Amylase enzyme is associated with preharvest sprouting (PHS) and late-maturity a amylase (LMA) in wheat, and reduces wheat and flour quality. Various means have been developed to measure the presence of a-amylase, thereby predicting end-use quality; most are based on enzyme activity. An alternativ...

Copy number polymorphism of the salivary amylase gene: implications in human nutrition research.

PubMed

Santos, J L; Saus, E; Smalley, S V; Cataldo, L R; Alberti, G; Parada, J; Gratacòs, M; Estivill, X

2012-01-01

The salivary α-amylase is a calcium-binding enzyme that initiates starch digestion in the oral cavity. The α-amylase genes are located in a cluster on the chromosome that includes salivary amylase genes (AMY1), two pancreatic α-amylase genes (AMY2A and AMY2B) and a related pseudogene. The AMY1 genes show extensive copy number variation which is directly proportional to the salivary α-amylase content in saliva. The α-amylase amount in saliva is also influenced by other factors, such as hydration status, psychosocial stress level, and short-term dietary habits. It has been shown that the average copy number of AMY1 gene is higher in populations that evolved under high-starch diets versus low-starch diets, reflecting an intense positive selection imposed by diet on amylase copy number during evolution. In this context, a number of different aspects can be considered in evaluating the possible impact of copy number variation of the AMY1 gene on nutrition research, such as issues related to human diet gene evolution, action on starch digestion, effect on glycemic response after starch consumption, modulation of the action of α-amylases inhibitors, effect on taste perception and satiety, influence on psychosocial stress and relation to oral health. Copyright © 2012 S. Karger AG, Basel.

Application of microbial α-amylase in industry - A review.

PubMed

de Souza, Paula Monteiro; de Oliveira Magalhães, Pérola

2010-10-01

Amylases are one of the main enzymes used in industry. Such enzymes hydrolyze the starch molecules into polymers composed of glucose units. Amylases have potential application in a wide number of industrial processes such as food, fermentation and pharmaceutical industries. α-Amylases can be obtained from plants, animals and microorganisms. However, enzymes from fungal and bacterial sources have dominated applications in industrial sectors. The production of α-amylase is essential for conversion of starches into oligosaccharides. Starch is an important constituent of the human diet and is a major storage product of many economically important crops such as wheat, rice, maize, tapioca, and potato. Starch-converting enzymes are used in the production of maltodextrin, modified starches, or glucose and fructose syrups. A large number of microbial α-amylases has applications in different industrial sectors such as food, textile, paper and detergent industries. The production of α-amylases has generally been carried out using submerged fermentation, but solid state fermentation systems appear as a promising technology. The properties of each α-amylase such as thermostability, pH profile, pH stability, and Ca-independency are important in the development of fermentation process. This review focuses on the production of bacterial and fungal α-amylases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications.

The potato amylase inhibitor gene SbAI regulates cold-induced sweetening in potato tubers by modulating amylase activity.

PubMed

Zhang, Huiling; Liu, Jun; Hou, Juan; Yao, Ying; Lin, Yuan; Ou, Yongbin; Song, Botao; Xie, Conghua

2014-09-01

Potato cold-induced sweetening (CIS) is critical for the postharvest quality of potato tubers. Starch degradation is considered to be one of the key pathways in the CIS process. However, the functions of the genes that encode enzymes related to starch degradation in CIS and the activity regulation of these enzymes have received less attention. A potato amylase inhibitor gene known as SbAI was cloned from the wild potato species Solanum berthaultii. This genetic transformation confirmed that in contrast to the SbAI suppression in CIS-resistant potatoes, overexpressing SbAI in CIS-sensitive potatoes resulted in less amylase activity and a lower rate of starch degradation accompanied by a lower reducing sugar (RS) content in cold-stored tubers. This finding suggested that the SbAI gene may play crucial roles in potato CIS by modulating the amylase activity. Further investigations indicated that pairwise protein-protein interactions occurred between SbAI and α-amylase StAmy23, β-amylases StBAM1 and StBAM9. SbAI could inhibit the activities of both α-amylase and β-amylase in potato tubers primarily by repressing StAmy23 and StBAM1, respectively. These findings provide the first evidence that SbAI is a key regulator of the amylases that confer starch degradation and RS accumulation in cold-stored potato tubers. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Copy number variation of human AMY1 is a minor contributor to variation in salivary amylase expression and activity.

PubMed

Carpenter, Danielle; Mitchell, Laura M; Armour, John A L

2017-02-20

Salivary amylase in humans is encoded by the copy variable gene AMY1 in the amylase gene cluster on chromosome 1. Although the role of salivary amylase is well established, the consequences of the copy number variation (CNV) at AMY1 on salivary amylase protein production are less well understood. The amylase gene cluster is highly structured with a fundamental difference between odd and even AMY1 copy number haplotypes. In this study, we aimed to explore, in samples from 119 unrelated individuals, not only the effects of AMY1 CNV on salivary amylase protein expression and amylase enzyme activity but also whether there is any evidence for underlying difference between the common haplotypes containing odd numbers of AMY1 and even copy number haplotypes. AMY1 copy number was significantly correlated with the variation observed in salivary amylase production (11.7% of variance, P < 0.0005) and enzyme activity (13.6% of variance, P < 0.0005) but did not explain the majority of observed variation between individuals. AMY1-odd and AMY1-even haplotypes showed a different relationship between copy number and expression levels, but the difference was not statistically significant (P = 0.052). Production of salivary amylase is correlated with AMY1 CNV, but the majority of interindividual variation comes from other sources. Long-range haplotype structure may affect expression, but this was not significant in our data.

Sequence similarities and evolutionary relationships of microbial, plant and animal alpha-amylases.

PubMed

Janecek, S

1994-09-01

Amino acid sequence comparison of 37 alpha-amylases from microbial, plant and animal sources was performed to identify their mutual sequence similarities in addition to the five already described conserved regions. These sequence regions were examined from structure/function and evolutionary perspectives. An unrooted evolutionary tree of alpha-amylases was constructed on a subset of 55 residues from the alignment of sequence similarities along with conserved regions. The most important new information extracted from the tree was as follows: (a) the close evolutionary relationship of Alteromonas haloplanctis alpha-amylase (thermolabile enzyme from an antarctic psychrotroph) with the already known group of homologous alpha-amylases from streptomycetes, Thermomonospora curvata, insects and mammals, and (b) the remarkable 40.1% identity between starch-saccharifying Bacillus subtilis alpha-amylase and the enzyme from the ruminal bacterium Butyrivibrio fibrisolvens, an alpha-amylase with an unusually large polypeptide chain (943 residues in the mature enzyme). Due to a very high degree of similarity, the whole amino acid sequences of three groups of alpha-amylases, namely (a) fungi and yeasts, (b) plants, and (c) A. haloplanctis, streptomycetes, T. curvata, insects and mammals, were aligned independently and their unrooted distance trees were calculated using these alignments. Possible rooting of the trees was also discussed. Based on the knowledge of the location of the five disulfide bonds in the structure of pig pancreatic alpha-amylase, the possible disulfide bridges were established for each of these groups of homologous alpha-amylases.

α-Amylase: an enzyme specificity found in various families of glycoside hydrolases.

PubMed

Janeček, Štefan; Svensson, Birte; MacGregor, E Ann

2014-04-01

α-Amylase (EC 3.2.1.1) represents the best known amylolytic enzyme. It catalyzes the hydrolysis of α-1,4-glucosidic bonds in starch and related α-glucans. In general, the α-amylase is an enzyme with a broad substrate preference and product specificity. In the sequence-based classification system of all carbohydrate-active enzymes, it is one of the most frequently occurring glycoside hydrolases (GH). α-Amylase is the main representative of family GH13, but it is probably also present in the families GH57 and GH119, and possibly even in GH126. Family GH13, known generally as the main α-amylase family, forms clan GH-H together with families GH70 and GH77 that, however, contain no α-amylase. Within the family GH13, the α-amylase specificity is currently present in several subfamilies, such as GH13_1, 5, 6, 7, 15, 24, 27, 28, 36, 37, and, possibly in a few more that are not yet defined. The α-amylases classified in family GH13 employ a reaction mechanism giving retention of configuration, share 4-7 conserved sequence regions (CSRs) and catalytic machinery, and adopt the (β/α)8-barrel catalytic domain. Although the family GH57 α-amylases also employ the retaining reaction mechanism, they possess their own five CSRs and catalytic machinery, and adopt a (β/α)7-barrel fold. These family GH57 attributes are likely to be characteristic of α-amylases from the family GH119, too. With regard to family GH126, confirmation of the unambiguous presence of the α-amylase specificity may need more biochemical investigation because of an obvious, but unexpected, homology with inverting β-glucan-active hydrolases.

Partial characterization of a novel amylase activity isolated from Tunisian Ficus carica latex.

PubMed

Aref, Houda Lazreg; Mosbah, Habib; Louati, Hanen; Said, Khaled; Selmi, Boulbaba

2011-11-01

A large number of plants still need to be investigated through screening of amylases suitable for industry. In the present study, and for the first time, we describe the amylolytic activity of Saint Pedro Ficus carica L. (Moraceae) crude latex of Kahli and Bidhi varieties. Effects of temperature, pH, metal ions, and inhibitors and compatibility with some commercial detergents were investigated for amylase activity. Amylase activity was screened in crude latex using the DNS method and potato starch as a substrate. Analyses of amylolytic reaction products by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) were performed. Bidhi and Kahli amylases were active in optimal pH of 6.5 and 7 at 45°C, respectively, displaying a half life of 85 and 60 min, respectively, at 80°C, and they were very stable in a wide range of pH (4-12). Bidhi amylase activity increased to 260% by addition of 10(-3) mM Fe(2+) or 10(-2) mM Cu(2+), and was strongly inhibited by Mg(2+) and EDTA. In the presence of Ca(2+) and Mg(2+), Kahli amylase activity was dramatically enhanced by 220 and 260%, respectively. The compatibility of both amylases with certain commercial detergents was also shown to be good as enzymes retained up to 98% of their activities after 30 min of incubation at 80°C. Analysis of amylolytic reaction products by TLC and HPLC suggested that Kahli amylase was an amyloglucosidase and Bidhi amylase was β-fructose, α(1-4) glucose. Bidhi amylase is a good choice for application in starch, food, detergents and medical industries.

Study of conformation and thermodynamics of α-amylase interaction with ethylene in vitro.

PubMed

Hu, Yiwei; Zhang, Guangxian; Zhang, Fengxiu

2016-10-01

In this article, the conformation and thermodynamics of α-amylase interaction with ethylene in vitro were investigated. The ultraviolet (UV) absorption showed a strong peak of α-amylase treated with 6.04, 29.32 and 262.11μmolL(-1) ethylene appears at ~222nm and a weak peak at 278nm blue-shifted 1nm. Circular dichroism (CD) spectra indicated that the conformations of α-amylase treated with 29.32 and 262.11μmolL(-1) ethylene were obviously changed in which α-helix content were decreased by 20 and 31% respectively, and β-sheet, β-turn and random coil contents were increased by contrast. Fluorescence spectra suggested that the peak intensities of α-amylase at 342nm were obviously increased with the ethylene increase from 6.04 to 525.75μmolL(-1) and more than control group. The binding constants K between ethylene and α-amylase were 3.318×10(6), 4.407×10(6) and 5.125×10(6)Lmol(-1) at 288, 298 and 308K, respectively. And the calculated values of ΔH(0) and ΔS(0) are positive, which suggests that the interaction between ethylene and α-amylase is an endothermic reaction. The negative ΔG(0) values implied that the direct effect of ethylene on α-amylase conformation was spontaneous. The possible reason is that ethylene molecules were easily embedded into the interior of α-amylase in term of the hydrophobic force between α-amylase and ethylene, causing the conformation and thermodynamics changes of α-amylase. Copyright © 2016 Elsevier B.V. All rights reserved.

Selection for low dormancy in annual ryegrass (Lolium rigidum) seeds results in high constitutive expression of a glucose-responsive α-amylase isoform

PubMed Central

Goggin, Danica E.; Powles, Stephen B.

2012-01-01

Background and Aims α-Amylase in grass caryopses (seeds) is usually expressed upon commencement of germination and is rarely seen in dry, mature seeds. A heat-stable α-amylase activity was unexpectedly selected for expression in dry annual ryegrass (Lolium rigidum) seeds during targeted selection for low primary dormancy. The aim of this study was to characterize this constitutive activity biochemically and determine if its presence conferred insensitivity to the germination inhibitors abscisic acid and benzoxazolinone. Methods α-Amylase activity in developing, mature and germinating seeds from the selected (low-dormancy) and a field-collected (dormant) population was characterized by native activity PAGE. The response of seed germination and α-amylase activity to abscisic acid and benzoxazolinone was assessed. Using an alginate affinity matrix, α-amylase was purified from dry and germinating seeds for analysis of its enzymatic properties. Key Results The constitutive α-amylase activity appeared late during seed development and was mainly localized in the aleurone; in germinating seeds, this activity was responsive to both glucose and gibberellin. It migrated differently on native PAGE compared with the major activities in germinating seeds of the dormant population, but the enzymatic properties of α-amylase purified from the low-dormancy and dormant seeds were largely indistinguishable. Seed imbibition on benzoxazolinone had little effect on the low-dormancy seeds but greatly inhibited germination and α-amylase activity in the dormant population. Conclusions The constitutive α-amylase activity in annual ryegrass seeds selected for low dormancy is electrophoretically different from that in germinating seeds and its presence confers insensitivity to benzoxazolinone. The concurrent selection of low dormancy and constitutive α-amylase activity may help to enhance seedling establishment under competitive conditions. PMID:23002268

Halophilic Amylase from a Moderately Halophilic Micrococcus

PubMed Central

Onishi, Hiroshi

1972-01-01

A moderately halophilic Micrococcus sp., isolated from unrefined solar salt, produced a considerable amount of extracellular dextrinogenic amylase when cultivated aerobically in media containing 1 to 3 m NaCl. The Micrococcus amylase had maximal activity at pH 6 to 7 in 1.4 to 2 m NaCl or KCl at 50 C. Calcium ion and a high concentration of NaCl or KCl were essential for activity and stability of the amylase. The salt response of the amylase depended greatly on the pH and temperature of the enzyme assay. PMID:5058445

Inhibition of Sunn pest, Eurygaster integriceps, α-amylases by α-amylase inhibitors (T-αAI) from Triticale.

PubMed

Mehrabadi, Mohammad; Bandani, Ali R; Saadati, Fatemeh

2010-01-01

The effect of triticale α-amylases inhibitors on starch hydrolysis catalyzed by the Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae) midgut amylases was examined. Biochemical studgawies showed that inhibitors from Triticale (a hybrid of wheat and rye) had inhibitiory effects on E. integriceps α-amylases. The effects of the triticale α-amylase inhibitor (T-αAI) on α-amylase of E. integriceps showed a dose dependent manner of inhibition, e.g. less inhibition of enzyme activity (around 10%) with a lower dose (0.25 mg protein) and high inhibition of enzyme activity (around 80%) when a high dose of inhibitor was used (1.5 mg protein). The enzyme kinetic studies using Michaelis-Menten and Lineweaver-Burk equations showed the K(m) remained constant (0.58%) but the maximum velocity (V(max)) decreased in the presence of a crude extract of Triticale inhibitors, indicating mixed inhibition. The temperature giving 50% inactivation of enzyme (T(50)) during a 30-min incubation at pH 7.0 was 73° C. The maximum inhibitory activity was achieved at 35° C and pH 5.0. Gel assays showed the meaningful inhibition of E. integriceps α-amylases by various concentrations of Triticale inhibitors. Based on the data presented in this study, it could be said that the T-αAI has good inhibitory activity on E. integriceps gut α-amylase.

Optimization, Purification, and Starch Stain Wash Application of Two New α-Amylases Extracted from Leaves and Stems of Pergularia tomentosa

PubMed Central

El Abed, Hanen; Belghith, Hafedh; Ben Abdallah, Ferjani; Belghith, Karima

2017-01-01

A continuous research is attempted to fulfil the highest industrial demands of natural amylases presenting special properties. New α-amylases extracted from stems and leaves of Pergularia tomentosa, which is widespread and growing spontaneously in Tunisia, were studied by the means of their activities optimization and purification. Some similarities were recorded for the two identified enzymes: (i) the highest amylase activity showed a promoted thermal stability at 50°C; (ii) the starch substrate at 1% enhanced the enzyme activity; (iii) the two α-amylases seem to be calcium-independent; (iv) Zn2+, Cu2+, and Ag2+ were considered as important inhibitors of the enzyme activity. Following the increased gradient of elution on Mono Q-Sepharose column, an increase in the specific activity of 11.82-fold and 10.92-fold was recorded, respectively, for leaves and stems with the presence of different peaks on the purification profiles. Pergularia amylases activities were stable and compatible with the tested commercial detergents. The combination of plant amylase and detergent allowed us to enhance the wash performance with an increase of 35.24 and 42.56%, respectively, for stems and leaves amylases. Characterized amylases were reported to have a promoted potential for their implication notably in detergent industry as well as biotechnological sector. PMID:29392138

Oral Fusobacterium nucleatum subsp. polymorphum binds to human salivary α-amylase.

PubMed

Zulfiqar, M; Yamaguchi, T; Sato, S; Oho, T

2013-12-01

Fusobacterium nucleatum acts as an intermediate between early and late colonizers in the oral cavity. In this study, we showed that F. nucleatum subsp. polymorphum can bind to a salivary component with a molecular weight of approximately 110 kDa and identified the protein and another major factor of 55 kDa, as salivary α-amylase by time-of-flight mass spectrometry and immuno-reactions. Salivary α-amylase is present in both monomeric and dimeric forms and we found that formation of the dimer depends on copper ions. The F. nucleatum adhered to both monomeric and dimeric salivary α-amylases, but the numbers of bacteria bound to the dimeric form were more than those bound to the monomeric form. The degree of adherence of F. nucleatum to four α-amylases from different sources was almost the same, however its binding to β-amylase was considerably decreased. Among four α-amylase inhibitors tested, acarbose and type 1 and 3 inhibitors derived from wheat flour showed significant activity against the adhesion of F.nucleatum to monomeric and dimeric amylases, however voglibose had little effect. Moreover F. nucleatum cells inhibited the enzymatic activity of salivary α-amylase in a dose-dependent manner. These results suggest that F. nucleatum plays more important and positive role as an early colonizer for maturation of oral microbial colonization. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Inhibition of Sunn Pest, Eurygaster integriceps, α-Amylases by α-Amylase Inhibitors (T-αAI) from Triticale

PubMed Central

Mehrabadi, Mohammad; Bandani, Ali R.; Saadati, Fatemeh

2010-01-01

The effect of triticale α-amylases inhibitors on starch hydrolysis catalyzed by the Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae) midgut amylases was examined. Biochemical studgawies showed that inhibitors from Triticale (a hybrid of wheat and rye) had inhibitiory effects on E. integriceps α-amylases. The effects of the triticale α-amylase inhibitor (T-αAI) on α-amylase of E. integriceps showed a dose dependent manner of inhibition, e.g. less inhibition of enzyme activity (around 10%) with a lower dose (0.25 mg protein) and high inhibition of enzyme activity (around 80%) when a high dose of inhibitor was used (1.5 mg protein). The enzyme kinetic studies using Michaelis-Menten and Lineweaver-Burk equations showed the Km remained constant (0.58%) but the maximum velocity (Vmax) decreased in the presence of a crude extract of Triticale inhibitors, indicating mixed inhibition. The temperature giving 50% inactivation of enzyme (T50) during a 30-min incubation at pH 7.0 was 73° C. The maximum inhibitory activity was achieved at 35° C and pH 5.0. Gel assays showed the meaningful inhibition of E. integriceps α-amylases by various concentrations of Triticale inhibitors. Based on the data presented in this study, it could be said that the T-αAI has good inhibitory activity on E. integriceps gut α-amylase. PMID:21062146

Application of microbial α-amylase in industry – A review

PubMed Central

de Souza, Paula Monteiro; de Oliveira Magalhães, Pérola

2010-01-01

Amylases are one of the main enzymes used in industry. Such enzymes hydrolyze the starch molecules into polymers composed of glucose units. Amylases have potential application in a wide number of industrial processes such as food, fermentation and pharmaceutical industries. α-Amylases can be obtained from plants, animals and microorganisms. However, enzymes from fungal and bacterial sources have dominated applications in industrial sectors. The production of α-amylase is essential for conversion of starches into oligosaccharides. Starch is an important constituent of the human diet and is a major storage product of many economically important crops such as wheat, rice, maize, tapioca, and potato. Starch-converting enzymes are used in the production of maltodextrin, modified starches, or glucose and fructose syrups. A large number of microbial α-amylases has applications in different industrial sectors such as food, textile, paper and detergent industries. The production of α-amylases has generally been carried out using submerged fermentation, but solid state fermentation systems appear as a promising technology. The properties of each α-amylase such as thermostability, pH profile, pH stability, and Ca-independency are important in the development of fermentation process. This review focuses on the production of bacterial and fungal α-amylases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications. PMID:24031565

Macroamylasemia

MedlinePlus

... amylase. However, macroamylasemia can look similar to acute pancreatitis , which also causes high levels of amylase in ... urine can help tell macroamylasemia apart from acute pancreatitis. Urine levels of amylase are low in people ...

Phenotypic Variation for Diastatic power, ß-Amylase Activity, and ß-Amylase Thermostability vs. Allelic Variation at the Bmy1 Locus in a Sample of North American Barley Germplasm

USDA-ARS?s Scientific Manuscript database

Malting quality data including diastatic power, ß-amylase activity, and ß-amylase thermostability, were collected on malts from three barley (Hordeum vulgare L.) breeding program trials containing two growth habits and 165 lines grown in multiple environments. We attempted to identify causal polymor...

Potential of the bean alpha-amylase inhibitor alpha-AI-1 to inhibit alpha-amylase activity in true bugs(Hemiptera)

USDA-ARS?s Scientific Manuscript database

True bugs (Hemiptera) are an important pest complex not controlled by Bt crops. An alternative source of resistance includes inhibitors of digestive enzymes. aAI-1, an a-amylase inhibitor from the common bean, has been shown to inhibit a-amylases of bruchid pests of grain legumes. Here we quantify t...

High-throughput hydrolysis of starch during permeation across α-amylase-immobilized porous hollow-fiber membranes

NASA Astrophysics Data System (ADS)

Miura, Suguru; Kubota, Noboru; Kawakita, Hidetaka; Saito, Kyoichi; Sugita, Kazuyuki; Watanabe, Kohei; Sugo, Takanobu

2002-02-01

Two kinds of supporting porous membranes, ethanolamine (EA) and phenol (Ph) fibers, for immobilization of α-amylase were prepared by radiation-induced graft polymerization of an epoxy-group-containing monomer, glycidyl methacrylate, onto a porous hollow-fiber membrane, and subsequent ring-opening with EA and Ph, respectively. An α-amylase solution was forced to permeate radially outward through the pores of the EA and Ph fibers. α-Amylase was captured at a density of 0.15 and 6.6 g/L of the membrane by the graft chain containing 2-hydroxyethylamino and phenyl groups, respectively. A permeation pressure of 0.10 MPa provided a space velocity of 780 and 1500 h -1 for the α-amylase-immobilized EA and Ph fibers, respectively. Quantitative hydrolysis of starch during permeation of a 20 g/L starch solution in the buffer across the α-amylase-immobilized Ph fiber was attained up to a space velocity of about 2000 h -1; this was achieved because of negligible diffusional mass-transfer resistance of the starch to the α-amylase due to convective flow, whereas an enzyme reaction-controlled system was observed for the α-amylase-immobilized EA fiber.

Thermostable 𝜶-Amylase Activity from Thermophilic Bacteria Isolated from Bora Hot Spring, Central Sulawesi

NASA Astrophysics Data System (ADS)

Gazali, F. M.; Suwastika, I. N.

2018-03-01

α-Amylase is one of the most important enzyme in biotechnology field, especially in industrial application. Thermostability of α-Amylase produced by thermophilic bacteria improves industrial process of starch degradation in starch industry. The present study were concerned to the characterization of α-Amylase activity from indigenous thermophilic bacteria isolated from Bora hot spring, Central Sulawesi. There were 18 isolates which had successfully isolated from 90°C sediment samples of Bora hot spring and 13 of them showed amylolytic activity. The α-Amylase activity was measured qualitatively at starch agar and quantitatively based on DNS (3,5-Dinitrosalicylic acid) methods, using maltose as standard solution. Two isolates (out of 13 amylolytic bacteria), BR 002 and BR 015 showed amylolytic index of 0.8 mm and 0.5 mm respectively, after being incubated at 55°C in the 0.002% Starch Agar Medium. The α-Amylase activity was further characterized quantitatively which includes the optimum condition of pH and temperature of α-Amylase crude enzyme from each isolate. To our knowledge, this is the first report on isolation and characterization of a thermostable α-Amylase from thermophilic bacteria isolated from Central Sulawesi particularly from Bora hot spring.

Evaluation of alpha- amylase inhibition by Urtica dioica and Juglans regia extracts

PubMed Central

Rahimzadeh, Mahsa; Jahanshahi, Samaneh; Moein, Soheila; Moein, Mahmood Reza

2014-01-01

Objective(s): One strategy for the treatment of diabetes is inhibition of pancreatic α- amylase. Plants contains different chemical constituents with potential for inhibition of α-amylase and hence maybe used as therapeutic. Materials and Methods: Urtica dioica and Juglans regia Linn were tested for α-amylase inhibition. Different concentrations of leaf aqueous extracts were incubated with enzyme substrate solution and the activity of enzyme was measured. For determination of the type of inhibition, Dixon plot was depicted. Acarbose was used as the standard inhibitor. Results: Both plant extracts showed time and concentration dependent inhibition of α-amylase. 60% inhibition was seen with 2 mg/ml of U. dioica and 0.4 mg/ml of J. regia aqueous extract. Dixon plots revealed the type of α-amylase inhibition by these two extracts as competitive inhibition. Conclusion: Determination of the type of α-amylase inhibition by these plant extracts could provide by successful use of plant chemicals as drug targets. PMID:25140210

Physical and catalytic properties of alpha-amylase from Tenebrio molitor L. larvae.

PubMed Central

Buonocore, V; Poerio, E; Silano, V; Tomasi, M

1976-01-01

The amylase from Tenebrio molitor L. larvae (yellow mealworm) was characterized according to a number of its molecular and catalytic properties. The insect amylase is a single polypeptide chain with mol.wt. 68000, an isoelectric point of 4.0 and a very low content of sulphur-containing amino acids. The enzyme is a Ca2+-protein and behaves as an alpha-amylase. Removal of Ca2+ by exhaustive dialysis against water causes the irreversible inactivation of the enzyme. Moreover, the enzyme is activated by the presence in the assay mixture of Cl-, or some other inorganic anions that are less effective than Cl-, and is inhibited by F-. Optimal conditions of pH and temperature for the enzymic activity are 5.8 and 37 degrees C. The insect amylase exhibits an identical kinetic behaviour toward starch, amylose and amylopectin; the enzyme hydrolyses glycogen with a higher affinity constant. Compared with the non-insect alpha-amylases described in the literature, Tenebrio molitor amylase has a lower affinity for starch. PMID:942374

Amylase production potentials of bacterial isolates obtained from the gut of Oryctes rhinoceros larvae

NASA Astrophysics Data System (ADS)

Aryati, P. C.; Pangastuti, A.; Sari, S. L. A.

2017-04-01

Amylase is one of the main enzymes used in industry, such as food, detergent, textile, and pharmaceutical industry. Amylase can be produced by plants, animals, and microorganisms. However, bacterial and fungal amylases have dominated application in industries. This research was aimed to determine amylolytic activity of bacteria isolated from the gut of Oryctes rhinoceros larvae. Based on clear zone formation, 9 from 11 isolates showed amylolytic activity. Isolates with the widest clear zone, i.e Bacillus subtilis GOR1, Bacillus cereus GOR3, and Bacillus pumilus GOR2, were screened for amylolytic activity based on reduction sugar production. The result showed that Bacillus subtilis GOR1 was the most potential as amylase producer, showed by the widest clear zone 5.224 cm2 and highest reduction sugar production 0.0235 mg/ml. Highest amylase specific activity (0.1447 U/mg protein) was obtained at 60°C and pH 7. Amylase activity was stable for 3 hours at 60°C with residual activity respectively was 59.7%.

Encouragement of Enzyme Reaction Utilizing Heat Generation from Ferromagnetic Particles Subjected to an AC Magnetic Field

PubMed Central

Suzuki, Masashi; Aki, Atsushi; Mizuki, Toru; Maekawa, Toru; Usami, Ron; Morimoto, Hisao

2015-01-01

We propose a method of activating an enzyme utilizing heat generation from ferromagnetic particles under an ac magnetic field. We immobilize α-amylase on the surface of ferromagnetic particles and analyze its activity. We find that when α-amylase/ferromagnetic particle hybrids, that is, ferromagnetic particles, on which α-amylase molecules are immobilized, are subjected to an ac magnetic field, the particles generate heat and as a result, α-amylase on the particles is heated up and activated. We next prepare a solution, in which α-amylase/ferromagnetic particle hybrids and free, nonimmobilized chitinase are dispersed, and analyze their activities. We find that when the solution is subjected to an ac magnetic field, the activity of α-amylase immobilized on the particles increases, whereas that of free chitinase hardly changes; in other words, only α-amylase immobilized on the particles is selectively activated due to heat generation from the particles. PMID:25993268

alpha-Amylase gene of Streptomyces limosus: nucleotide sequence, expression motifs, and amino acid sequence homology to mammalian and invertebrate alpha-amylases.

PubMed Central

Long, C M; Virolle, M J; Chang, S Y; Chang, S; Bibb, M J

1987-01-01

The nucleotide sequence of the coding and regulatory regions of the alpha-amylase gene (aml) of Streptomyces limosus was determined. High-resolution S1 mapping was used to locate the 5' end of the transcript and demonstrated that the gene is transcribed from a unique promoter. The predicted amino acid sequence has considerable identity to mammalian and invertebrate alpha-amylases, but not to those of plant, fungal, or eubacterial origin. Consistent with this is the susceptibility of the enzyme to an inhibitor of mammalian alpha-amylases. The amino-terminal sequence of the extracellular enzyme was determined, revealing the presence of a typical signal peptide preceding the mature form of the alpha-amylase. Images PMID:3500166

Starch Degradation in the Cotyledons of Germinating Lentils

PubMed Central

Tárrago, Jorge Fernández; Nicolás, Gregorio

1976-01-01

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. Images PMID:16659730

Cloning of a novel thermostable glucoamylase from thermophilic fungus Rhizomucor pusillus and high-level co-expression with α-amylase in Pichia pastoris.

PubMed

He, Zhenggui; Zhang, Lujia; Mao, Youzhi; Gu, Jingchao; Pan, Qi; Zhou, Sixing; Gao, Bei; Wei, Dongzhi

2014-12-24

Fungal amylase, mainly constitute of fungal α-amylase and glucoamylase, are utilized in a broad range of industries, such as starch hydrolysis, food and brewing. Although various amylases have been found in fungi, the amylases from Aspergillus dominate the commercial application. One of main problems exist with regard to these commercial use of amylases is relatively low thermal and acid stability. In order to maximize the efficiency of starch process, developing fungal amylases with increased thermostability and acid stability has been attracting researchers' interest continually. Besides, synergetic action of glucoamylase and α-amylase could facilitate the degradation of starch. And co-expressing glucoamylase with α-amylase in one host could avoid the need to ferment repeatedly and improves cost-effectiveness of the process. A novel fungal glucoamylase (RpGla) gene encoding a putative protein of 512 amino acid residues was cloned from Rhizomucor pusillus. BLAST analysis revealed that RpGla shared highest identity of 51% with the Rhizopus oryzae glucoamylase (ABB77799.1). The fungal glucoamylase RpGla was expressed in Pichia pastoris (KM71/9KGla) with maximum activity of 1237 U ml(-1). The optimum pH and temperature of RpGla were pH 4.0 and 70 °C, respectively. Fungal α-amylase (RpAmy) gene was also cloned from R. pusillus and transformed into KM71/9KGla, resulted in recombinant yeast KM71/9KGla-ZαAmy harboring the RpGla and RpAmy genes simultaneously. The maximum saccharogenic activity of KM71/9KGla-ZαAmy was 2218 U ml(-1), which improved 79% compared to KM71/9KGla. Soluble starch hydrolyzed by purified RpGla achieved 43% glucose and 34% maltose. Higher productivity was achieved with a final yield of 48% glucose and 47% maltose catalyzed by purified enzyme preparation produced by KM71/9KGla-ZαAmy. A novel fungal glucoamylase and fungal α-amylase genes were cloned from Rhizomucor pusillus. The two enzymes showed good thermostability and acid stability, and similar biochemical properties facilitated synergetic action of the two enzymes. A dramatic improvement was seen in amylase activity through co-expressing RpGla with RpAmy in Pichia pastoris. This is the first report of improving activity through co-expression glucoamylase with α-amylase in P. pastoris. Besides, fungal glucoamylase and α-amylase from R. pusillus were shown as promising candidates for further application in starch hydrolysis.

Effects of a dietary Aspergillus oryzae extract containing alpha-amylase activity on performance and carcass characteristics of finishing beef cattle.

PubMed

Tricarico, J M; Abney, M D; Galyean, M L; Rivera, J D; Hanson, K C; McLeod, K R; Harmon, D L

2007-03-01

Three experiments were conducted to examine the effects of an Aspergillus oryzae extract containing alpha-amylase activity on performance and carcass characteristics of finishing beef cattle. In Exp. 1, 120 crossbred steers were used in a randomized complete block design to evaluate the effects of roughage source (alfalfa hay vs. cottonseed hulls) and supplemental alpha-amylase at 950 dextrinizing units (DU)/kg of DM. Significant roughage source x alpha-amylase interactions (P < 0.05) were observed for performance. In steers fed cottonseed hulls, supplemental alpha-amylase increased ADG through d 28 and 112 and tended (P < 0.15) to increase ADG in all other periods. The increases in ADG were related to increased DMI and efficiency of gain during the initial 28-d period but were primarily related to increased DMI as the feeding period progressed. Supplemental alpha-amylase increased (P = 0.02) the LM area across both roughage sources. In Exp. 2, 96 crossbred heifers were used in a randomized complete block design with a 2 x 3 factorial arrangement of treatments to evaluate the effects of corn processing (dry cracked vs. high moisture) and supplemental alpha-amylase concentration (0, 580, or 1,160 DU/kg of DM). Alpha-amylase supplementation increased DMI (P = 0.05) and ADG (P = 0.03) during the initial 28 d on feed and carcass-adjusted ADG (P = 0.04) across corn processing methods. Longissimus muscle area was greatest (quadratic effect, P = 0.04), and yield grade was least (quadratic effect, P = 0.02) in heifers fed 580 DU of alpha-amylase/kg of DM across corn processing methods. In Exp. 3, 56 crossbred steers were used in a randomized complete block design to evaluate the effects of supplemental alpha-amylase (930 DU/kg of DM) on performance when DMI was restricted to yield a programmed ADG. Alpha-amylase supplementation did not affect performance when DMI was restricted. We conclude that dietary alpha-amylase supplementation of finishing beef diets may result in increased ADG through increased DMI under certain dietary conditions and that further research is warranted to explain its mode of action and interactions with dietary ingredients.

Dose- and tissue-specific interaction of monoterpenes with the gibberellin-mediated release of potato tuber bud dormancy, sprout growth and induction of α-amylases and β-amylases.

PubMed

Rentzsch, Sonja; Podzimska, Dagmara; Voegele, Antje; Imbeck, Madeleine; Müller, Kerstin; Linkies, Ada; Leubner-Metzger, Gerhard

2012-01-01

Gibberellins (GA) are involved in bud dormancy release in several species. We show here that GA-treatment released bud dormancy, initiated bud sprouting and promoted sprout growth of excised potato tuber bud discs ('eyes'). Monoterpenes from peppermint oil (PMO) and S-(+)-carvone (CAR) interact with the GA-mediated bud dormancy release in a hormesis-type response: low monoterpene concentrations enhance dormancy release and the initiation of bud sprouting, whereas high concentrations inhibit it. PMO and CAR did, however, not affect sprout growth rate after its onset. We further show that GA-induced dormancy release is associated with tissue-specific regulation of α- and β-amylases. Molecular phylogenetic analysis shows that potato α-amylases cluster into two distinct groups: α-AMY1 and α-AMY2. GA-treatment induced transcript accumulation of members of both α-amylase groups, as well as α- and β-amylase enzyme activity in sprout and 'sub-eye' tissues. In sprouts, CAR interacts with the GA-mediated accumulation of α-amylase transcripts in an α-AMY2-specific and dose-dependent manner. Low CAR concentrations enhance the accumulation of α-AMY2-type α-amylase transcripts, but do not affect the α-AMY1-type transcripts. Low CAR concentrations also enhance the accumulation of α- and β-amylase enzyme activity in sprouts, but not in 'sub-eye' tissues. In contrast, high CAR concentrations have no appreciable effect in sprouts on the enzyme activities and the α-amylase transcript abundances of either group. The dose-dependent effects on the enzyme activities and the α-AMY2-type α-amylase transcripts in sprouts are specific for CAR but not for PMO. Different monoterpenes therefore may have specific targets for their interaction with hormone signalling pathways.

Dry-grind processing using amylase corn and superior yeast to reduce the exogenous enzyme requirements in bioethanol production.

PubMed

Kumar, Deepak; Singh, Vijay

2016-01-01

Conventional corn dry-grind ethanol production process requires exogenous alpha and glucoamylases enzymes to breakdown starch into glucose, which is fermented to ethanol by yeast. This study evaluates the potential use of new genetically engineered corn and yeast, which can eliminate or minimize the use of these external enzymes, improve the economics and process efficiencies, and simplify the process. An approach of in situ ethanol removal during fermentation was also investigated for its potential to improve the efficiency of high-solid fermentation, which can significantly reduce the downstream ethanol and co-product recovery cost. The fermentation of amylase corn (producing endogenous α-amylase) using conventional yeast and no addition of exogenous α-amylase resulted in ethanol concentration of 4.1 % higher compared to control treatment (conventional corn using exogenous α-amylase). Conventional corn processed with exogenous α-amylase and superior yeast (producing glucoamylase or GA) with no exogenous glucoamylase addition resulted in ethanol concentration similar to control treatment (conventional yeast with exogenous glucoamylase addition). Combination of amylase corn and superior yeast required only 25 % of recommended glucoamylase dose to complete fermentation and achieve ethanol concentration and yield similar to control treatment (conventional corn with exogenous α-amylase, conventional yeast with exogenous glucoamylase). Use of superior yeast with 50 % GA addition resulted in similar increases in yield for conventional or amylase corn of approximately 7 % compared to that of control treatment. Combination of amylase corn, superior yeast, and in situ ethanol removal resulted in a process that allowed complete fermentation of 40 % slurry solids with only 50 % of exogenous GA enzyme requirements and 64.6 % higher ethanol yield compared to that of conventional process. Use of amylase corn and superior yeast in the dry-grind processing industry can reduce the total external enzyme usage by more than 80 %, and combining their use with in situ removal of ethanol during fermentation allows efficient high-solid fermentation.

Significantly enhancing recombinant alkaline amylase production in Bacillus subtilis by integration of a novel mutagenesis-screening strategy with systems-level fermentation optimization.

PubMed

Ma, Yingfang; Shen, Wei; Chen, Xianzhong; Liu, Long; Zhou, Zhemin; Xu, Fei; Yang, Haiquan

2016-01-01

Alkaline amylase has significant potential for applications in the textile, paper and detergent industries, however, low yield of which cannot meet the requirement of industrial application. In this work, a novel ARTP mutagenesis-screening method and fermentation optimization strategies were used to significantly improve the expression level of recombinant alkaline amylase in B. subtilis 168. The activity of alkaline amylase in mutant B. subtilis 168 mut-16# strain was 1.34-fold greater than that in the wild-type, and the highest specific production rate was improved from 1.31 U/(mg·h) in the wild-type strain to 1.57 U/(mg·h) in the mutant strain. Meanwhile, the growth of B. subtilis was significantly enhanced by ARTP mutagenesis. When the agitation speed was 550 rpm, the highest activity of recombinant alkaline amylase was 1.16- and 1.25-fold of the activities at 450 and 650 rpm, respectively. When the concentration of soluble starch and soy peptone in the initial fermentation medium was doubled, alkaline amylase activity was increased 1.29-fold. Feeding hydrolyzed starch and soy peptone mixture or glucose significantly improved cell growth, but inhibited the alkaline amylase production in B. subtilis 168 mut-16#. The highest alkaline amylase activity by feeding hydrolyzed starch reached 591.4 U/mL, which was 1.51-fold the activity by feeding hydrolyzed starch and soy peptone mixture. Single pulse feeding-based batch feeding at 10 h favored the production of alkaline amylase in B. subtilis 168 mut-16#. The results indicated that this novel ARTP mutagenesis-screening method could significantly improve the yield of recombinant proteins in B. subtilis . Meanwhile, fermentation optimization strategies efficiently promoted expression of recombinant alkaline amylase in B. subtilis 168 mut-16#. These findings have great potential for facilitating the industrial-scale production of alkaline amylase and other enzymes, using B. subtilis cultures as microbial cell factories.

Predictive value of α-amylase in tracheal aspirates for ventilator-associated pneumonia in elderly patients.

PubMed

Qu, Ge-Ping; Fang, Xiang-Qun; Xu, Ya-Ping; Shi, Min; Wang, Yang; Gong, Mei-Liang; Fang, Hao-Ming

2018-04-01

This study aims to investigate the correlation between α-amylase in tracheal aspirates and risk factors of aspiration, as well as ventilator-associated pneumonia (VAP), in elderly patients undergoing mechanical ventilation and explore the clinical value of α-amylase for predicting VAP. Tracheal aspirates were collected from elderly patients within 2 weeks after tracheal intubation in mechanical ventilation, and α-amylase was detected. Patients were grouped according to the presence of VAP. The correlation between α-amylase and risk factors of aspiration before intubation, as well as VAP, were analyzed. The sample of this study comprised 147 patients. The average age of these patients was 86.9 years. The incidence of VAP was 21% during the study period. Tracheal aspirate α-amylase level increased with the increase in the number of risk factors for aspiration before intubation, α-amylase level was significantly higher in the VAP group than in the non-VAP group, the area under the receiver operating characteristic curve (ROC) of the diagnostic value of α-amylase for VAP was 0.813 (95% CI: 0.721-0.896), threshold value was 4,681.5 U/L, sensitivity was 0.801 and specificity was 0.793. Logistic multivariate analysis revealed the following risk factors for VAP: a number of risk factors before intubation of ≥3, a Glasgow score of <8 points, the absence of continuous aspiration of subglottic secretion and a tracheal aspirate α-amylase level of >4681.5 U/L. Tracheal aspirate α-amylase can serve as a biomarker for predicting VAP in elderly patients undergoing mechanical ventilation. © 2017 John Wiley & Sons Ltd.

Molecular cloning and characterization of an α-amylase cDNA highly expressed in major feeding stages of the coffee berry borer, Hypothenemus hampei.

PubMed

Bezerra, C A; Macedo, L L P; Amorim, T M L; Santos, V O; Fragoso, R R; Lucena, W A; Meneguim, A M; Valencia-Jimenez, A; Engler, G; Silva, M C M; Albuquerque, E V S; Grossi-de-Sa, M F

2014-12-10

α-Amylases are common enzymes responsible for hydrolyzing starch. Insect-pests, whose larvae develop in seeds, rely obligatorily on α-amylase activity to digest starch, as their major food source. Considering the relevance of insect α-amylases and the natural α-amylase inhibitors present in seeds to protect from insect damage, we report here the molecular cloning and nucleotide sequence of the full-length AmyHha cDNA of the coffee berry borer, Hypothenemus hampei, a major insect-pest of coffee crops. The AmyHha sequence has 1879 bp, containing a 1458 bp open reading frame, which encodes a predicted protein with 485 amino acid residues, with a predicted molecular mass of 51.2 kDa. The deduced protein showed 55-79% identity to other insect α-amylases, including Anthonomus grandis, Ips typographus and Sitophilus oryzae α-amylases. In depth analysis revealed that the highly conserved three amino acid residues (Asp184, Glu220, and Asp285), which compose the catalytic site are also presented in AmyHha amylase. The AmyHha gene seems to be a single copy in the haploid genome and AmyHha transcription levels were found higher in L2 larvae and adult insects, both corresponding to major feeding phases. Modeling of the AmyHha predicted protein uncovered striking structural similarities to the Tenebrio molitor α-amylase also displaying the same amino acid residues involved in enzyme catalysis (Asp184, Glu220 and Asp285). Since AmyHha gene was mostly transcribed in the intestinal tract of H. hampei larvae, the cognate α-amylase could be considered a high valuable target to coffee bean insect control by biotechnological strategies. Copyright © 2014. Published by Elsevier B.V.

Taking the Starch out of Oral Biofilm Formation: Molecular Basis and Functional Significance of Salivary α-Amylase Binding to Oral Streptococci

PubMed Central

Nikitkova, Anna E.; Haase, Elaine M.

2013-01-01

α-Amylase-binding streptococci (ABS) are a heterogeneous group of commensal oral bacterial species that comprise a significant proportion of dental plaque microfloras. Salivary α-amylase, one of the most abundant proteins in human saliva, binds to the surface of these bacteria via specific surface-exposed α-amylase-binding proteins. The functional significance of α-amylase-binding proteins in oral colonization by streptococci is important for understanding how salivary components influence oral biofilm formation by these important dental plaque species. This review summarizes the results of an extensive series of studies that have sought to define the molecular basis for α-amylase binding to the surface of the bacterium as well as the biological significance of this phenomenon in dental plaque biofilm formation. PMID:23144140

Expression and Characterization of Geobacillus stearothermophilus SR74 Recombinant α-Amylase in Pichia pastoris

PubMed Central

Gandhi, Sivasangkary; Salleh, Abu Bakar; Rahman, Raja Noor Zaliha Raja Abd; Chor Leow, Thean; Oslan, Siti Nurbaya

2015-01-01

Geobacillus stearothermophilus SR74 is a locally isolated thermophilic bacteria producing thermostable and thermoactive α-amylase. Increased production and commercialization of thermostable α-amylase strongly warrant the need of a suitable expression system. In this study, the gene encoding the thermostable α-amylase in G. stearothermophilus SR74 was amplified, sequenced, and subcloned into P. pastoris GS115 strain under the control of a methanol inducible promoter, alcohol oxidase (AOX). Methanol induced recombinant expression and secretion of the protein resulted in high levels of extracellular amylase production. YPTM medium supplemented with methanol (1% v/v) was the best medium and once optimized, the maximum recombinant α-amylase SR74 achieved in shake flask was 28.6 U mL−1 at 120 h after induction. The recombinant 59 kDa α-amylase SR74 was purified 1.9-fold using affinity chromatography with a product yield of 52.6% and a specific activity of 151.8 U mg−1. The optimum pH of α-amylase SR74 was 7.0 and the enzyme was stable between pH 6.0–8.0. The purified enzyme was thermostable and thermoactive, exhibiting maximum activity at 65°C with a half-life (t 1/2) of 88 min at 60°C. In conclusion, thermostable α-amylase SR74 from G. stearothermophilus SR74 would be beneficial for industrial applications, especially in liquefying saccrification. PMID:26090417

Production of raw-starch-digesting α-amylase isoform from Bacillus sp. under solid-state fermentation and biochemical characterization.

PubMed

Božić, Nataša; Slavić, Marinela Šokarda; Gavrilović, Anja; Vujčić, Zoran

2014-07-01

α-Amylase production by solid-state fermentation of different Bacillus sp. was studied previously on different fermentation media. However, no study has been reported on the influence of selected media on expression of desired amylase isoforms such as raw-starch-digesting amylase (RSDA). In this paper, the influence of different inexpensive and available agro-resources as solid media (corn, wheat and triticale) on α-amylase isoform induction from three wild-type Bacillus sp., selected among one hundred strains tested, namely 9B, 12B and 24A was investigated. For all three strains, tested amylases were detected in the multiple forms; however, number and intensity of each form differed depending on the solid media used for growth. To determine which isoform from Bacillus sp. 12B was RSDA, the suspected isoform was purified. The optimum pH for the purified α-amylase isoform was 6.0-8.0, while the optimum temperature was 60-90 °C. Isoform was considerably thermostable and Ca(2+)-independent, and actually the only α-amylase active towards raw starch. Purification and characterization of RSDA showed that not all of the solid media tested induced RSDA. From an economic point of view, it might be significant to obtain pure isoenzyme for potential use in the raw-starch hydrolysis, since it was 5 times more efficient in raw corn starch hydrolysis than the crude amylase preparation.

Characterization of the Activity and Stability of Amylase from Saliva and Detergent: Laboratory Practicals for Studying the Activity and Stability of Amylase from Saliva and Various Commercial Detergents

ERIC Educational Resources Information Center

Valls, Cristina; Rojas, Cristina; Pujadas, Gerard; Garcia-Vallve, Santi; Mulero, Miquel

2012-01-01

This article presents two integrated laboratory exercises intended to show students the role of [alpha]-amylases (AAMYs) in saliva and detergents. These laboratory practicals are based on the determination of the enzymatic activity of amylase from saliva and different detergents using the Phadebas test (quantitative) and the Lugol test…

α-Amylase sensor based on the degradation of oligosaccharide hydrogel films monitored with a quartz crystal sensor.

PubMed

Gibbs, Martin John; Biela, Anna; Krause, Steffi

2015-05-15

α-Amylase hydrolyses starch molecules to produce smaller oligosaccharides and sugars. Amylases are of great importance in biotechnology and find application in fermentation, detergents, food and the paper industry. The measurement of α-amylase activity in serum and urine has been used in the diagnosis of acute pancreatitis. Salivary amylase has also been shown to be a stress indicator. Sensor coatings suitable for the detection of α-amylase activity have been developed. Oligosaccharides such as glycogen and amylopectin were spin-coated onto gold coated quartz crystals with a base frequency of 10 MHz. The films were subsequently cross-linked with hexamethylene diisocyanate. Film degradation was monitored with a quartz crystal microbalance (QCM) and electrochemical impedance measurements. The films were shown to be stable in phosphate buffered saline (PBS). Addition of α-amylase to the solution resulted in the rapid degradation of the films. The maximum rate of degradation was found to be strongly dependent on the amylase activity in the range typically found in serum when diagnosing pancreatitis (0.08-8 U/ml). Sensor responses in serum were found to be very similar to those obtained in buffer indicating the absence of non-specific binding. Copyright © 2014 Elsevier B.V. All rights reserved.

[Determination of exogenous gamma-amylase residue in honey].

PubMed

Fei, Xiaoqing; Wu, Bin; Shen, Chongyu; Zhang, Rui; Ding, Tao; Li, Lihua

2012-08-01

A novel method for the determination of exogenous gamma-amylase residue in honey using liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) was established. After pre-separation by gel column chromatography, the gamma-amylase in honey samples was separated from the sugars. The gamma-amylase was then used to catalyze maltose into glucose. This enzymatic reaction was under the conditions of 55 degrees C and 0.03 mol/L phosphate buffer solution (pH 4.5) for 48 h. The maltose and glucose in the above enzymatic reaction solution were separated using liquid chromatography. By measuring the content of glucose with isotope ratio mass spectrometry, the gamma-amylase in honey can be determined. The linear range of gamma-amylase was 5 - 200 U/kg with the quantification limit of 5 U/kg. The recoveries were between 89.6% and 108.2% with the relative standard deviations from 3.3% to 4.9%. This method was used to analyze 38 honey and rice syrup samples, and the detection rate of gamma-amylase was 76.3%. To further verify the detection capability of this method, an authentic honey was adulterated with 15% (mass fraction) rice syrup. The gamma-amylase content in this sample was 10.2 U/kg. This method can effectively identify honey adulteration with rice syrups from the perspective of enzymology.

Activity and cellular localization of amylases of rabbit cecal bacteria.

PubMed

Sirotek, K; Marounek, M; Suchorská, O

2006-01-01

Five 11-week-old rabbits, fed a commercial granulated feed, were slaughtered and cecal starch-degrading bacteria enumerated; total concentration of cultivable bacteria utilizing starch averaged 5.5 x 10(10) CFU/g. The activity and cellular localization of amylases was determined in 9 bacteria identified as Actinomyces israeli (strains AA2 and AD4), Bacteroides spp. (strain AA3), Dichelobacter nodosus (strain AA4), Mitsuokella multiacidus (strain AA6), Eubacterium spp. (strains AA7 and AB2), Clostridium spp. (strains AD1 and AA5). Four strains (AA3, AA4, AA5, AD4) produced extracellular amylases with an activity of 26-35 micromol of reducing sugars per h per mg of protein; in five strains (AA2, AA6, AA7, AB2, AD1) amylases were membrane-bound with an activity of 14-18 micromol of reducing sugars per h per mg of protein. All strains exhibited a low intracellular amylolytic activity. The pH optimum of amylases was 6.8-7.0. In strains producing extracellular amylases a substantial loss of viscosity was observed during incubations of cultivation supernatant with starch, similar to viscosity reduction in starch solutions treated with alpha-amylase; this indicates an endo-type (random cleavage) of extracellular amylase reaction in the bacteria under study. No strain possessed glucoamylase activity.

Drain amylase aids detection of anastomotic leak after esophagectomy.

PubMed

Baker, Erin H; Hill, Joshua S; Reames, Mark K; Symanowski, James; Hurley, Susie C; Salo, Jonathan C

2016-04-01

Anastomotic leak following esophagectomy is associated with significant morbidity and mortality. As hospital length of stay decreases, the timely diagnosis of leak becomes more important. We evaluated CT esophagram, white blood count (WBC), and drain amylase levels in the early detection of anastomotic leak. The diagnostic performance of CT esophagram, drain amylase >800 IU/L, and WBC >12,000/µL within the first 10 days after surgery in predicting leak at any time after esophagectomy was calculated. Anastomotic leak occurred in 13 patients (13%). CT esophagram performed within 10 days of surgery diagnosed six of these leaks with a sensitivity of 0.54. Elevation in drain amylase level within 10 days of surgery diagnosed anastomotic leak with a sensitivity of 0.38. When the CT esophagram and drain amylase were combined, the sensitivity rose to 0.69 with a specificity of 0.98. WBC elevation had a sensitivity of 0.92, with a specificity of 0.34. Among 30 patients with normal drain amylase and a normal WBC, one developed an anastomotic leak. Drain amylase adds to the sensitivity of CT esophagram in the early detection of anastomotic leak. Selected patients with normal drain amylase levels and normal WBC may be able to safely forgo CT esophagram.

Dynamic rheological properties of dough as affected by amylases from various sources.

PubMed

Doğan, Ismail S

2002-12-01

The effect of alpha-amylases from cereal, fungal, and bacterial sources on dough dynamic rheological properties was investigated. Dynamic rheological study of flour-and-water doughs during resting period showed significant changes in dough rheological properties as a function of alpha-amylases. Addition of alpha-amylases caused a time-dependent decrease in G', storage modulus. The enzyme action on starch during baking increased viscous flow properties. These changes were temperature-dependent. The thermal inactivation temperature of alpha-amylase plays an important role in modification of starch. Rheological changes in dough will alter the machinability of the dough and the quality of end products.

Screening, Gene Cloning, and Characterizations of an Acid-Stable α-Amylase.

PubMed

Liu, Xinyu; Jia, Wei; An, Yi; Cheng, Kun; Wang, Mingdao; Yang, Sen; Chen, Hongge

2015-06-01

Based on its α-amylase activity at pH 5.0 and optimal pH of the crude enzyme, a strain (named B-5) with acid α-amylase production was screened. The B-5 strain was identified as Bacillus amyloliquefaciens through morphological, physiological, and biochemical characteristics analysis, as well as 16S rDNA phylogenetic analysis. Its α-amylase gene of GenBank Accession No. GU318401 was cloned and expressed in Escherichia coli. The purified recombinant α-amylase AMY-Ba showed the optimal pH of 5.0, and was stable at a pH range of 4.0-6.0. When hydrolyzing soluble starch, amylose, and amylopectin, AMY-Ba released glucose and maltose as major end products. The α-amylase AMY-Ba in this work was a different type from the well-investigated J01542 (GenBank Accession No.)-type α-amylase from the same species. AMY-Ba exhibited notable adsorption and hydrolysis ability towards various raw starches. Structure analysis of AMY-Ba suggested the presence of a new starch-binding domain at its C-terminal region.

Lignin from bamboo shoot shells as an activator and novel immobilizing support for α-amylase.

PubMed

Gong, Weihua; Ran, Zhanxiang; Ye, Fayin; Zhao, Guohua

2017-08-01

This study examined the feasibility of α-amylase activation and immobilization, using lignin from bamboo shoot shells (BSS). Our results demonstrated that BSS lignin is an excellent α-amylase activator and it elevated α-amylase activity more than two-fold at a concentration of 5mg/ml. For immobilization of α-amylase via adsorption, BSS lignin was incubated in an α-amylase solution (5mg/ml) for 20min, and the maximum specific activity, amount of loaded protein and activity recovery were 92.4U/mg, 19.0mg/g and 111%, respectively. In contrast to its free counterpart, immobilized α-amylase improved the catalytic efficiency and storage stability, under comparable working conditions (temperature and pH). Regarding its convenient usage, immobilized enzyme can be suspended in advance, but a suspension incubated at 60°C should be used within 30min. The residual activity after 14 re-uses remained at a reasonable level (53.2%). In conclusion, this study reveals a novel support for enzyme immobilization. Copyright © 2017 Elsevier Ltd. All rights reserved.

Synthesis and study of the α-amylase inhibitory potential of thiadiazole quinoline derivatives.

PubMed

Taha, Muhammad; Tariq Javid, Muhammad; Imran, Syahrul; Selvaraj, Manikandan; Chigurupati, Sridevi; Ullah, Hayat; Rahim, Fazal; Khan, Fahad; Islam Mohammad, Jahidul; Mohammed Khan, Khalid

2017-10-01

α-Amylase is a target for type-2 diabetes mellitus treatment. However, small molecule inhibitors of α-amylase are currently scarce. In the course of developing small molecule α-amylase inhibitors, we designed and synthesized thiadiazole quinoline analogs (1-30), characterized by different spectroscopic techniques such as 1 HNMR and EI-MS and screened for α-amylase inhibitory potential. Thirteen analogs 1, 2, 3, 4, 5, 6, 22, 23, 25, 26, 27, 28 and 30 showed outstanding α-amylase inhibitory potential with IC 50 values ranges between 0.002±0.60 and 42.31±0.17μM which is many folds better than standard acarbose having IC 50 value 53.02±0.12μM. Eleven analogs 7, 9, 10, 11, 12, 14, 15, 17, 18, 19 and 24 showed good to moderate inhibitory potential while seven analogs 8, 13, 16, 20, 21 and 29 were found inactive. Our study identifies novel series of potent α-amylase inhibitors for further investigation. Structure activity relationship has been established. Copyright © 2017 Elsevier Inc. All rights reserved.

A kinetic model to explain the maximum in alpha-amylase activity measurements in the presence of small carbohydrates.

PubMed

Baks, Tim; Janssen, Anja E M; Boom, Remko M

2006-06-20

The effect of the presence of several small carbohydrates on the measurement of the alpha-amylase activity was determined over a broad concentration range. At low carbohydrate concentrations, a distinct maximum in the alpha-amylase activity versus concentration curves was observed in several cases. At higher concentrations, all carbohydrates show a decreasing alpha-amylase activity at increasing carbohydrate concentrations. A general kinetic model has been developed that can be used to describe and explain these phenomena. This model is based on the formation of a carbohydrate-enzyme complex that remains active. It is assumed that this complex is formed when a carbohydrate binds to alpha-amylase without blocking the catalytic site and its surrounding subsites. Furthermore, the kinetic model incorporates substrate inhibition and substrate competition. Depending on the carbohydrate type and concentration, the measured alpha-amylase activity can be 75% lower than the actual alpha-amylase activity. The model that has been developed can be used to correct for these effects in order to obtain the actual amount of active enzyme. 2006 Wiley Periodicals, Inc.

Utilization of a maltotetraose-producing amylase as a whole wheat bread improver: dough rheology and baking performance.

PubMed

Bae, Woosung; Lee, Sung Ho; Yoo, Sang-Ho; Lee, Suyong

2014-08-01

A maltotetraose-producing enzyme (G4-amylase) was utilized to improve the baking performance of whole-grain wheat flour. Whole-grain bread dough prepared with G4-amylase showed reduced water absorption and increased development time, while the dough stability was not affected. Also, the G4-amylase-treated samples exhibited lower Mixolab torque values than the control upon heating and cooling. Rheological measurements showed the decreased ratio of Rmax /E and increased tan δ, clearly demonstrating that the viscous characteristics of whole-grain bread dough became dominant with increasing levels of G4-amylase. The use of G4-amylase produced whole-grain wheat breads with a variety of maltooligosaccharides, primarily maltotetraose that positively contributed to the bread volume (1.2-fold higher than the control). Moreover, G4-amylase delayed the crumb firming of whole-grain wheat bread during a 7-d storage period, showing that it can function as an antiretrogradation agent to enhance the quality attributes of whole-grain wheat bread. © 2014 Institute of Food Technologists®

α-Amylase inhibitors: a review of raw material and isolated compounds from plant source.

PubMed

Sales, Paloma Michelle; Souza, Paula Monteiro; Simeoni, Luiz Alberto; Silveira, Damaris

2012-01-01

Inhibition of α-amylase, enzyme that plays a role in digestion of starch and glycogen, is considered a strategy for the treatment of disorders in carbohydrate uptake, such as diabetes and obesity, as well as, dental caries and periodontal diseases. Plants are an important source of chemical constituents with potential for inhibition of α-amylase and can be used as therapeutic or functional food sources. A review about crude extracts and isolated compounds from plant source that have been tested for α-amylase inhibitory activity has been done. The analysis of the results shows a variety of crude extracts that present α-amylase inhibitory activity and some of them had relevant activity when compared with controls used in the studies. Amongst the phyto-constituents that have been investigated, flavonoids are one of them that demonstrated the highest inhibitory activities with the potential of inhibition related to number of hydroxyl groups in the molecule of the compound. Several phyto-constituents and plant species as α-amylase inhibitors are being reported in this article. Majority of studies have focused on the anti-amylase phenolic compounds.

Amylases and Their Importance during Glycan Degradation: Genome Sequence Release of Salmonella Amylase Knockout Strains.

PubMed

Arabyan, Narine; Huang, Bihua C; Weimer, Bart C

2017-05-18

Amylases catalyze the cleavage of α-d-1,4 and α-d-1,6-glycosidic bonds in starch and related carbohydrates. Amylases are widely distributed in nature and are important in carbohydrate metabolism. This is the release of four single and two double deletions in Salmonella enterica serovar Typhimurium LT2 that are important for glycan degradation during infection. Copyright © 2017 Arabyan et al.

Stable, inducible thermoacidophilic alpha-amylase from Bacillus acidocaldarius.

PubMed Central

Buonocore, V; Caporale, C; De Rosa, M; Gambacorta, A

1976-01-01

Bacillus acidocaldarius Agnano 101 produces an inducible thermoacidophilic alpha-amylase. The enzyme production occurs during the stationary phase of growth in the presence of compounds with alpha-1,4-glucosidic linkages. The enzymatic activity is both present in the culture medium and associated with the cells; the enzymes purified from both sources show identical molecular and catalytic properties. The purified amylase has a single polypeptide chain of molecular weight 68,000 and behaves like an alpha-amylase with affinity constants for starch and related substances of 0.8 to 0.9 mg/ml. The pH and temperature optima for activity are 3.5 and 75degreesC, respectively. The amylase is stable at acidic pH (below 4.5). Its thermal stability is strictly dependent upon protein concentration; the half-life at 60degreesC of the amylase in a 70-mug/ml solution is about 5 days. PMID:10276

Effect of neohesperidin dihydrochalcone on the activity and stability of alpha-amylase: a comparative study on bacterial, fungal, and mammalian enzymes.

PubMed

Kashani-Amin, Elaheh; Ebrahim-Habibi, Azadeh; Larijani, Bagher; Moosavi-Movahedi, Ali Akbar

2015-10-01

Neohesperidin dihydrochalcone (NHDC) was recently introduced as an activator of mammalian alpha-amylase. In the current study, the effect of NHDC has been investigated on bacterial and fungal alpha-amylases. Enzyme assays and kinetic analysis demonstrated the capability of NHDC to significantly activate both tested alpha-amylases. The ligand activation pattern was found to be more similar between the fungal and mammalian enzyme in comparison with the bacterial one. Further, thermostability experiments indicated a stability increase in the presence of NHDC for the bacterial enzyme. In silico (docking) test locates a putative binding site for NHDC on alpha-amylase surface in domain B. This domain shows differences in various alpha-amylase types, and the different behavior of the ligand toward the studied enzymes may be attributed to this fact. Copyright © 2015 John Wiley & Sons, Ltd.

Isolation, optimization, and partial purification of amylase from Chrysosporium asperatum by submerged fermentation.

PubMed

Sanghvi, Gaurav V; Koyani, Rina; Rajput, Kishore S

2011-05-01

A potent fungus for amylase production, Chrysosporium asperatum, was isolated from among 30 different cultures obtained from wood samples collected in the Junagadh forest, India. All of the isolated cultures were screened for their ability to produce amylase by submerged fermentation. Among the selected cultures, C. asperatum (Class Euascomycetes; Onygenales; Onygenaceae) gave maximum amylase production. In all of the different media tested, potato starch was found to be a good substrate for production of amylase enzyme at 30 degrees C and pH 5.0. Production of enzyme reached the maximum when a combination of starch and 2% xylose, and organic nitrogen (1% yeast extract) and ammonium sulfate were used as carbon and nitrogen sources, respectively. There was no significant effect of metal ions on enzyme activity. The enzyme was relatively stable at 50 degrees C for 20 min, and no inhibitory effect of Ca+2 ions on amylase production was observed.

Classification of microbial α-amylases for food manufacturing using proteinase digestion.

PubMed

Akiyama, Takumi; Yamazaki, Takeshi; Tada, Atsuko; Ito, Yusai; Otsuki, Noriko; Akiyama, Hiroshi

2014-09-01

Enzymes produced by microorganisms and plants are used as food additives to aid the processing of foods. Identification of the origin of these enzyme products is important for their proper use. Proteinase digestion of α-amylase products, followed by high performance liquid chromatography (HPLC) analysis, was applied to α-amylase from the mold Aspergillus species, the bacteria Bacillus species, and the actinomycetes Saccharomonospora species. Eighteen commercial products of α-amylase were digested with trypsin and endoproteinase Lys-C and HPLC analyzed. For some proteinase/sample combinations, the area of the intact α-amylase peak decreased and new peaks were detected after digestion. The presence and retention times of the novel peaks were used to group the products. The results from this method, called the proteinase digestion-HPLC method, allowed the classification of the α-amylase products into 10 groups, whereas the results from sodium dodecyl sulfate polyacrylamide gel electrophoresis allowed their classification into seven groups.

LaaA, a novel high-active alkalophilic alpha-amylase from deep-sea bacterium Luteimonas abyssi XH031(T).

PubMed

Song, Qinghao; Wang, Yan; Yin, Chong; Zhang, Xiao-Hua

2016-08-01

Alpha-amylase is a kind of broadly used industrial enzymes, most of which have been exploited from terrestrial organism. Comparatively, alpha-amylase from marine environment was largely undeveloped. In this study, a novel alkalophilic alpha-amylase with high activity, Luteimonas abyssi alpha-amylase (LaaA), was cloned from deep-sea bacterium L. abyssi XH031(T) and expressed in Escherichia coli BL21. The gene has a length of 1428bp and encodes 475 amino acids with a 35-residue signal peptide. The specific activity of LaaA reached 8881U/mg at the optimum pH 9.0, which is obvious higher than other reported alpha-amylase. This enzyme can remain active at pH levels ranging from 6.0 to 11.0 and temperatures below 45°C, retaining high activity even at low temperatures (almost 38% residual activity at 10°C). In addition, 1mM Na(+), K(+), and Mn(2+) enhanced the activity of LaaA. To investigate the function of potential active sites, R227G, D229K, E256Q/H, H327V and D328V mutants were generated, and the results suggested that Arg227, Asp229, Glu256 and Asp328 were total conserved and essential for the activity of alpha-amylase LaaA. This study shows that the alpha-amylase LaaA is an alkali-tolerant and high-active amylase with strong potential for use in detergent industry. Copyright © 2016 Elsevier Inc. All rights reserved.

Improving Bread Quality with the Application of a Newly Purified Thermostable α-Amylase from Rhizopus oryzae FSIS4

PubMed Central

Ait Kaki El-Hadef El-Okki, Amel; Gagaoua, Mohammed; Bourekoua, Hayat; Hafid, Kahina; Bennamoun, Leila; Djekrif-Dakhmouche, Shahrazed; El-Hadef El-Okki, Mohamed; Meraihi, Zahia

2017-01-01

A new thermostable α-amylase from Rhizopus oryzae FSIS4 was purified for first time and recovered in a single step using a three-phase partitioning (TPP) system. The fungal α-amylase, at a concentration of 1.936 U per kg of flour, was used in bread-making and compared to the commercial enzyme. The results showed a significant effect of the recovered α-amylase in the prepared bread and allowed us to improve the quality of the bread. The study indicated clearly that the recovered α-amylase is a potential candidate for future applications in the bread-making industry and in other food biotechnology applications. PMID:28231081

Construction of promoter-probe shuttle vectors for Escherichia coli and corynebacteria on the basis of promoterless alpha-amylase gene.

PubMed

Ugorcáková, J; Bukovská, G; Timko, J

2000-01-01

We constructed new promoter-probe vectors for E. coli and corynebacteria based on the promoterless alpha-amylase gene originating from Bacillus subtilis. Vectors pJUPAE1 and pJUPAE2 are suitable for isolation of transcriptionally active fragments from plasmids, phages or genomic DNA. alpha-Amylase activity can be easily visually detected on agar plates containing a chromogenic substrate, or by direct measurement of alpha-amylase activity.

Tracking amylolytic enzyme activities during congress mashing with North American barley cultivars: Comparisons of patterns of activity and ß-amylases with differing Bmy1 ...correlations of amylolytic enzyme activities

USDA-ARS?s Scientific Manuscript database

This study was conducted to test three hypotheses: 1) that a-amylase will have less consistent patterns of activity during mashing than ß-amylase and limit dextrinase 2) that differing ß-amylase 1 intron III alleles (Bmy1.a and Bmy1.b) would not be useful in predicting high or low activities or th...

A new group of glycoside hydrolase family 13 α-amylases with an aberrant catalytic triad

PubMed Central

Sarian, Fean D.; Janeček, Štefan; Pijning, Tjaard; Ihsanawati; Nurachman, Zeily; Radjasa, Ocky K.; Dijkhuizen, Lubbert; Natalia, Dessy; van der Maarel, Marc J. E. C.

2017-01-01

α-Amylases are glycoside hydrolase enzymes that act on the α(1→4) glycosidic linkages in glycogen, starch, and related α-glucans, and are ubiquitously present in Nature. Most α-amylases have been classified in glycoside hydrolase family 13 with a typical (β/α)8-barrel containing two aspartic acid and one glutamic acid residue that play an essential role in catalysis. An atypical α-amylase (BmaN1) with only two of the three invariant catalytic residues present was isolated from Bacillus megaterium strain NL3, a bacterial isolate from a sea anemone of Kakaban landlocked marine lake, Derawan Island, Indonesia. In BmaN1 the third residue, the aspartic acid that acts as the transition state stabilizer, was replaced by a histidine. Three-dimensional structure modeling of the BmaN1 amino acid sequence confirmed the aberrant catalytic triad. Glucose and maltose were found as products of the action of the novel α-amylase on soluble starch, demonstrating that it is active in spite of the peculiar catalytic triad. This novel BmaN1 α-amylase is part of a group of α-amylases that all have this atypical catalytic triad, consisting of aspartic acid, glutamic acid and histidine. Phylogenetic analysis showed that this group of α-amylases comprises a new subfamily of the glycoside hydrolase family 13. PMID:28287181

Effects of Pulsed Electric Field (PEF) Treatment on Enhancing Activity and Conformation of α-Amylase.

PubMed

Tian, Mei-ling; Fang, Ting; Du, Mu-ying; Zhang, Fu-sheng

2016-04-01

To explore an efficient, safe, and speedy application of pulsed electric field (PEF) technology for enzymatic modification, effects of PEF treatment on the enzymatic activity, property and kinetic parameters of α-amylase were investigated. Conformational transitions were also studied with the aid of circular dichroism (CD) and fluorescence spectra. The maximum enzymatic activity of α-amylase was obtained under 15 kV/cm electric field intensity and 100 mL/min flow velocity PEF treatment, in which the enzymatic activity increased by 22.13 ± 1.14% compared with control. The activation effect could last for 18 h at 4 °C. PEF treatment could widen the range of optimum temperature for α-amylase, however, it barely exerted any effect on the optimum pH. On the other hand, α-amylase treated by PEF showed an increase of Vmax, t1/2 and ΔG, whereas a decrease of Km and k were observed. Furthermore, it can be observed from fluorescence and CD spectra that PEF treatment had increased the number of amino acid residues, especially that of tryptophan, on α-amylase surface with enhanced α-helices by 34.76% and decreased random coil by 12.04% on α-amylase when compared with that of untreated. These changes in structure had positive effect on enhancing α-amylase activity and property.

Characterization of two coleopteran α-amylases and molecular insights into their differential inhibition by synthetic α-amylase inhibitor, acarbose.

PubMed

Channale, Sonal M; Bhide, Amey J; Yadav, Yashpal; Kashyap, Garima; Pawar, Pankaj K; Maheshwari, V L; Ramasamy, Sureshkumar; Giri, Ashok P

2016-07-01

Post-harvest insect infestation of stored grains makes them unfit for human consumption and leads to severe economic loss. Here, we report functional and structural characterization of two coleopteran α-amylases viz. Callosobruchus chinensis α-amylase (CcAmy) and Tribolium castaneum α-amylase (TcAmy) along with their interactions with proteinaceous and non-proteinaceous α-amylase inhibitors. Secondary structural alignment of CcAmy and TcAmy with other coleopteran α-amylases revealed conserved motifs, active sites, di-sulfide bonds and two point mutations at spatially conserved substrate or inhibitor-binding sites. Homology modeling and molecular docking showed structural differences between these two enzymes. Both the enzymes had similar optimum pH values but differed in their optimum temperature. Overall, pattern of enzyme stabilities were similar under various temperature and pH conditions. Further, CcAmy and TcAmy differed in their substrate affinity and catalytic efficiency towards starch and amylopectin. HPLC analysis detected common amylolytic products like maltose and malto-triose while glucose and malto-tetrose were unique in CcAmy and TcAmy catalyzed reactions respectively. At very low concentrations, wheat α-amylase inhibitor was found to be superior over the acarbose as far as complete inhibition of amylolytic activities of CcAmy and TcAmy was concerned. Mechanism underlying differential amylolytic reaction inhibition by acarbose was discussed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Biochemical characterisation of α-amylase inhibitors from Achyranthes aspera and their interactions with digestive amylases of coleopteran and lepidopteran insects.

PubMed

Hivrale, Vandana K; Chougule, Nanasaheb P; Giri, Ashok P; Chhabda, Pavan J; Kachole, Manvendra S

2011-08-15

Starchy seeds are an important food and a source of dietary ingredients in many countries. However, they suffer from extensive predation by bruchids (weevils) and other pests. α-Amylase inhibitors are attractive candidates for the control of seed weevils, as these insects are highly dependent on starch as an energy source. A proteinaceous α-amylase inhibitor from the seeds of Achyranthes aspera was identified, purified and characterised. In electrophoretic analysis, two prominent amylase inhibitor activity bands (AI1 and AI2) were detected. The inhibitor was purified 9.99-fold with 1206.95 total amylase inhibitor units mg⁻¹ protein. The molecular weight of the purified inhibitor was around 6 kDa. The isolated α-amylase inhibitor was found to be resistant to heat and proteolysis. Feeding analysis of Callosobruchus maculatus larvae on a diet containing seed powder of A. aspera revealed that survival of the larvae was severely affected, with the highest mortality rate occurring on the fifth day of feeding. The isolated inhibitor inhibited the majority of amylase isoforms of C. maculatus, Tribolium confusum and Helicoverpa armigera in electrophoretic analysis and solution assays. The information obtained in the present investigation could be useful for a genetic engineering approach that would make seeds resistant to storage pest infestations. Copyright © 2011 Society of Chemical Industry.

Polyaniline-graphene based α-amylase biosensor with a linear dynamic range in excess of 6 orders of magnitude.

PubMed

Teixeira, Sofia Rodrigues; Lloyd, Catherine; Yao, Seydou; Andrea Salvatore Gazze; Whitaker, Iain S; Francis, Lewis; Conlan, R Steven; Azzopardi, Ernest

2016-11-15

α-amylase is an established marker for diagnosis of pancreatic and salivary disease, and recent research has seen a substantial expansion of its use in therapeutic and diagnostic applications for infection, cancer and wound healing. The lack of bedside monitoring devices for α-amylase detection has hitherto restricted the clinical progress of such applications. We have developed a highly sensitive α-amylase immunosensor platform, produced via in situ electropolymerization of aniline onto a screen-printed graphene support (SPE). Covalently binding an α-amylase specific antibody to a polyaniline (PANI) layer and controlling device assembly using electrochemical impedance spectroscopy (EIS), we have achieved a highly linear response against α-amylase concentration. Each stage of the assembly was characterized using a suite of high-resolution topographical, chemical and mechanical techniques. Quantitative, highly sensitive detection was demonstrated using an artificially spiked human blood plasma samples. The device has a remarkably wide limit of quantification (0.025-1000IU/L) compared to α-amylase assays in current clinical use. With potential for simple scale up to volume manufacturing though standard semiconductor production techniques and subsequently clinical application, this biosensor will enable clinical benefit through early disease detection, and better informed administration of correct therapeutic dose of drugs used to treat α-amylase related diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

[Correlation index amylase-creatinine clearance to endogenous creatinine clearance in severe preeclampsia].

PubMed

Vázquez Rodríguez, Juan Gustavo; Cruz Cruz, Polita del Rocío; Márquez Hubert, Elizabeth

2009-07-01

Tubular lesion may cause acute renal insufficiency in pregnant patients with severe preeclampsia. To describe the correlation between the amylase/creatinine clearance ratio and endogenous creatinine depuration in pregnant patients with severe preeclampsia. Transversal study (pilot study) twenty eight women with pregnancies of 20 to 40 weeks complicated by severe preeclampsia were studied. Subjects had serum and urine creatinine and amylase determinations to calculate the amylase/creatinine clearance ratio (%). According to the results, two groups were formed: group A (> 3%) and group B (< or = 3%). The correlation between amylase/creatinine clearance ratio and endogenous creatinine depuration was evaluated. measures of central tendency and dispersion, Student's t-test, Pearson correlation coefficient (r) and linear regression were used. Group A included 23 cases (82%) and group B included 5 cases (18%). Amylase/creatinine clearance ratio (%) for group A was 5.22 +/- 1.6 and for group B was 2.41 +/- 0.41 (p = 0.001). The endogenous creatinine depuration (mL /min. /1.73 m2 SC) for group A was 105.6 +/- 9.71 and for group B was 132.10 +/- 7.95 (p = 0.54). The r between amylase/creatinine clearance ratio and endogenous creatinine depuration for group A was -0.43 and for group B was -0.25. A moderately significant negative correlation exists between amylase/creatinine clearance ratio and endogenous creatinine depuration.

Employing in vitro directed molecular evolution for the selection of α-amylase variant inhibitors with activity toward cotton boll weevil enzyme.

PubMed

da Silva, Maria Cristina Mattar; Del Sarto, Rafael Perseghini; Lucena, Wagner Alexandre; Rigden, Daniel John; Teixeira, Fabíola Rodrigues; Bezerra, Caroline de Andrade; Albuquerque, Erika Valéria Saliba; Grossi-de-Sa, Maria Fatima

2013-09-20

Numerous species of insect pests attack cotton plants, out of which the cotton boll weevil (Anthonomus grandis) is the main insect in Brazil and must be controlled to avert large economic losses. Like other insect pests, A. grandis secretes a high level of α-amylases in the midgut lumen, which are required for digestion of carbohydrates. Thus, α-amylase inhibitors (α-AIs) represent a powerful tool to apply in the control of insect pests. Here, we applied DNA shuffling and phage display techniques and obtained a combinatorial library containing 10⁸ α-AI variant forms. From this library, variants were selected exhibiting in vitro affinity for cotton boll weevil α-amylases. Twenty-six variant sequences were cloned into plant expression vectors and expressed in Arabidopsis thaliana. Transformed plant extracts were assayed in vitro to select specific and potent α-amylase inhibitors against boll weevil amylases. While the wild type inhibitors, used to create the shuffled library, did not inhibit the A. grandis α-amylases, three α-AI mutants, named α-AIC3, α-AIA11 and α-AIG4 revealed high inhibitory activities against A. grandis α-amylases in an in vitro assay. In summary, data reported here shown the potential biotechnology of new α-AI variant genes for cotton boll weevil control. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Molecular cloning of alpha-amylases from cotton boll weevil, Anthonomus grandis and structural relations to plant inhibitors: an approach to insect resistance.

PubMed

Oliveira-Neto, Osmundo B; Batista, João A N; Rigden, Daniel J; Franco, Octávio L; Falcão, Rosana; Fragoso, Rodrigo R; Mello, Luciane V; dos Santos, Roseane C; Grossi-de-Sá, Maria F

2003-01-01

Anthonomus grandis, the cotton boll weevil, causes severe cotton crop losses in North and South America. Here we demonstrate the presence of starch in the cotton pollen grains and young ovules that are the main A. grandis food source. We further demonstrate the presence of alpha-amylase activity, an essential enzyme of carbohydrate metabolism for many crop pests, in A. grandis midgut. Two alpha-amylase cDNAs from A. grandis larvae were isolated using RT-PCR followed by 5' and 3' RACE techniques. These encode proteins with predicted molecular masses of 50.8 and 52.7kDa, respectively, which share 58% amino acid identity. Expression of both genes is induced upon feeding and concentrated in the midgut of adult insects. Several alpha-amylase inhibitors from plants were assayed against A. grandis alpha-amylases but, unexpectedly, only the BIII inhibitor from rye kernels proved highly effective, with inhibitors generally active against other insect amylases lacking effect. Structural modeling of Amylag1 and Amylag2 showed that different factors seem to be responsible for the lack of effect of 0.19 and alpha-AI1 inhibitors on A. grandis alpha-amylase activity. This work suggests that genetic engineering of cotton to express alpha-amylase inhibitors may offer a novel route to A. grandis resistance.

Production and Partial Characterization of α-Amylase Enzyme from Bacillus sp. BCC 01-50 and Potential Applications

PubMed Central

Qureshi, Abdul Sattar; Khushk, Imrana; Ali, Chaudhry Haider; Lashari, Safia; Bhutto, Muhammad Aqeel; Mangrio, Ghulam Sughra; Lu, Changrui

2017-01-01

Amylase is an industrially important enzyme and applied in many industrial processes such as saccharification of starchy materials, food, pharmaceutical, detergent, and textile industries. This research work deals with the optimization of fermentation conditions for α-amylase production from thermophilic bacterial strain Bacillus sp. BCC 01-50 and characterization of crude amylase. The time profile of bacterial growth and amylase production was investigated in synthetic medium and maximum enzyme titer was observed after 60 h. In addition, effects of different carbon sources were tested as a substrate for amylase production and molasses was found to be the best. Various organic and inorganic compounds, potassium nitrate, ammonium chloride, sodium nitrate, urea, yeast extract, tryptone, beef extract, and peptone, were used and beef extract was found to be the best among the nitrogen sources used. Temperature, pH, agitation speed, and size of inoculum were also optimized. Highest enzyme activity was obtained when the strain was cultured in molasses medium for 60 h in shaking incubator (150 rpm) at 50°C and pH 8. Crude amylase showed maximal activity at pH 9 and 65°C. Enzyme remained stable in alkaline pH range 9-10 and 60–70°C. Crude amylase showed great potential for its application in detergent industry and saccharification of starchy materials. PMID:28168200

CdTe/CdSe quantum dots improve the binding affinities between α-amylase and polyphenols.

PubMed

Ni, Xiaoling

2012-03-01

People exposed to engineered nanomaterials have potential health risks associated. Human α-amylase is one of the key enzymes in the digestive system. There are few reports about the influence of quantum dots (QDs) on the digestive enzymes and their inhibition system. This work focused on the toxic effect of CdTe/CdSe QDs on the interactions between α-amylase and its natural inhibitors. Thirty-six dietary polyphenols, natural α-amylase inhibitors from food, were studied for their affinities for α-amylase in the absence and presence of CdTe/CdSe QDs by a fluorescence quenching method. The magnitudes of apparent binding constants of polyphenols for α-amylase were almost in the range of 10(5)-10(7) L mol(-1) in the presence of CdTe/CdSe QDs, which were higher than the magnitudes of apparent binding constants in the absence of CdTe/CdSe QDs (10(4)-10(6) L mol(-1)). CdTe/CdSe QDs obviously improved the affinities of dietary polyphenols for α-amylase up to 389.04 times. It is possible that the binding interaction between polyphenols and α-amylase in the presence of CdTe/CdSe QDs was mainly caused by electrostatic interactions. QDs significantly influence the digestive enzymes and their inhibition system. This journal is © The Royal Society of Chemistry 2012

Production and Partial Characterization of α-Amylase Enzyme from Bacillus sp. BCC 01-50 and Potential Applications.

PubMed

Simair, Altaf Ahmed; Qureshi, Abdul Sattar; Khushk, Imrana; Ali, Chaudhry Haider; Lashari, Safia; Bhutto, Muhammad Aqeel; Mangrio, Ghulam Sughra; Lu, Changrui

2017-01-01

Amylase is an industrially important enzyme and applied in many industrial processes such as saccharification of starchy materials, food, pharmaceutical, detergent, and textile industries. This research work deals with the optimization of fermentation conditions for α -amylase production from thermophilic bacterial strain Bacillus sp. BCC 01-50 and characterization of crude amylase. The time profile of bacterial growth and amylase production was investigated in synthetic medium and maximum enzyme titer was observed after 60 h. In addition, effects of different carbon sources were tested as a substrate for amylase production and molasses was found to be the best. Various organic and inorganic compounds, potassium nitrate, ammonium chloride, sodium nitrate, urea, yeast extract, tryptone, beef extract, and peptone, were used and beef extract was found to be the best among the nitrogen sources used. Temperature, pH, agitation speed, and size of inoculum were also optimized. Highest enzyme activity was obtained when the strain was cultured in molasses medium for 60 h in shaking incubator (150 rpm) at 50°C and pH 8. Crude amylase showed maximal activity at pH 9 and 65°C. Enzyme remained stable in alkaline pH range 9-10 and 60-70°C. Crude amylase showed great potential for its application in detergent industry and saccharification of starchy materials.

Method for using a yeast alpha-amylase promoter

DOEpatents

Gao, Johnway; Skeen, Rodney S.; Hooker, Brian S.; Anderson, Daniel B.

2003-04-22

The present invention provides the promoter clone discovery of an alpha-amylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated alpha-amylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

Pancreatic α-Amylase Controls Glucose Assimilation by Duodenal Retrieval through N-Glycan-specific Binding, Endocytosis, and Degradation*

PubMed Central

Date, Kimie; Satoh, Ayano; Iida, Kaoruko; Ogawa, Haruko

2015-01-01

α-Amylase, a major pancreatic protein and starch hydrolase, is essential for energy acquisition. Mammalian pancreatic α-amylase binds specifically to glycoprotein N-glycans in the brush-border membrane to activate starch digestion, whereas it significantly inhibits glucose uptake by Na+/glucose cotransporter 1 (SGLT1) at high concentrations (Asanuma-Date, K., Hirano, Y., Le, N., Sano, K., Kawasaki, N., Hashii, N., Hiruta, Y., Nakayama, K., Umemura, M., Ishikawa, K., Sakagami, H., and Ogawa, H. (2012) Functional regulation of sugar assimilation by N-glycan-specific interaction of pancreatic α-amylase with glycoproteins of duodenal brush border membrane. J. Biol. Chem. 287, 23104–23118). However, how the inhibition is stopped was unknown. Here, we show a new mechanism for the regulation of intestinal glucose absorption. Immunohistochemistry revealed that α-amylase in the duodena of non-fasted, but not fasted, pigs was internalized from the pancreatic fluid and immunostained. We demonstrated that after N-glycan binding, pancreatic α-amylase underwent internalization into lysosomes in a process that was inhibited by α-mannoside. The internalized α-amylase was degraded, showing low enzymatic activity and molecular weight at the basolateral membrane. In a human intestinal Caco-2 cell line, Alexa Fluor 488-labeled pancreatic α-amylase bound to the cytomembrane was transported to lysosomes through the endocytic pathway and then disappeared, suggesting degradation. Our findings indicate that N-glycan recognition by α-amylase protects enterocytes against a sudden increase in glucose concentration and restores glucose uptake by gradual internalization, which homeostatically controls the postprandial blood glucose level. The internalization of α-amylase may also enhance the supply of amino acids required for the high turnover of small intestine epithelial cells. This study provides novel and significant insights into the control of blood sugar during the absorption stage in the intestine. PMID:26023238

Comprehensive enzymatic analysis of the amylolytic system in the digestive fluid of the sea hare, Aplysia kurodai: Unique properties of two α-amylases and two α-glucosidases.

PubMed

Tsuji, Akihiko; Nishiyama, Nami; Ohshima, Miki; Maniwa, Saori; Kuwamura, Shuji; Shiraishi, Masataka; Yuasa, Keizo

2014-01-01

Sea lettuce (Ulva pertusa) is a nuisance species of green algae that is found all over the world. East-Asian species of the marine gastropod, the sea hare Aplysia kurodai, shows a clear feeding preference for sea lettuce. Compared with cellulose, sea lettuce contains a higher amount of starch as a storage polysaccharide. However, the entire amylolytic system in the digestive fluid of A. kurodai has not been studied in detail. We purified α-amylases and α-glucosidases from the digestive fluid of A. kurodai and investigated the synergistic action of these enzymes on sea lettuce. A. kurodai contain two α-amylases (59 and 80 kDa) and two α-glucosidases (74 and 86 kDa). The 59-kDa α-amylase, but not the 80-kDa α-amylase, was markedly activated by Ca(2+) or Cl(-). Both α-amylases degraded starch and maltoheptaose, producing maltotriose, maltose, and glucose. Glucose production from starch was higher with 80-kDa α-amylase than with 59-kDa α-amylase. Kinetic analysis indicated that 74-kDa α-glucosidase prefers short α-1,4-linked oligosaccharide, whereas 86-kDa α-glucosidase prefers large α-1,6 and α-1,4-linked polysaccharides such as glycogen. When sea lettuce was used as a substrate, a 2-fold greater amount of glucose was released by treatment with 59-kDa α-amylase and 74-kDa α-glucosidase than by treatment with 45-kDa cellulase and 210-kDa β-glucosidase of A. kurodai. Unlike mammals, sea hares efficiently digest sea lettuce to glucose by a combination of two α-amylases and two α-glucosidases in the digestive fluids without membrane-bound maltase-glucoamylase and sucrase-isomaltase complexes.

Screening and characterization of amylase and cellulase activities in psychrotolerant yeasts.

PubMed

Carrasco, Mario; Villarreal, Pablo; Barahona, Salvador; Alcaíno, Jennifer; Cifuentes, Víctor; Baeza, Marcelo

2016-02-19

Amylases and cellulases have great potential for application in industries such as food, detergent, laundry, textile, baking and biofuels. A common requirement in these fields is to reduce the temperatures of the processes, leading to a continuous search for microorganisms that secrete cold-active amylases and cellulases. Psychrotolerant yeasts are good candidates because they inhabit cold-environments. In this work, we analyzed the ability of yeasts isolated from the Antarctic region to grow on starch or carboxymethylcellulose, and their potential extracellular amylases and cellulases. All tested yeasts were able to grow with soluble starch or carboxymethylcellulose as the sole carbon source; however, not all of them produced ethanol by fermentation of these carbon sources. For the majority of the yeast species, the extracellular amylase or cellulase activity was higher when cultured in medium supplemented with glucose rather than with soluble starch or carboxymethylcellulose. Additionally, higher amylase activities were observed when tested at pH 5.4 and 6.2, and at 30-37 °C, except for Rhodotorula glacialis that showed elevated activity at 10-22 °C. In general, cellulase activity was high until pH 6.2 and between 22-37 °C, while the sample from Mrakia blollopis showed high activity at 4-22 °C. Peptide mass fingerprinting analysis of a potential amylase from Tetracladium sp. of about 70 kDa, showed several peptides with positive matches with glucoamylases from other fungi. Almost all yeast species showed extracellular amylase or cellulase activity, and an inducing effect by the respective substrate was observed in a minor number of yeasts. These enzymatic activities were higher at 30 °C in most yeast, with highest amylase and cellulase activity in Tetracladium sp. and M. gelida, respectively. However, Rh. glacialis and M. blollopis displayed high amylase or cellulase activity, respectively, under 22 °C. In this sense, these yeasts are interesting candidates for industrial processes that require lower temperatures.

Comprehensive enzymatic analysis of the amylolytic system in the digestive fluid of the sea hare, Aplysia kurodai: Unique properties of two α-amylases and two α-glucosidases

PubMed Central

Tsuji, Akihiko; Nishiyama, Nami; Ohshima, Miki; Maniwa, Saori; Kuwamura, Shuji; Shiraishi, Masataka; Yuasa, Keizo

2014-01-01

Sea lettuce (Ulva pertusa) is a nuisance species of green algae that is found all over the world. East-Asian species of the marine gastropod, the sea hare Aplysia kurodai, shows a clear feeding preference for sea lettuce. Compared with cellulose, sea lettuce contains a higher amount of starch as a storage polysaccharide. However, the entire amylolytic system in the digestive fluid of A. kurodai has not been studied in detail. We purified α-amylases and α-glucosidases from the digestive fluid of A. kurodai and investigated the synergistic action of these enzymes on sea lettuce. A. kurodai contain two α-amylases (59 and 80 kDa) and two α-glucosidases (74 and 86 kDa). The 59-kDa α-amylase, but not the 80-kDa α-amylase, was markedly activated by Ca2+ or Cl−. Both α-amylases degraded starch and maltoheptaose, producing maltotriose, maltose, and glucose. Glucose production from starch was higher with 80-kDa α-amylase than with 59-kDa α-amylase. Kinetic analysis indicated that 74-kDa α-glucosidase prefers short α-1,4-linked oligosaccharide, whereas 86-kDa α-glucosidase prefers large α-1,6 and α-1,4-linked polysaccharides such as glycogen. When sea lettuce was used as a substrate, a 2-fold greater amount of glucose was released by treatment with 59-kDa α-amylase and 74-kDa α-glucosidase than by treatment with 45-kDa cellulase and 210-kDa β-glucosidase of A. kurodai. Unlike mammals, sea hares efficiently digest sea lettuce to glucose by a combination of two α-amylases and two α-glucosidases in the digestive fluids without membrane-bound maltase–glucoamylase and sucrase–isomaltase complexes. PMID:25161866

Production of itaconic acid in Escherichia coli expressing recombinant α-amylase using starch as substrate.

PubMed

Okamoto, Shusuke; Chin, Taejun; Nagata, Keisuke; Takahashi, Tetsuya; Ohara, Hitomi; Aso, Yuji

2015-05-01

Several studies on fermentative production of a vinyl monomer itaconic acid from hydrolyzed starch using Aspergillus terreus have been reported. Herein, we report itaconic acid production by Escherichia coli expressing recombinant α-amylase, using soluble starch as its sole carbon source. To express α-amylase in E. coli, we first constructed recombinant plasmids expressing α-amylases by using cell surface display technology derived from two amylolytic bacteria, Bacillus amyloliquefaciens NBRC 15535(T) and Streptococcus bovis NRIC 1535. The recombinant α-amylase from S. bovis (SBA) showed activity at 28°C, which is the optimal temperature for production of itaconic acid, while α-amylase from B. amyloliquefaciens displayed no noticeable activity. E. coli cells expressing SBA produced 0.15 g/L itaconic acid after 69 h cultivation under pH-stat conditions, using 1% starch as the sole carbon source. In fact, E. coli cells expressing SBA had similar growth rates when grown in the presence of 1% glucose or starch, thereby highlighting the expression of an active α-amylase that enabled utilization of starch to produce itaconic acid in E. coli. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Engineering α-amylase levels in wheat grain suggests a highly sophisticated level of carbohydrate regulation during development

PubMed Central

Whan, Alex; Dielen, Anne-Sophie; Mieog, Jos; Bowerman, Andrew F.; Robinson, Hannah M.; Byrne, Keren; Colgrave, Michelle; Larkin, Philip J.; Howitt, Crispin A.; Morell, Matthew K.; Ral, Jean-Philippe

2014-01-01

Wheat starch degradation requires the synergistic action of different amylolytic enzymes. Our spatio-temporal study of wheat α-amylases throughout grain development shows that AMY3 is the most abundant isoform compared with the other known α-amylases. Endosperm-specific over-expression of AMY3 resulted in an increase of total α-amylase activity in harvested grains. Unexpectedly, increased activity did not have a significant impact on starch content or composition but led to an increase of soluble carbohydrate (mainly sucrose) in dry grain. In AMY3 overexpression lines (A3OE), germination was slightly delayed and triacylglycerol (TAG) content was increased in the endosperm of mature grain. Despite increased AMY3 transcript and protein content throughout grain development, alterations of α-amylase activity and starch granule degradation were not detected until grain maturation, suggesting a post-translational inhibition of α-amylase activity in the endosperm during the starch filling period. These findings show unexpected effects of a high level of α-amylase on grain development and composition, notably in carbon partitioning and TAG accumulation, and suggest the presence of a hitherto unknown regulatory pathway during grain filling. PMID:25053646

Inhibitory activity of α-amylase and α-glucosidase by plant extracts from the Brazilian cerrado.

PubMed

Souza, Paula Monteiro de; Sales, Paloma Michelle de; Simeoni, Luiz Alberto; Silva, Elton Clementino; Silveira, Dâmaris; Magalhães, Pérola de Oliveira

2012-03-01

Diabetes mellitus is the most common disease in the world. One therapeutic approach for treating diabetes is inhibition of α-amylase and α-glucosidase activities to reduce postprandial blood glucose levels. In vitro tests showed that several plant extracts from Brazilian cerrado species can inhibit the activity of α-amylase and α-glucosidase. The extracts of Eugenia dysenterica, Stryphnodendron adstringens, Pouteria caimito, Pouteria ramiflora, and Pouteria torta showed strong α-amylase and α-glucosidase inhibitory activity. Eugenia dysenterica, P. caimito, P. ramiflora, and P. torta aqueous extracts exerted the highest activity against α-amylase (IC₅₀) values of 14.93, 13.6, 7.08, and 5.67 µg/mL, respectively) and α-glucosidase (IC₅₀ values of 0.46, 2.58, 0.35, and 0.22 µg/mL, respectively). Stryphnodendron adstringens ethanol extract also exhibited inhibitory activity against both enzymes (IC₅₀) 1.86 µg/mL against α-amylase and 0.61 µg/mL against α-glucosidase). The results suggest that the activity of these cerrado plants on α-amylase and α-glucosidase represents a potential tool for development of new strategies for treatment of diabetes. © Georg Thieme Verlag KG Stuttgart · New York.

Effect of certain plant extracts on alpha-amylase activity.

PubMed

Prashanth, D; Padmaja, R; Samiulla, D S

2001-02-01

Ethanolic extracts of Punica granatum, Mangifera indica, Boerhaavia diffusa, Embelia ribes, Phyllanthus maderaspatensis, and Withania somnifera, were tested for their effect on alpha-amylase activity (in vitro). P. granatum and M. indica were found to exhibit interesting alpha-amylase inhibitory activity.

Three camelid VHH domains in complex with porcine pancreatic alpha-amylase. Inhibition and versatility of binding topology.

PubMed

Desmyter, Aline; Spinelli, Silvia; Payan, Francoise; Lauwereys, Marc; Wyns, Lode; Muyldermans, Serge; Cambillau, Christian

2002-06-28

Camelids produce functional antibodies devoid of light chains and CH1 domains. The antigen-binding fragment of such heavy chain antibodies is therefore comprised in one single domain, the camelid heavy chain antibody VH (VHH). Here we report on the structures of three dromedary VHH domains in complex with porcine pancreatic alpha-amylase. Two VHHs bound outside the catalytic site and did not inhibit or inhibited only partially the amylase activity. The third one, AMD9, interacted with the active site crevice and was a strong amylase inhibitor (K(i) = 10 nm). In contrast with complexes of other proteinaceous amylase inhibitors, amylase kept its native structure. The water-accessible surface areas of VHHs covered by amylase ranged between 850 and 1150 A(2), values similar to or even larger than those observed in the complexes between proteins and classical antibodies. These values could certainly be reached because a surprisingly high extent of framework residues are involved in the interactions of VHHs with amylase. The framework residues that participate in the antigen recognition represented 25-40% of the buried surface. The inhibitory interaction of AMD9 involved mainly its complementarity-determining region (CDR) 2 loop, whereas the CDR3 loop was small and certainly did not protrude as it does in cAb-Lys3, a VHH-inhibiting lysozyme. AMD9 inhibited amylase, although it was outside the direct reach of the catalytic residues; therefore it is to be expected that inhibiting VHHs might also be elicited against proteases. These results illustrate the versatility and efficiency of VHH domains as protein binders and enzyme inhibitors and are arguments in favor of their use as drugs against diabetes.

New perspectives on the role of α- and β-amylases in transient starch synthesis.

PubMed

Wu, Alex Chi; Ral, Jean-Philippe; Morell, Matthew K; Gilbert, Robert G

2014-01-01

Transient starch in leaves is synthesized by various biosynthetic enzymes in the chloroplasts during the light period. This paper presents the first mathematical model for the (bio)synthesis of the chain-length distribution (CLD) of transient starch to aid the understanding of this synthesis. The model expresses the rate of change of the CLD in terms of the actions of the enzymes involved. Using this to simulate the experimental CLD with different enzyme combinations is a new means to test for enzymes that are significant to the rate of change of the CLD during synthesis. Comparison between the simulated CLD from different enzyme combinations and the experimental CLD in the leaves of the model plant Arabidopsis thaliana indicate α-amylase, in addition to the core starch biosynthetic enzymes, is also involved in the modification of glucans for the synthesis of insoluble starch granules. The simulations suggest involvement of β-amylase, in the absence of α-amylase in mutants, slows the rate of attaining a crystalline-competent CLD for crystallization of glucans to form insoluble starch. This suggests a minor role of β-amylase in shaping normal starch synthesis. The model simulation predicts that debranching of glucans is an efficient mechanism for the attainment of crystalline-competent CLD; however, attaining this is still possible, albeit slower, through combinations of α- and β-amylase in the absence of isoamylase-type debranching enzyme. In Arabidopsis defective in one of the isoamylase-type debranching enzymes, the impact of α-amylase in starch synthesis is reduced, while β-amylase becomes significantly involved, slowing the rate of synthesis in this mutant. Modeling of transient starch CLD brings to light previously unrecognized but significant effects of α- and β-amylase on the rate of transient starch synthesis.

New Perspectives on the Role of α- and β-Amylases in Transient Starch Synthesis

PubMed Central

Wu, Alex Chi; Ral, Jean-Philippe; Morell, Matthew K.; Gilbert, Robert G.

2014-01-01

Transient starch in leaves is synthesized by various biosynthetic enzymes in the chloroplasts during the light period. This paper presents the first mathematical model for the (bio)synthesis of the chain-length distribution (CLD) of transient starch to aid the understanding of this synthesis. The model expresses the rate of change of the CLD in terms of the actions of the enzymes involved. Using this to simulate the experimental CLD with different enzyme combinations is a new means to test for enzymes that are significant to the rate of change of the CLD during synthesis. Comparison between the simulated CLD from different enzyme combinations and the experimental CLD in the leaves of the model plant Arabidopsis thaliana indicate α-amylase, in addition to the core starch biosynthetic enzymes, is also involved in the modification of glucans for the synthesis of insoluble starch granules. The simulations suggest involvement of β-amylase, in the absence of α-amylase in mutants, slows the rate of attaining a crystalline-competent CLD for crystallization of glucans to form insoluble starch. This suggests a minor role of β-amylase in shaping normal starch synthesis. The model simulation predicts that debranching of glucans is an efficient mechanism for the attainment of crystalline-competent CLD; however, attaining this is still possible, albeit slower, through combinations of α- and β-amylase in the absence of isoamylase-type debranching enzyme. In Arabidopsis defective in one of the isoamylase-type debranching enzymes, the impact of α-amylase in starch synthesis is reduced, while β-amylase becomes significantly involved, slowing the rate of synthesis in this mutant. Modeling of transient starch CLD brings to light previously unrecognized but significant effects of α- and β-amylase on the rate of transient starch synthesis. PMID:24971464

Alterations in Kernel Proteome after Infection with Fusarium culmorum in Two Triticale Cultivars with Contrasting Resistance to Fusarium Head Blight

PubMed Central

Perlikowski, Dawid; Wiśniewska, Halina; Kaczmarek, Joanna; Góral, Tomasz; Ochodzki, Piotr; Kwiatek, Michał; Majka, Maciej; Augustyniak, Adam; Kosmala, Arkadiusz

2016-01-01

Highlight: The level of pathogen alpha-amylase and plant beta-amylase activities could be components of plant-pathogen interaction associated with the resistance of triticale to Fusarium head blight. Triticale was used here as a model to recognize new components of molecular mechanism of resistance to Fusarium head blight (FHB) in cereals. Fusarium-damaged kernels (FDK) of two lines distinct in levels of resistance to FHB were applied into a proteome profiling using two-dimensional gel electrophoresis (2-DE) to create protein maps and mass spectrometry (MS) to identify the proteins differentially accumulated between the analyzed lines. This proteomic research was supported by a measurement of alpha- and beta-amylase activities, mycotoxin content, and fungal biomass in the analyzed kernels. The 2-DE analysis indicated a total of 23 spots with clear differences in a protein content between the more resistant and more susceptible triticale lines after infection with Fusarium culmorum. A majority of the proteins were involved in a cell carbohydrate metabolism, stressing the importance of this protein group in a plant response to Fusarium infection. The increased accumulation levels of different isoforms of plant beta-amylase were observed for a more susceptible triticale line after inoculation but these were not supported by a total level of beta-amylase activity, showing the highest value in the control conditions. The more resistant line was characterized by a higher abundance of alpha-amylase inhibitor CM2 subunit and simultaneously a lower activity of alpha-amylase after inoculation. We suggest that the level of pathogen alpha-amylase and plant beta-amylase activities could be components of plant-pathogen interaction associated with the resistance of triticale to FHB. PMID:27582751

Cloning of a yeast alpha-amylase promoter and its regulated heterologous expression

DOEpatents

Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR; Hooker, Brian S [Kennewick, WA; Anderson, Daniel B [Pasco, WA

2003-04-01

The present invention provides the promoter clone discovery of an alpha-amylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated alpha-amylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

Long-term stability of diurnal salivary cortisol and alpha-amylase secretion patterns.

PubMed

Skoluda, Nadine; La Marca, Roberto; Gollwitzer, Mario; Müller, Andreas; Limm, Heribert; Marten-Mittag, Birgitt; Gündel, Harald; Angerer, Peter; Nater, Urs M

2017-06-01

This study aimed to investigate long-term stability and variability of diurnal cortisol and alpha-amylase patterns. Diurnal cortisol and alpha-amylase secretion patterns were assessed on a single workday with three waves of measurement across a total time period of 24months in 189 participants. Separate hierarchical linear models were analyzed, with and without a number of potential predictor variables (age, BMI, smoking, chronic stress, stress reactivity). While low long-term stability was found in diurnal cortisol, the stability of diurnal alpha-amylase was moderate across the time period of 24months. Several predictor variables had a positive impact on diurnal cortisol and alpha-amylase secretion patterns averaged across waves. Our findings underpin the notion that long-term stability is not necessarily warranted in longitudinal studies. It is important to choose an appropriate study design when attempting to disentangle clinically and biologically relevant changes from naturally occurring variations in diurnal cortisol and alpha-amylase. Copyright © 2017 Elsevier Inc. All rights reserved.

Important Aspects of Post-Prandial Antidiabetic Drug, Acarbose.

PubMed

Singla, Rajeev Kumar; Singh, Radha; Dubey, Ashok Kumar

2016-01-01

Acarbose, a well known and efficacious α-amylase and α-glucosidase inhibitor, is a postprandial acting antidiabetic drug. DNS-based α-amylase inhibitory assays showed that use of acarbose at concentrations above 125 µg/ml resulted in release of reducing sugar in the reaction, an unexpected observation. Objective of the present study was to design experimental strategies to address this unusual finding. Acarbose was found to be susceptible to thermo-lysis. Further, besides being an inhibitor, it could also be hydrolyzed by porcine pancreatic α-amylase, but had weaker affinity for α - amylase compared to starch. GRIP docking was done for the mechanistic analysis of the active site in the enzyme for substrate, inhibitor and, inhibitor's metabolite (K2). Interaction between acarbose and α-amylase involved significant hydrogen binding compared to that of starch, producing a stronger enzyme-inhibitor complex. Further, docking analysis led us to predict the site on α-amylase where the inhibitor (acarbose) bound more tightly, which possibly affected the binding and hydrolysis of starch exerting its effective anti-diabetic function.

Three-dimensional structure of a variant `Termamyl-like' Geobacillus stearothermophilus α-amylase at 1.9 Å resolution.

PubMed

Offen, Wendy A; Viksoe-Nielsen, Anders; Borchert, Torben V; Wilson, Keith S; Davies, Gideon J

2015-01-01

The enzyme-catalysed degradation of starch is central to many industrial processes, including sugar manufacture and first-generation biofuels. Classical biotechnological platforms involve steam explosion of starch followed by the action of endo-acting glycoside hydrolases termed α-amylases and then exo-acting α-glucosidases (glucoamylases) to yield glucose, which is subsequently processed. A key enzymatic player in this pipeline is the `Termamyl' class of bacterial α-amylases and designed/evolved variants thereof. Here, the three-dimensional structure of one such Termamyl α-amylase variant based upon the parent Geobacillus stearothermophilus α-amylase is presented. The structure has been solved at 1.9 Å resolution, revealing the classical three-domain fold stabilized by Ca2+ and a Ca2+-Na+-Ca2+ triad. As expected, the structure is similar to the G. stearothermophilus α-amylase but with main-chain deviations of up to 3 Å in some regions, reflecting both the mutations and differing crystal-packing environments.

Enzymatic detergent formulation containing amylase from Aspergillus niger: a comparative study with commercial detergent formulations.

PubMed

Mitidieri, Sydnei; Souza Martinelli, Anne Helene; Schrank, Augusto; Vainstein, Marilene Henning

2006-07-01

There is a wide range of biotechnological applications for amylases, including the textile, pharmaceutical, food and laundry industries. Hydrolytic enzymes are 100% biodegradable and enzymatic detergents can achieve effective cleaning with lukewarm water. Microorganisms and culture media were tested for amylase production and the best producer was Aspergillus niger L119 (3.9 U ml(-1) +/- 0.2) in submerged culture and its amylase demonstrated excellent activity at 50-55 degrees C and pH 4.0, remaining stable at 53 degrees C for up to 200 h. In order to establish the potential uses of this enzyme in detergents, different formulations were tested using the A. niger amylase extract. Enzyme activity was compared with three commercial formulations. The detergents are used in hospitals to clean surgical and endoscopy equipment. The presence of amylase in the formulation is because of its action within hospital drainage system, whether or not it has any function in cleaning the equipment.

Production of surfactant and detergent-stable, halophilic, and alkalitolerant alpha-amylase by a moderately halophilic Bacillus sp. Strain TSCVKK.

PubMed

Kiran, Kondepudi Kanthi; Chandra, T S

2008-01-01

A moderately halophilic alkalitolerant Bacillus sp. Strain TSCVKK, with an ability to produce extracellular halophilic, alkalitolerant, surfactant, and detergent-stable alpha-amylase was isolated from soil samples obtained from a salt-manufacturing industry in Chennai. The culture conditions for higher amylase production were optimized with respect to NaCl, substrate, pH, and temperature. Maximum amylase production of 592 mU/ml was achieved in the medium at 48 h with 10% NaCl, 1% dextrin, 0.4% yeast extract, 0.2% tryptone, and 0.2% CaCl(2) at pH 8.0 at 30 degrees C. The enzyme activity in the culture supernatant was highest with 10% NaCl at pH 7.5 and 55 degrees C. The amylase that was partially purified by acetone precipitation was highly stable in various surfactants and detergents. Glucose, maltose, and maltooligosaccharides were the main end products of starch hydrolysis indicating that it is an alpha-amylase.

A glycoprotein α-amylase inhibitor from Withania somnifera differentially inhibits various α-amylases and affects the growth and development of Tribolium castaneum.

PubMed

Kasar, Sainath S; Marathe, Kiran R; Bhide, Amey J; Herwade, Abhijeet P; Giri, Ashok P; Maheshwari, Vijay L; Pawar, Pankaj K

2017-07-01

Identification and characterisation of plant defensive molecules enrich our resources to design crop protection strategies. In particular, plant-derived proteinaceous inhibitor(s) of insect digestive enzymes appear to be a safe, sustainable and attractive option. A glycoprotein having non-competitive α-amylase inhibitory activity with a molecular weight of 8.3 kDa was isolated and purified from seeds of Withania somnifera α-amylase inhibitor (WSAI). Its mass spectrometry analysis revealed 59% sequence coverage with Wrightide II-type α-amylase inhibitor from Wrightia religiosa. A dose-dependent inhibition of α-amylases from Aspergillus oryzae, Bacillus subtilis, Helicoverpa armigera and Tribolium castaneum was recorded. Interestingly, WSAI did not inhibit human salivary α-amylase significantly. When adults of T. castaneum were fed with WSAI (1.6 mg g -1 ), decrease in consumption, growth and efficiency of conversion of ingested food was evident, along with over fourfold increases in feeding deterrence index. A decline in larval residual α-amylase activity after feeding of WSAI resulted in a reduction in longevity of T. castaneum. The study reflects the significance of WSAI in affecting the overall growth and development of T. castaneum. Pre- and post-harvest pest resistive capability makes WSAI a potential candidate for insect pest management. Further, the effectiveness of this inhibitor could be explored either in formulations or through a transgenic approach. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Interactions between α-amylase and an acidic branched polysaccharide from green tea.

PubMed

Wu, Shuyun; Lai, Minghua; Luo, Jiahao; Pan, Jingwen; Zhang, Li-Ming; Yang, Liqun

2017-01-01

To understand the mechanism responsible for the α-amylase inhibitory activity of tea polysaccharides, the interaction between α-amylase and an acidic branched tea polysaccharide (TPSA) was investigated using fluorescence spectroscopy and resonance light scattering analysis. TPSA, exhibiting inhibitory activity towards α-amylase (the maximum inhibition percentage of 65%), was isolated from green tea (Camellia sinensis) and characterized by nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, ultraviolet-visible spectroscopy, and gas chromatography. Synchronous fluorescence spectroscopy revealed that the binding interaction between the tryptophan residues of α-amylase and TPSA was predominant. Based on the fluorescence quenching effect of tryptophan residues induced by TPSA, the binding constants between α-amylase and TPSA were determined to be 18.6×10 6 , 8.0×10 6 and 4.6×10 6 L·mol -1 at 20, 30 and 37°C, respectively. The calculated Gibbs free-energy changes were negative, indicating that the bonding interaction was a spontaneous process. The enthalpy and the entropy changes were -62.13 KJ·mol -1 and -0.0728 KJ·mol -1 ·K -1 , suggesting that hydrogen bonding interactions might play a major role in the binding process. The formation of an α-amylase/TPSA complex was evidenced by fluorescence quenching and resonance light scattering analysis, and this complex could be the main contributor to the α-amylase inhibitory activity of TPSA. Copyright © 2016 Elsevier B.V. All rights reserved.

Thapsigargin defines the roles of cellular calcium in secretagogue-stimulated enzyme secretion from pancreatic acini.

PubMed

Metz, D C; Patto, R J; Mrozinski, J E; Jensen, R T; Turner, R J; Gardner, J D

1992-10-15

In the present study we used thapsigargin (TG), an inhibitor of microsomal calcium ATPase, to evaluate the roles of free cytoplasmic calcium and intracellular stored calcium in secretagogue-stimulated enzyme secretion from rat pancreatic acini. Using microspectrofluorimetry of fura-2-loaded pancreatic acini, we found that TG caused a sustained increase in free cytoplasmic calcium by mobilizing calcium from inositol 1,4,5-trisphosphate-sensitive intracellular stores and by increasing influx of extracellular calcium. TG also caused a small increase in basal amylase secretion, inhibited the stimulation of amylase secretion caused by secretagogues that increase inositol 1,4,5-trisphosphate, and potentiated the stimulation of amylase secretion caused by 12-O-tetradecanoylphorbol-13-acetate or secretagogues that increase cyclic adenosine 3',5'-monophosphate. Bombesin, which like TG increased free cytoplasmic calcium, also potentiated the stimulation of amylase secretion caused by secretagogues that increase cyclic adenosine 3',5'-monophosphate, but did not inhibit the stimulation of amylase secretion caused by secretagogues that increase inositol 1,4,5-trisphosphate. Finally, TG inhibited the sustained phase of cholecystokinin-stimulated amylase secretion and potentiated the time course of vasoactive intestinal peptide-stimulated amylase secretion. The present findings indicate that stimulation of amylase secretion by secretagogues that increase inositol 1,4,5-trisphosphate does not depend on increased free cytoplasmic calcium per se. In contrast, TG-induced potentiation of the stimulation of secretagogues that increase cellular cyclic adenosine 3',5'-monophosphate appears to result from increased free cytoplasmic calcium per se.

Rethinking the starch digestion hypothesis for AMY1 copy number variation in humans.

PubMed

Fernández, Catalina I; Wiley, Andrea S

2017-08-01

Alpha-amylase exists across taxonomic kingdoms with a deep evolutionary history of gene duplications that resulted in several α-amylase paralogs. Copy number variation (CNV) in the salivary α-amylase gene (AMY1) exists in many taxa, but among primates, humans appear to have higher average AMY1 copies than nonhuman primates. Additionally, AMY1 CNV in humans has been associated with starch content of diets, and one known function of α-amylase is its involvement in starch digestion. Thus high AMY1 CNV is considered to result from selection favoring more efficient starch digestion in the Homo lineage. Here, we present several lines of evidence that challenge the hypothesis that increased AMY1 CNV is an adaptation to starch consumption. We observe that α- amylase plays a very limited role in starch digestion, with additional steps required for starch digestion and glucose metabolism. Specifically, we note that α-amylase hydrolysis only produces a minute amount of free glucose with further enzymatic digestion and glucose absorption being rate-limiting steps for glucose availability. Indeed α-amylase is nonessential for starch digestion since sucrase-isomaltase and maltase-glucoamylase can hydrolyze whole starch granules while releasing glucose. While higher AMY1 CN and CNV among human populations may result from natural selection, existing evidence does not support starch digestion as the major selective force. We report that in humans α-amylase is expressed in several other tissues where it may have potential roles of evolutionary significance. © 2017 Wiley Periodicals, Inc.

Improved α-amylase production by Aspergillus oryzae after a double deletion of genes involved in carbon catabolite repression.

PubMed

Ichinose, Sakurako; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

2014-01-01

In filamentous fungi, the expression of secretory glycoside hydrolase encoding genes, such as those for amylases, cellulases, and xylanases, is generally repressed in the presence of glucose. CreA and CreB have been observed to be regulating factors for carbon catabolite repression. In this study, we generated single and double deletion creA and/or creB mutants in Aspergillus oryzae. The α-amylase activities of each strain were compared under various culture conditions. For the wild-type strain, mRNA levels of α-amylase were markedly decreased in the later stage of submerged culture under inducing conditions, whereas this reduced expression was not observed for single creA and double creA/creB deletion mutants. In addition, α-amylase activity of the wild-type strain was reduced in submerged culture containing high concentrations of inducing sugars, whereas all constructed mutants showed higher α-amylase activities. In particular, the α-amylase activity of the double deletion mutant in a medium containing 5% starch was >10-fold higher than that of the wild-type strain under the same culture conditions. In solid-state cultures using wheat bran as a substrate, the α-amylase activities of single creA and double deletion mutants were >2-fold higher than that of the wild-type strain. These results suggested that deleting both creA and creB resulted in dramatic improvements in the production of secretory glycoside hydrolases in filamentous fungi.

Taxonomy of the Streptomyces strain ZG0656 that produces acarviostatin alpha-amylase inhibitors and analysis of their effects on blood glucose levels in mammalian systems.

PubMed

Geng, P; Bai, G; Shi, Q; Zhang, L; Gao, Z; Zhang, Q

2009-02-01

To clarify the taxonomic status of strain ZG0656 and analyse the effects of its acarviostatin products on blood glucose levels in mammalian systems. Our program to screen for new alpha-amylase inhibitors led to the isolation of strain ZG0656. The polyphasic taxonomic study revealed that strain ZG0656 represents a novel variation of Streptomyces coelicoflavus, for which we propose the name S. coelicoflavus var. nankaiensis. Four chemically distinct alpha-amylase inhibitors, acarviostatins I03, II03, III03 and IV03, were isolated from strain ZG0656. Acarviostatins III03 and IV03 are both novel oligomers. All four acarviostatins are mixed noncompetitive porcine pancreas alpha-amylase inhibitors. Acarviostatin III03 is the most potent alpha-amylase inhibitor known to date. Moreover, in the in vitro and in vivo experiments, acarviostatins III03 showed significant inhibition of starch hydrolysis and glucose transfer to blood. Strain ZG0656 is a novel variation of S. coelicoflavus, whose products are novel effective alpha-amylase inhibitors. Among the products, acarviostatins III03 could significantly depress blood glucose levels in mammalian systems and be developed towards a possible therapeutic agent for diabetes. Acarviostatin III03 is the most potent alpha-amylase inhibitor known to date. The oligomer will benefit the research on the relationship between alpha-amylase and various inhibitors and will offer more choices in diabetes treatments.

Purification and biochemical characterization of a thermostable and acid-stable alpha-amylase from Bacillus licheniformis B4-423.

PubMed

Wu, Xiangrong; Wang, Yuxia; Tong, Bending; Chen, Xianghua; Chen, Jianhua

2018-04-01

Novel thermostable amylase need to be continuously explored with the improvement of industrial requirements. A new acidophilic and thermostable amylase producing bacterium isolated from spring was identified as Bacillus strain on the basis of 16S rDNA. The amylase was purified by ammonium sulphate precipitation, gel chromatography and anion exchange chromatography. SDS-PAGE revealed that the enzyme was monomeric with a molecular weight of 58 kDa. The amylase exhibited optimal activity at pH 5.0 and temperature 100 °C. Then the enzyme showed high stability in pH ranges 4.0-10.0 and more than 90% of maximal activity was found from 20 °C to 80 °C. Apart from good stability toward SDS and non-ionic detergent, the purified enzyme exhibited high compatibility with some inhibitors such as urea and EDTA. The results demonstrated the stability of the enzyme in different organic solvents. Moreover, we determined the amylase gene, compared the structure with α-amylase BAA and BLA and found some thermostability determinants in our enzyme. Overall, presenting various properties were including high thermostability, Ca 2+ -independency, broad temperature and pH profiles, organic-solvent tolerance as well as excellent stability with detergents. Such characteristics have not been reported for this type of enzyme, and the α-amylase will be a suitable candidate in industrial fields. Copyright © 2017 Elsevier B.V. All rights reserved.

27 CFR 24.247 - Materials authorized for the treatment of distilling material.

Code of Federal Regulations, 2014 CFR

2014-04-01

... fermentable carbohydrates The amylase enzyme activity shall be derived from Aspergillus niger, Aspergillus... convent starches to fermentable carbohydrates The amylase enzyme activity shall be derived from barley... starches to fermentable carbohydrates The amylase enzyme actvity shall be derived from Aspergillus niger or...

27 CFR 24.247 - Materials authorized for the treatment of distilling material.

Code of Federal Regulations, 2011 CFR

2011-04-01

... fermentable carbohydrates The amylase enzyme activity shall be derived from Aspergillus niger, Aspergillus... convent starches to fermentable carbohydrates The amylase enzyme activity shall be derived from barley... starches to fermentable carbohydrates The amylase enzyme actvity shall be derived from Aspergillus niger or...

27 CFR 24.247 - Materials authorized for the treatment of distilling material.

Code of Federal Regulations, 2012 CFR

2012-04-01

... fermentable carbohydrates The amylase enzyme activity shall be derived from Aspergillus niger, Aspergillus... convent starches to fermentable carbohydrates The amylase enzyme activity shall be derived from barley... starches to fermentable carbohydrates The amylase enzyme actvity shall be derived from Aspergillus niger or...

27 CFR 24.247 - Materials authorized for the treatment of distilling material.

Code of Federal Regulations, 2013 CFR

2013-04-01

... fermentable carbohydrates The amylase enzyme activity shall be derived from Aspergillus niger, Aspergillus... convent starches to fermentable carbohydrates The amylase enzyme activity shall be derived from barley... starches to fermentable carbohydrates The amylase enzyme actvity shall be derived from Aspergillus niger or...

Use of activated carbon to remove undesirable residual amylase from refinery streams

USDA-ARS?s Scientific Manuscript database

In recent years, there has been increased world-wide concern over residual (carry-over)activity of mostly high temperature (HT) and very high temperature (VHT) stable amylases in white, refined sugars from refineries to various food and end-user industries. HT and VHT stable amylases were developed ...

Further Experiments on Gibberellin-Stimulated Amylase Production in Cereal Grains

ERIC Educational Resources Information Center

Coppage, Jo; Hill, T. A.

1973-01-01

Experiments conducted on wheat and barley grains to analyze activities of alpha- and beta-amylase enzymes. Gibberellins were used exogenously. Techniques are described in detail. Results on different cultivars revealed that beta-amylase was not an invariable result of imbibition. Techniques employed can be used by school students. (PS)

Mixed-mode resins: taking shortcut in downstream processing of raw-starch digesting α-amylases

PubMed Central

Lončar, Nikola; Slavić, Marinela Šokarda; Vujčić, Zoran; Božić, Nataša

2015-01-01

Bacillus licheniformis 9945a α-amylase is known as a potent enzyme for raw starch hydrolysis. In this paper, a mixed mode Nuvia cPrime™ resin is examined with the aim to improve the downstream processing of raw starch digesting amylases and exploit the hydrophobic patches on their surface. This resin combines hydrophobic interactions with cation exchange groups and as such the presence of salt facilitates hydrophobic interactions while the ion-exchange groups enable proper selectivity. α-Amylase was produced using an optimized fed-batch approach in a defined media and significant overexpression of 1.2 g L−1 was achieved. This single step procedure enables simultaneous concentration, pigment removal as well as purification of amylase with yields of 96% directly from the fermentation broth. PMID:26492875

Cloning and Characterization of Cold-Adapted α-Amylase from Antarctic Arthrobacter agilis.

PubMed

Kim, Su-Mi; Park, Hyun; Choi, Jong-Il

2017-03-01

In this study, the gene encoding an α-amylase from a psychrophilic Arthrobacter agilis PAMC 27388 strain was cloned into a pET-28a(+) vector and heterologously expressed in Escherichia coli BL21(DE3). The recombinant α-amylase with a molecular mass of about 80 kDa was purified by using Ni 2+ -NTA affinity chromatography. This recombinant α-amylase exhibited optimal activity at pH 3.0 and 30 °C and was highly stable at varying temperatures (30-60 °C) and within the pH range of 4.0-8.0. Furthermore, α-amylase activity was enhanced in the presence of FeCl 3 (1 mM) and β-mercaptoethanol (5 mM), while CoCl 2 (1 mM), ammonium persulfate (5 mM), SDS (10 %), Triton X-100 (10 %), and urea (1 %) inhibited the enzymatic activity. Importantly, the presence of Ca 2+ ions and phenylmethylsulfonyl fluoride (PMSF) did not affect enzymatic activity. Thin layer chromatography (TLC) analysis showed that recombinant A. agilis α-amylase hydrolyzed starch, maltotetraose, and maltotriose, producing maltose as the major end product. These results make recombinant A. agilis α-amylase an attractive potential candidate for industrial applications in the textile, paper, detergent, and pharmaceutical industries.

Cloning, enhanced expression and characterization of an α-amylase gene from a wild strain in B. subtilis WB800.

PubMed

Chen, Jing; Chen, Xianghua; Dai, Jun; Xie, Guangrong; Yan, Luying; Lu, Lina; Chen, Jianhua

2015-09-01

A Bacillus strain with high productivity of α-amylase isolated from a starch farm was identified as Bacillus amyloliquefaciens. The α-amylase encoding gene amy1 was cloned into pMD18-T vector and amplified in E. coli DH5α. Shuttle vector pP43MNX was reconstructed to obtain vector pP43X for heterologous expression of the α-amylase in B. subtilis WB800. Recombinant enzyme was sufficiently purified by precipitation, gel filtration and anion exchange with a specific activity of 5566 U/mg. The α-amylase sequence contains an open reading frame of 1545 bp, which encodes a protein of 514 amino acid residues with a predicted molecular mass of 58.4 kDa. The enzyme exhibited maximal activity at pH 6.0 and 60 °C. Catalytic efficiency of the recombinant α-amylase was inhibited by Hg(2+), Pb(2+) and Cu(2+), but stimulated by Li(+), Mn(2+) and Ca(2+). The purified enzyme showed decreased activity toward detergents (SDS, Tween 20 and Triton X-100). Compared with production by the wild strain, there was a 1.48-fold increase in the productivity of α-amylase in recombinant B. subtilis WB800. Copyright © 2015 Elsevier B.V. All rights reserved.

Recurrent Rearrangements of Human Amylase Genes Create Multiple Independent CNV Series.

PubMed

Shwan, Nzar A A; Louzada, Sandra; Yang, Fengtang; Armour, John A L

2017-05-01

The human amylase gene cluster includes the human salivary (AMY1) and pancreatic amylase genes (AMY2A and AMY2B), and is a highly variable and dynamic region of the genome. Copy number variation (CNV) of AMY1 has been implicated in human dietary adaptation, and in population association with obesity, but neither of these findings has been independently replicated. Despite these functional implications, the structural genomic basis of CNV has only been defined in detail very recently. In this work, we use high-resolution analysis of copy number, and analysis of segregation in trios, to define new, independent allelic series of amylase CNVs in sub-Saharan Africans, including a series of higher-order expansions of a unit consisting of one copy each of AMY1, AMY2A, and AMY2B. We use fiber-FISH (fluorescence in situ hybridization) to define unexpected complexity in the accompanying rearrangements. These findings demonstrate recurrent involvement of the amylase gene region in genomic instability, involving at least five independent rearrangements of the pancreatic amylase genes (AMY2A and AMY2B). Structural features shared by fundamentally distinct lineages strongly suggest that the common ancestral state for the human amylase cluster contained more than one, and probably three, copies of AMY1. © 2017 WILEY PERIODICALS, INC.

α-Amylase inhibitor activity of endophytic bacteria isolated from Annona muricata L

NASA Astrophysics Data System (ADS)

Pujiyanto, Sri; Resdiani, Merysa; Raharja, Budi; Siti Ferniah, Rejeki

2018-05-01

α-amylase (α-1,4-glucan-4-glucohydrolase, EC 3.2.1.1) is an enzyme that catalyzes the degradation of starch into its monomers. Most people use medicinal plants for keeping normal level of blood glucose, for example, the Annona muricata. The objectives of this study are to obtain endophytic bacteria from the plant, knowing the activity of the α-amylase inhibitor of selected isolates. Endophytic bacteria are isolated from the roots, stems, and leaves of the plant have been sterilized surface and grown in NA medium. A total of 11 isolates were found to produce α-amylase inhibitor compounds. The isolates obtained were tested for their α-amylase inhibitor activity, and isolates with the highest activity tested further. Isolate DS21 show the best activity with 72,22% inhibition. The experimental design used in this research is Completely Randomized Design (RAL). The best isolates treated by a variety of carbon sources, and the best carbon source treated with various pH. The data obtained were analyzed usingAnalysis of Variance (ANOVA). The results of statistical tests show the treatment of starch and lactose has a significant effect on the production of α-amylase inhibitors (P <0.05) and the pH 5 and 6,0 significantly affected the production of α-amylase inhibitors (P <0.05).

Tobacco plants transformed with the bean. alpha. ai gene express an inhibitor of insect. alpha. -amylase in their seeds. [Nicotiana tabacum; Tenebrio molitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Altabella, T.; Chrispeels, M.J.

Bean (Phaseolus vulgaris L.) seeds contain a putative plant defense protein that inhibits insect and mammalian but not plant {alpha}-amylases. We recently presented strong circumstantial evidence that this {alpha}-amylase inhibitor ({alpha}Al) is encoded by an already-identified lectin gene whose product is referred to as lectin-like-protein (LLP). We have now made a chimeric gene consisting of the coding sequence of the lectin gene that encodes LLP and the 5{prime} and 3{prime} flanking sequences of the lectin gene that encodes phytohemagglutinin-L. When this chimeric gene was expressed in transgenic tobacco (Nicotiana tabacum), we observed in the seeds a series of polypeptides (M{submore » r} 10,000-18,000) that cross-react with antibodies to the bean {alpha}-amylase inhibitor. Most of these polypeptides bind to a pig pancreas {alpha}-amylase affinity column. An extract of the seeds of the transformed tobacco plants inhibits pig pancreas {alpha}-amylase activity as well as the {alpha}-amylase present in the midgut of Tenebrio molitor. We suggest that introduction of this lectin gene (to be called {alpha}ai) into other leguminous plants may be a strategy to protect the seeds from the seed-eating larvae of Coleoptera.« less

Patterns of cortisol and alpha-amylase reactivity to psychosocial stress in maltreated women

PubMed Central

Mielock, Alyssa S.; Morris, Matthew C.; Rao, Uma

2016-01-01

Background Childhood maltreatment can trigger enduring changes in major stress response systems, particularly in the context of major depressive disorder (MDD). However, the relative impact of maltreatment versus MDD on hypothalamic-pituitary-adrenal axis and sympathetic-adrenal-medullary system stress reactivity is not well understood. Method This study examined salivary cortisol and alpha-amylase responses to the Trier Social Stress Test (TSST) in 26 maltreated (15 with current MDD) and 26 non-maltreated (17 with current MDD) women. Results Maltreated women showed greater anticipatory cortisol reactivity during the TSST protocol compared to non-maltreated women. Maltreated women also showed rapid deceleration in cortisol levels. Whereas non-maltreated women showed initial declines in alpha-amylase levels but rapidly increasing alpha-amylase levels during the TSST protocol, maltreated women did not exhibit changes in alpha-amylase levels during the TSST protocol. Contrary to expectation, MDD did not impact cortisol or alpha-amylase responses. Limitations The present study is limited by retrospective report of childhood maltreatment, cross-sectional design, and modest sample sizes. Conclusions These findings suggest that childhood maltreatment plays a greater role driving alterations in cortisol and alpha-amylase stress reactivity than MDD. Understanding the biological embedding of maltreatment is critical for elucidating mechanisms linking these experiences to risk for negative mental and physical health outcomes. PMID:27875756

Engineering α-amylase levels in wheat grain suggests a highly sophisticated level of carbohydrate regulation during development.

PubMed

Whan, Alex; Dielen, Anne-Sophie; Mieog, Jos; Bowerman, Andrew F; Robinson, Hannah M; Byrne, Keren; Colgrave, Michelle; Larkin, Philip J; Howitt, Crispin A; Morell, Matthew K; Ral, Jean-Philippe

2014-10-01

Wheat starch degradation requires the synergistic action of different amylolytic enzymes. Our spatio-temporal study of wheat α-amylases throughout grain development shows that AMY3 is the most abundant isoform compared with the other known α-amylases. Endosperm-specific over-expression of AMY3 resulted in an increase of total α-amylase activity in harvested grains. Unexpectedly, increased activity did not have a significant impact on starch content or composition but led to an increase of soluble carbohydrate (mainly sucrose) in dry grain. In AMY3 overexpression lines (A3OE), germination was slightly delayed and triacylglycerol (TAG) content was increased in the endosperm of mature grain. Despite increased AMY3 transcript and protein content throughout grain development, alterations of α-amylase activity and starch granule degradation were not detected until grain maturation, suggesting a post-translational inhibition of α-amylase activity in the endosperm during the starch filling period. These findings show unexpected effects of a high level of α-amylase on grain development and composition, notably in carbon partitioning and TAG accumulation, and suggest the presence of a hitherto unknown regulatory pathway during grain filling. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Alpha-amylase serum levels in professional soccer players are not related with physical fitness.

PubMed

Sanchis-Gomar, Fabian; Alis, Rafael; Rampinini, Ermanno; Bosio, Andrea; Romagnoli, Marco; Lombardi, Giovanni; Lippi, Giuseppe

2017-03-01

Recent evidence has showed that serum or salivary values of α-amylase predict endurance running performance. In this study we investigate whether serum α-amylase concentration may be associated with training status during a competitive season and after a detraining period in professional soccer players. The study population consisted in 15 male professional soccer players from an Italian major league team (age [mean±SD] 27±5 years, weight 76.9±4.1 kg, height 1.82±0.05 m). Serum α-amylase levels were measured 3 times during the last part of a competitive season (January, March and May) and just before preseason training (July). Metabolic and cardiovascular fitness of soccer players was improved during the last part of the season. The levels of α-amylase did not change significantly throughout the study period (χ2=7.331, P=0.062), nor they were found to be associated with variation of physical fitness and training status. The α-amylase fluctuations throughout a competitive season and after vacation time were meaningless in professional soccer players. No significant associations with physical fitness variations could be observed. These results suggest that α-amylase concentration may be a useful parameter for identifying individual inclination to endurance exercise, but not for predicting actual training status.

Psychological stress-induced changes in salivary alpha-amylase and adrenergic activity.

PubMed

Kang, Younhee

2010-12-01

The aim of the study was to examine the relationships among salivary alpha-amylase, plasma catecholamines, blood pressure, and heart rate during psychological stress. This study used a pretest-post-test experimental design with a control group, using repeated measures. A total of 33 participants was divided into the experimental group (n = 16) that underwent a college academic final test as the psychological stress and the control group (n = 17) that did not undergo the test. The levels of salivary alpha-amylase and plasma catecholamines, blood pressure, and heart rate were measured seven times and stress and anxiety were measured once and twice, respectively, as subjective stress markers. Significant changes in the level of salivary alpha-amylase were found in response to psychological stress. However, the correlations of salivary alpha-amylase with the plasma catecholamines, blood pressure, and heart rate were only partially found to be statistically significant. In conclusion, it was shown that salivary alpha-amylase was sensitive to stress throughout this study. Thus, salivary alpha-amylase may be used to measure stress uninvasively in both clinical settings and nursing research where the effects of stress might be scrutinized. Furthermore, the mechanisms of illnesses that are induced by stress could be explored. © 2010 Blackwell Publishing Asia Pty Ltd.

Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii.

PubMed

Michelin, Michele; Silva, Tony M; Benassi, Vivian M; Peixoto-Nogueira, Simone C; Moraes, Luiz Alberto B; Leão, Juliana M; Jorge, João A; Terenzi, Héctor F; Polizeli, Maria de Lourdes T M

2010-11-02

An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60°C and 4.0, respectively. The enzyme was stable for 1 h at 55°C, showing a t₅₀ of 53 min at 60°C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K(m) of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase). Copyright © 2010 Elsevier Ltd. All rights reserved.

Characterization of glucoamylase and α-amylase from Monascus anka: enhanced production of α-amylase in red koji.

PubMed

Yoshizaki, Yumiko; Susuki, Tomoka; Takamine, Kazunori; Tamaki, Hisanori; Ito, Kiyoshi; Sameshima, Yoshihiro

2010-12-01

To enhance glucoamylase and α-amylase production from Monascus anka, we investigated the influence of different culture conditions on enzyme production and purified and characterized these enzymes. The effect of different raw materials was investigated by using solid-state plates of raw materials such as barley and non-waxy or waxy rice. The barley plate was the most suitable for mycelial growth, but glucoamylase and α-amylase production per growth area did not differ according to the raw material. Investigation of the effect of temperature showed that incubation at 37 °C promoted maximal cell growth, while incubation at 25 °C and at 40 °C resulted in enhanced α-amylase and glucoamylase production, respectively. Characterization of the purified enzymes revealed that α-amylase was unstable at acidic pH and less resistant to heat (stable at < 40 °C) than glucoamylase. When these culture conditions were applied to enzyme production in red koji, reducing the temperature from 35 °C to 25 °C for 48 h in the late stages of growth resulted in higher glucoamylase and α-amylase production (1.4 and 18 times, respectively) with a concomitant increase in protein stability. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Proglucagon-Derived Peptides Do Not Significantly Affect Acute Exocrine Pancreas in Rats

PubMed Central

Akalestou, Elina; Christakis, Ioannis; Solomou, Antonia M.; Minnion, James S.; Rutter, Guy A.; Bloom, Stephen R.

2015-01-01

Objectives Reports have suggested a link between treatment with glucagon-like peptide 1 (GLP-1) analogues and an increased risk of pancreatitis. Oxyntomodulin, a dual agonist of both GLP-1 and glucagon receptors, is currently being investigated as a potential anti-obesity therapy, but little is known about its pancreatic safety. The aim of this study was to investigate the acute effect of oxyntomodulin and other proglucagon-derived peptides on the rat exocrine pancreas. Methods GLP-1, oxyntomodulin, glucagon and exendin-4 were infused into anaesthetised rats to measure plasma amylase concentration changes. Additionally, the effect of each peptide on both amylase release and proliferation in rat pancreatic acinar (AR42J) and primary isolated ductal cells was determined. Results Plasma amylase did not increase post peptide infusion, compared to vehicle and cholecystokinin (CCK); however, oxyntomodulin inhibited plasma amylase when co-administered with CCK. None of the peptides caused a significant increase in proliferation rate or amylase secretion from acinar and ductal cells. Conclusions The investigated peptides do not have an acute effect on the exocrine pancreas with regard to proliferation and plasma amylase, when administered individually. Oxyntomodulin appears to be a potent inhibitor of amylase release, potentially making it a safer anti-obesity agent regarding pancreatitis, compared to GLP-1 agonists. PMID:26731187

Phylogenetic and Comparative Sequence Analysis of Thermostable Alpha Amylases of kingdom Archea, Prokaryotes and Eukaryotes.

PubMed

Huma, Tayyaba; Maryam, Arooma; Rehman, Shahid Ur; Qamar, Muhammad Tahir Ul; Shaheen, Tayyaba; Haque, Asma; Shaheen, Bushra

2014-01-01

Alpha amylase family is generally defined as a group of enzymes that can hydrolyse and transglycosylase α-(1, 4) or α-(1, 6) glycosidic bonds along with the preservation of anomeric configuration. For the comparative analysis of alpha amylase family, nucleotide sequences of seven thermo stable organisms of Kingdom Archea i.e. Pyrococcus furiosus (100-105°C), Kingdom Prokaryotes i.e. Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C), Bacillus amyloliquefaciens (72°C), Bacillus subtilis (70°C) and Bacillus KSM K38 (55°C) and Eukaryotes i.e. Aspergillus oryzae (60°C) were selected from NCBI. Primary structure composition analysis and Conserved sequence analysis were conducted through Bio Edit tools. Results from BioEdit shown only three conserved regions of base pairs and least similarity in MSA of the above mentioned alpha amylases. In Mega 5.1 Phylogeny of thermo stable alpha amylases of Kingdom Archea, Prokaryotes and Eukaryote was handled by Neighbor-Joining (NJ) algorithm. Mega 5.1 phylogenetic results suggested that alpha amylases of thermo stable organisms i.e. Pyrococcus furiosus (100-105°C), Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C) and Bacillus amyloliquefaciens (72°C) are more distantly related as compared to less thermo stable organisms. By keeping in mind the characteristics of most thermo stable alpha amylases novel and improved features can be introduced in less thermo stable alpha amylases so that they become more thermo tolerant and productive for industry.

Lipase or amylase for the diagnosis of acute pancreatitis?

PubMed

Ismail, Ola Z; Bhayana, Vipin

2017-12-01

Acute pancreatitis is a rapid onset of inflammation of the pancreas causing mild to severe life threatening conditions [1, 2]. In Canada, acute pancreatitis is the 5th most expensive digestive disease in Canada with a considerable economic burden on the health care system [3]. The diagnosis of acute pancreatitis is usually based on the presence of abdominal pain and elevated levels of serum amylase and/or lipase. Many health care centers use either serum amylase, lipase or both to diagnose acute pancreatitis without considering which one could provide a better diagnostic accuracy. The aim of this review is to investigate whether serum lipase alone is a sufficient biomarker for the diagnosis of acute pancreatitis. We have examined various studies looking at the utilization, sensitivity, specificity and cost associated savings of lipase and amylase in the diagnosis of acute pancreatitis. When comparing different studies, serum lipase offers a higher sensitivity than serum amylase in diagnosing acute pancreatitis. Lipase also offers a larger diagnostic window than amylase since it is elevated for a longer time, thus allowing it to be a useful diagnostic biomarker in early and late stages of acute pancreatitis. Several recent evidence-based guidelines recommend the use of lipase over amylase. Nevertheless, both lipase and amylase alone lack the ability to determine the severity and etiology of acute pancreatitis. The co-ordering of both tests has shown little to no increase in the diagnostic sensitivity and specificity. Thus, unnecessary testing and laboratory expenditures can be reduced by testing lipase alone. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

[Are amylases in bakery products and flour potential food allergens?].

PubMed

Baur, X; Sander, I; Jansen, A; Czuppon, A B

1994-05-21

The enzyme alpha-amylase from the mould Aspergillus oryzae (Asp o II) routinely used for the production of bread, cakes and pastries has in recent years been identified as an inhalative allergen for occupational diseases (bakers' asthma). It is doubtful whether this amylase in the final product, i.e. after the baking procedure, can still be regarded as an allergen. To clarify this question, detailed case histories on 138 subjects were recorded (98 allergics, 20 patients suffering form chronic intestinal diseases, 20 healthy controls). The clinical examinations included prick skin test and IgE antibody determination using one of the customary enzyme preparations. EAST showed a few of these 138 bread consumers to be weakly sensitized to the enzyme. One of the subjects displayed a significant reaction to alpha-amylase heated to 200 degrees C. As expected, eleven bakers sensitized to alpha-amylase by inhaling it in the workplace (positive prick test, positive case history) predominantly exhibited specific IgE antibodies to the native enzyme. Apart from one weakly positive finding, heated alpha-amylase yielded negative results in this collective. Baking conditions vary widely, especially with regard to single components, temperature and duration. Thus, further investigations as to residual allergenicity or the feasible occurrence of new antigenic determinants during the production of bread, cake and pastries are required. 27% of bakers examined and 9% of atopics showed antibodies to a flour inherent enzyme, a beta-amylase. On the whole, the selected conditions hinted at a weakly sensitizing potential inherent in baking flour and in added amylase.

Amylase activity is associated with AMY2B copy numbers in dog: implications for dog domestication, diet and diabetes

PubMed Central

Arendt, Maja; Fall, Tove; Lindblad-Toh, Kerstin; Axelsson, Erik

2014-01-01

High amylase activity in dogs is associated with a drastic increase in copy numbers of the gene coding for pancreatic amylase, AMY2B, that likely allowed dogs to thrive on a relatively starch-rich diet during early dog domestication. Although most dogs thus probably digest starch more efficiently than do wolves, AMY2B copy numbers vary widely within the dog population, and it is not clear how this variation affects the individual ability to handle starch nor how it affects dog health. In humans, copy numbers of the gene coding for salivary amylase, AMY1, correlate with both salivary amylase levels and enzyme activity, and high amylase activity is related to improved glycemic homeostasis and lower frequencies of metabolic syndrome. Here, we investigate the relationship between AMY2B copy numbers and serum amylase activity in dogs and show that amylase activity correlates with AMY2B copy numbers. We then describe how AMY2B copy numbers vary in individuals from 20 dog breeds and find strong breed-dependent patterns, indicating that the ability to digest starch varies both at the breed and individual level. Finally, to test whether AMY2B copy number is strongly associated with the risk of developing diabetes mellitus, we compare copy numbers in cases and controls as well as in breeds with varying diabetes susceptibility. Although we see no such association here, future studies using larger cohorts are needed before excluding a possible link between AMY2B and diabetes mellitus. PMID:24975239

Capillary isoelectric focusing with whole column imaging detection (iCIEF): A new approach to the characterization and quantification of salivary α-amylase.

PubMed

Zarabadi, Atefeh S; Huang, Tiemin; Mielke, John G

2017-05-15

Saliva is an easily collected biological fluid with potentially important diagnostic value. While gel electrophoresis is generally used for salivary analysis, we employed the capillary isoelectric focusing technique to allow for a rapid, automated mode of electrophoresis. Capillary isoelectric focusing coupled with UV whole column imaging detection (iCIEF) was used to develop a robust protocol to characterize salivary α-amylase collected from various glands. Notably, three sample preparation methods were examined: ultrafiltration, gel-filtration, and starch affinity interaction with salivary amylase. Salivary α-amylase separated into two major peaks before sample treatment; while both filtration methods and starch affinity interaction of salivary amylase enhanced the resolution of isozymes, desalting with gel-filtration displayed the best recovery and the highest resolution of isozymes. Good agreement existed between the observed isoelectric points and the values reported in the literature. In addition, a high level of precision was apparent, and the relative standard deviation for replicates was less than 0.5% for pIs (peak positions) and below 10% for peak area. Furthermore, saliva secreted from the parotid gland proved to have a higher amylase content compared to either secretions from the submandibular/sublingual complex, or whole saliva, as well as amylase enhancement under stimulation. The results suggest that the iCIEF technique can be used to accurately resolve and quantitate amylase isozymes in a rapid and automated fashion, and that gel-filtration should be applied to saliva samples beforehand to allow for optimal purification and characterization. Copyright © 2017 Elsevier B.V. All rights reserved.

Adipokinetic hormones control amylase activity in the cockroach (Periplaneta americana) gut.

PubMed

Bodláková, Karolina; Jedlička, Pavel; Kodrík, Dalibor

2017-04-01

This study examined the biochemical characteristics of α-amylase and hormonal (adipokinetic hormone: AKH) stimulation of α-amylase activity in the cockroach (Periplaneta americana) midgut. We applied two AKHs in vivo and in vitro, then measured resultant amylase activity and gene expression, as well as the expression of AKH receptor (AKHR). The results revealed that optimal amylase activity is characterized by the following: pH: 5.7, temperature: 38.4 °C, K m (Michaelis-Menten constant): 2.54 mg starch/mL, and V max (maximum reaction velocity): 0.185 μmol maltose/mL/min. In vivo application of AKHs resulted in significant increase of amylase activity: by two-fold in the gastric caeca and 4-7 fold in the rest of the midgut. In vitro experiments supported results seen in vivo: a 24-h incubation with the hormones resulted in the increase of amylase activity by 1.4 times in the caeca and 4-9 times in the midgut. Further, gene expression analyses reveal that AKHR is expressed in both the caeca and the rest of the midgut, although expression levels in the former were 23 times higher than levels in the latter. A similar pattern was found for the amylase (AMY) gene. Hormonal treatment did not affect the expression of either gene. This study is the first to provide evidence indicating direct AKH stimulation of digestive enzyme activity in the insect midgut, supported by specific AKHR gene expression in this organ. © 2016 Institute of Zoology, Chinese Academy of Sciences.

One-step production of immobilized alpha-amylase in recombinant Escherichia coli.

PubMed

Rasiah, Indira A; Rehm, Bernd H A

2009-04-01

Industrial enzymes are often immobilized via chemical cross-linking onto solid supports to enhance stability and facilitate repeated use in bioreactors. For starch-degrading enzymes, immobilization usually places constraints on enzymatic conversion due to the limited diffusion of the macromolecular substrate through available supports. This study describes the one-step immobilization of a highly thermostable alpha-amylase (BLA) from Bacillus licheniformis and its functional display on the surface of polyester beads inside engineered Escherichia coli. An optimized BLA variant (Termamyl) was N-terminally fused to the polyester granule-forming enzyme PhaC of Cupriavidus necator. The fusion protein lacking the signal sequence mediated formation of stable polyester beads exhibiting alpha-amylase activity. The alpha-amylase beads were assessed with respect to alpha-amylase activity, which was demonstrated qualitatively and quantitatively. The immobilized alpha-amylase showed Michaelis-Menten enzyme kinetics exerting a V(max) of about 506 mU/mg of bead protein with a K(m) of about 5 microM, consistent with that of free alpha-amylase. The stability of the enzyme at 85 degrees C and the capacity for repeated usage in a starch liquefaction process were also demonstrated. In addition, structural integrity and functionality of the beads at extremes of pH and temperature, demonstrating their suitability for industrial use, were confirmed by electron microscopy and protein/enzyme analysis. This study proposes a novel, cost-effective method for the production of immobilized alpha-amylase in a single step by using the polyester granules forming protein PhaC as a fusion partner in engineered E. coli.

One-Step Production of Immobilized α-Amylase in Recombinant Escherichia coli▿ †

PubMed Central

Rasiah, Indira A.; Rehm, Bernd H. A.

2009-01-01

Industrial enzymes are often immobilized via chemical cross-linking onto solid supports to enhance stability and facilitate repeated use in bioreactors. For starch-degrading enzymes, immobilization usually places constraints on enzymatic conversion due to the limited diffusion of the macromolecular substrate through available supports. This study describes the one-step immobilization of a highly thermostable α-amylase (BLA) from Bacillus licheniformis and its functional display on the surface of polyester beads inside engineered Escherichia coli. An optimized BLA variant (Termamyl) was N-terminally fused to the polyester granule-forming enzyme PhaC of Cupriavidus necator. The fusion protein lacking the signal sequence mediated formation of stable polyester beads exhibiting α-amylase activity. The α-amylase beads were assessed with respect to α-amylase activity, which was demonstrated qualitatively and quantitatively. The immobilized α-amylase showed Michaelis-Menten enzyme kinetics exerting a Vmax of about 506 mU/mg of bead protein with a Km of about 5 μM, consistent with that of free α-amylase. The stability of the enzyme at 85°C and the capacity for repeated usage in a starch liquefaction process were also demonstrated. In addition, structural integrity and functionality of the beads at extremes of pH and temperature, demonstrating their suitability for industrial use, were confirmed by electron microscopy and protein/enzyme analysis. This study proposes a novel, cost-effective method for the production of immobilized α-amylase in a single step by using the polyester granules forming protein PhaC as a fusion partner in engineered E. coli. PMID:19201981

Hand-held monitor of sympathetic nervous system using salivary amylase activity and its validation by driver fatigue assessment.

PubMed

Yamaguchi, Masaki; Deguchi, Mitsuo; Wakasugi, Junichi; Ono, Shin; Takai, Noriyasu; Higashi, Tomoyuki; Mizuno, Yasufumi

2006-01-15

In order to realize a hand-held monitor of the sympathetic nervous system, we fabricated a completely automated analytical system for salivary amylase activity using a dry-chemistry system. This was made possible by the fabrication of a disposable test-strip equipped with built-in collecting and reagent papers and an automatic saliva transfer device. In order to cancel out the effects of variations in environmental temperature and pH of saliva, temperature- and pH-adjusted equations were experimentally determined, and each theoretical value was input into the memory of the hand-held monitor. Within a range of salivary amylase activity between 10 and 140 kU/l, the calibration curve for the hand-held monitor showed a coefficient with R(2)=0.97. Accordingly, it was demonstrated that the hand-held monitor enabled a user to automatically measure the salivary amylase activity with high accuracy with only 30 microl sample of saliva within a minute from collection to completion of the measurement. In order to make individual variations of salivary amylase activity negligible during driver fatigue assessment, a normalized equation was proposed. The normalized salivary amylase activity correlated with the mental and physical fatigue states. Thus, this study demonstrated that an excellent hand-held monitor with an algorithm for normalization of individuals' differences in salivary amylase activity, which could be easily and quickly used for evaluating the activity of the sympathetic nervous system at any time. Furthermore, it is suggested that the salivary amylase activity might be used as a better index for psychological research.

Structure of waxy maize starch hydrolyzed by maltogenic alpha-amylase in relation to its retrogradation

USDA-ARS?s Scientific Manuscript database

Maltogenic a-amylase is widely used as an antistaling agent in bakery foods. The objective of this study was to determine the degree of hydrolysis (DH) and starch structure after maltogenic amylase treatments in relation to its retrogradation. Waxy maize starch was cooked and hydrolyzed to different...

Development of an industrial method to quantitatively measure carry-over amylase activity in raw and refined sugars

USDA-ARS?s Scientific Manuscript database

In recent years, there has been increased concern over carry-over activity of mostly high temperature (HT) and very high temperature (VHT) stable amylases in white, refined sugars from refineries to various food manufacturing industries and other end-users. HT and VHT stable amylases were developed...

Enhanced α-amylase production by a marine protist, Ulkenia sp. using response surface methodology and genetic algorithm.

PubMed

Shirodkar, Priyanka V; Muraleedharan, Usha Devi

2017-11-26

Amylases are a group of enzymes with a wide variety of industrial applications. Enhancement of α-amylase production from the marine protists, thraustochytrids has been attempted for the first time by applying statistical-based experimental designs using response surface methodology (RSM) and genetic algorithm (GA) for optimization of the most influencing process variables. A full factorial central composite experimental design was used to study the cumulative interactive effect of nutritional components viz., glucose, corn starch, and yeast extract. RSM was performed on two objectives, that is, growth of Ulkenia sp. AH-2 (ATCC® PRA-296) and α-amylase activity. When GA was conducted for maximization of the enzyme activity, the optimal α-amylase activity was found to be 71.20 U/mL which was close to that obtained by RSM (71.93 U/mL), both of which were in agreement with the predicted value of 72.37 U/mL. Optimal growth at the optimized process variables was found to be 1.89A 660nm . The optimized medium increased α-amylase production by 1.2-fold.

α-Glucosidase and α-amylase inhibitors from seed oil: A review of liposoluble substance to treat diabetes.

PubMed

Teng, Hui; Chen, Lei

2017-11-02

One of the effective managements of diabetes mellitus, in particular, noninsulin-dependent diabetes mellitus, is to retard the absorption of glucose by inhibition of carbohydrate hydrolyzing enzymes, such as α-glucosidase and α-amylase, in the digestive organs. Currently, there is renewed interest in plant-based medicines and functional foods modulating physiological effects in the inhibition of α-glucosidase and α-amylase. Accordingly, inhibitors of α-glucosidase or α-amylase derived from various sources have also been isolated, and majority of phenolic compounds and their effects have been investigated in animals as well. As such, when the presence of α-glucosidase inhibitor in many foodstuffs was screened for, we found that vegetable seed oil also strongly inhibited α-glucosidase and α-amylase. Seed oil is an important source of liposoluble constituents with potential for inhibition of these enzymes, hence can also be used as therapeutic or functional food sources. Therefore, this review is aimed at highlighting the main liposoluble classes of α-glucosidase and α-amylase inhibitors, but it is not intended to be an exhaustive review on the subject.

Macromolecular cross-linked enzyme aggregates (M-CLEAs) of α-amylase.

PubMed

Nadar, Shamraja S; Muley, Abhijeet B; Ladole, Mayur R; Joshi, Pranoti U

2016-03-01

Macromolecular cross-linked enzyme aggregates (M-CLEAs) of α-amylase were prepared by precipitation and subsequent cross-linking. The non-toxic, biodegradable, biocompatible, renewable polysaccharide based macromolecular cross-linkers viz. agar, chitosan, dextran, and gum arabic were used as a substitute for traditional glutaraldehyde to augment activity recovery toward macromolecular substrate. Macromolecular cross-linkers were prepared by periodate mediated controlled oxidation of polysaccharides. The effects of precipitating agent, concentration and different cross-linkers on activity recovery of α-amylase CLEAs were investigated. α-Amylase aggregated with ammonium sulphate and cross-linked by dextran showed 91% activity recovery, whereas glutaraldehyde CLEAs (G-CLEAs) exhibited 42% activity recovery. M-CLEAs exhibited higher thermal stability in correlation with α-amylase and G-CLEAs. Moreover, dextran and chitosan M-CLEAs showed same affinity for starch hydrolysis as of free α-amylase. The changes in secondary structures revealed the enhancements in structural and conformational rigidity attributed by cross-linkers. Finally, after five consecutive cycles dextran M-CLEAs retained 1.25 times higher initial activity than G-CLEAs. Copyright © 2015 Elsevier B.V. All rights reserved.

Purification and characterization of midgut α-amylase in a predatory bug, Andralus spinidens

PubMed Central

Sorkhabi-Abdolmaleki, Sahar; Zibaee, Arash; Hoda, Hassan; FazeliDinan, Mahmoud

2014-01-01

Abstract α-Amylases are widespread enzymes that catalyze endohydrolysis of long α-1,4-glucan chains such as starch and glycogen. The highest amylolytic activity was found in 5th instar nymphs and midgut of the predatory bug, Andrallus spinidens F. (Hemiptera: Pentatomidae). The α-amylase was purified following a three-step procedure. The purified α-amylase had a specific activity of 13.46 U/mg protein, recovery of 4.21, purification fold of 13.87, and molecular weight of 21.3 kDa. The enzyme had optimal pH and temperature of 7 and 45°C, respectively. Na+, Mn+, Mg2+, and Zn2+ significantly decreased activity of the purified α-amylase, but some concentrations of K+, Ca2+, and Cu2+ had the opposite effect. EDTA, EGTA, and DTC significantly decreased enzymatic activity, showing the presence of metal ions in the catalytic site of the enzyme. Kinetic parameters of the purified α-amylase showed a Km of 3.71% in starch and 4.96% for glycogen, suggesting that the enzyme had a higher affinity for starch. PMID:25373212

Impact of α-amylase during breadmaking on in vitro kinetics of starch hydrolysis and glycaemic index of enriched bread with bran.

PubMed

Sanz-Penella, Juan Mario; Laparra, José Moisés; Haros, Monika

2014-09-01

Nowadays, the use of enzymes has become a common practice in the bakery industry, as they can improve dough quality and texture of final product. However, the use of α-amylases could have a negative effect in the glycaemic load of product, due to the released sugars from the starch hydrolysis that are not used by yeasts during the fermentation process. This study evaluated the effect of the addition of α-amylase in bakery products with bran on in vitro kinetics of starch hydrolysis. The use of flour with a high degree of extraction or high bran amount could decrease the GI even with the inclusion of α-amylase in the formulation. It should be taken into account the amount of bran and α-amylase when formulating breads in order to obtain products with lower GI than white bread. However, the fact that kinetics of starch hydrolysis remained unaltered indicates that the use of α-amylase in bread-making processes could provide technological advantages improving quality of breads without markedly changes in their glycaemic index.

Synthesis of a Possible Precursor of α-Amylase in Wheat Aleurone Cells 1

PubMed Central

Okita, Thomas W.; Decaleya, Roberto; Rappaport, Lawrence

1979-01-01

α-Amylase from wheat aleurone (Triticum aestivum) was synthesized in a S-150 wheat germ readout system using polysomes, and a messenger RNA-dependent reticulocyte lysate system using polyadenylic acid [poly(A)]-enriched RNA. The product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, precipitation by specific λ-globulin for α-amylase, and proteolysis. Two immunoprecipitated products were synthesized from the readout system, the predominant species migrating coincidentally with authentic α-amylase on sodium dodecyl sulfate-polyacrylamide gels. A putative precursor, 1,500 daltons larger, was evident but was less abundant. The relationship between the two polypeptides was established by proteolytic analysis using Staphylococcus aureus V8 protease. At least nine fragments were generated and were identical in both species. The poly(A)-enriched RNA synthesized only the putative precursor in the reticulocyte lysate system. Attempts to process the precursor to the mature size of α-amylase failed. These findings are discussed in connection with the signal hypothesis (proposed for the transport of proteins across membranes) and the mode of secretion of α-amylase in aleurone cells. Images PMID:16660677

In vitro α -amylase and α-glucosidase inhibitory potential of Trigonella foenum-graecum leaves extract

PubMed Central

Ganeshpurkar, Aditya; Diwedi, Varsha; Bhardwaj, Yash

2013-01-01

Trigonella foenum-graecum is one of the widely used herbs in food and medicine. The seeds of the plants are investigated for antidiabetic potential; however, no efforts have been done to explore the potential of leaves to modify carbohydrate metabolizing enzymes viz. α-amylase and α-glucosidase. The present work was designed to investigate the inhibitory potential of ethyl acetate and water extract of T. foenum-graecum on enzymes α-amylase and α-glucosidase. Different concentrations of extracts were used to study inhibition of enzymatic activity of α-amylase and α-glucosidase. A dose dependent inhibitory effect on enzymes was observed. The current study, for the first time, revealed α-amylase and α-glucosidase inhibitory potential of T. foenum-graecum and the study could be helpful to isolate and characterize compounds responsible for it. PMID:24049415

Analysis of the extreme diversity of salivary alpha-amylase isoforms generated by physiological proteolysis using liquid chromatography-tandem mass spectrometry.

PubMed

Bailey, Ulla-Maja; Punyadeera, Chamindie; Cooper-White, Justin J; Schulz, Benjamin L

2012-12-12

Saliva is a crucial biofluid for oral health and is also of increasing importance as a non-invasive source of disease biomarkers. Salivary alpha-amylase is an abundant protein in saliva, and changes in amylase expression have been previously associated with a variety of diseases and conditions. Salivary alpha-amylase is subject to a high diversity of post-translational modifications, including physiological proteolysis in the oral cavity. Here we developed methodology for rapid sample preparation and non-targeted LC-ESI-MS/MS analysis of saliva from healthy subjects and observed an extreme diversity of alpha-amylase proteolytic isoforms. Our results emphasize the importance of consideration of post-translational events such as proteolysis in proteomic studies, biomarker discovery and validation, particularly in saliva. Copyright © 2012 Elsevier B.V. All rights reserved.

Distinct mechanism of activation of two transcription factors, AmyR and MalR, involved in amylolytic enzyme production in Aspergillus oryzae.

PubMed

Suzuki, Kuta; Tanaka, Mizuki; Konno, Yui; Ichikawa, Takanori; Ichinose, Sakurako; Hasegawa-Shiro, Sachiko; Shintani, Takahiro; Gomi, Katsuya

2015-02-01

The production of amylolytic enzymes in Aspergillus oryzae is induced in the presence of starch or maltose, and two Zn2Cys6-type transcription factors, AmyR and MalR, are involved in this regulation. AmyR directly regulates the expression of amylase genes, and MalR controls the expression of maltose-utilizing (MAL) cluster genes. Deletion of malR gene resulted in poor growth on starch medium and reduction in α-amylase production level. To elucidate the activation mechanisms of these two transcription factors in amylase production, the expression profiles of amylases and MAL cluster genes under carbon catabolite derepression condition and subcellular localization of these transcription factors fused with a green fluorescent protein (GFP) were examined. Glucose, maltose, and isomaltose induced the expression of amylase genes, and GFP-AmyR was translocated from the cytoplasm to nucleus after the addition of these sugars. Rapid induction of amylase gene expression and nuclear localization of GFP-AmyR by isomaltose suggested that this sugar was the strongest inducer for AmyR activation. In contrast, GFP-MalR was constitutively localized in the nucleus and the expression of MAL cluster genes was induced by maltose, but not by glucose or isomaltose. In the presence of maltose, the expression of amylase genes was preceded by MAL cluster gene expression. Furthermore, deletion of the malR gene resulted in a significant decrease in the α-amylase activity induced by maltose, but had apparently no effect on the expression of α-amylase genes in the presence of isomaltose. These results suggested that activation of AmyR and MalR is regulated in a different manner, and the preceding activation of MalR is essential for the utilization of maltose as an inducer for AmyR activation.

Biotechnological Processes in Microbial Amylase Production

PubMed Central

Arshad, M. K. Md; Lakshmipriya, Thangavel; Hashim, Uda; Chinni, Suresh V.

2017-01-01

Amylase is an important and indispensable enzyme that plays a pivotal role in the field of biotechnology. It is produced mainly from microbial sources and is used in many industries. Industrial sectors with top-down and bottom-up approaches are currently focusing on improving microbial amylase production levels by implementing bioengineering technologies. The further support of energy consumption studies, such as those on thermodynamics, pinch technology, and environment-friendly technologies, has hastened the large-scale production of the enzyme. Herein, the importance of microbial (bacteria and fungi) amylase is discussed along with its production methods from the laboratory to industrial scales. PMID:28280725

Biotechnological Processes in Microbial Amylase Production.

PubMed

Gopinath, Subash C B; Anbu, Periasamy; Arshad, M K Md; Lakshmipriya, Thangavel; Voon, Chun Hong; Hashim, Uda; Chinni, Suresh V

2017-01-01

Amylase is an important and indispensable enzyme that plays a pivotal role in the field of biotechnology. It is produced mainly from microbial sources and is used in many industries. Industrial sectors with top-down and bottom-up approaches are currently focusing on improving microbial amylase production levels by implementing bioengineering technologies. The further support of energy consumption studies, such as those on thermodynamics, pinch technology, and environment-friendly technologies, has hastened the large-scale production of the enzyme. Herein, the importance of microbial (bacteria and fungi) amylase is discussed along with its production methods from the laboratory to industrial scales.

Characterization of salivary alpha-amylase binding to Streptococcus sanguis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scannapieco, F.A.; Bergey, E.J.; Reddy, M.S.

1989-09-01

The purpose of this study was to identify the major salivary components which interact with oral bacteria and to determine the mechanism(s) responsible for their binding to the bacterial surface. Strains of Streptococcus sanguis, Streptococcus mitis, Streptococcus mutans, and Actinomyces viscosus were incubated for 2 h in freshly collected human submandibular-sublingual saliva (HSMSL) or parotid saliva (HPS), and bound salivary components were eluted with 2% sodium dodecyl sulfate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer, alpha-amylase was the prominent salivary component eluted from S. sanguis. Studies with {sup 125}I-labeled HSMSL or {sup 125}I-labeled HPS also demonstrated a componentmore » with an electrophoretic mobility identical to that of alpha-amylase which bound to S. sanguis. Purified alpha-amylase from human parotid saliva was radiolabeled and found to bind to strains of S. sanguis genotypes 1 and 3 and S. mitis genotype 2, but not to strains of other species of oral bacteria. Binding of ({sup 125}I)alpha-amylase to streptococci was saturable, calcium independent, and inhibitable by excess unlabeled alpha-amylases from a variety of sources, but not by secretory immunoglobulin A and the proline-rich glycoprotein from HPS. Reduced and alkylated alpha-amylase lost enzymatic and bacterial binding activities. Binding was inhibited by incubation with maltotriose, maltooligosaccharides, limit dextrins, and starch.« less

Molecular Identification of a Newly Isolated Bacillus subtilis BI19 and Optimization of Production Conditions for Enhanced Production of Extracellular Amylase

PubMed Central

Dash, Biplab Kumar; Rahman, M. Mizanur; Sarker, Palash Kumar

2015-01-01

A study was carried out with a newly isolated bacterial strain yielding extracellular amylase. The phylogenetic tree constructed on the basis of 16S rDNA gene sequences revealed this strain as clustered with the closest members of Bacillus sp. and identified as Bacillus subtilis BI19. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Rice flour (1.25%) as a cheap natural carbon source was found to induce amylase production mostly. A combination of peptone and tryptone as organic and ammonium sulfate as inorganic nitrogen sources gave highest yield. Maximum production was obtained after 24 h of incubation at 37°C with an initial medium pH 8.0. Addition of surfactants like Tween 80 (0.25 g/L) and sodium lauryl sulfate (0.2 g/L) resulted in 28% and 15% increase in enzyme production, respectively. Amylase production was 3.06 times higher when optimized production conditions were used. Optimum reaction temperature and pH for crude amylase activity were 50°C and 6.0, respectively. The crude enzyme showed activity and stability over a fair range of temperature and pH. These results suggest that B. subtilis BI19 could be exploited for production of amylase at relatively low cost and time. PMID:26180814

Spatio-temporal appearance of α-amylase and limit dextrinase in barley aleurone layer in response to gibberellic acid, abscisic acid and salicylic acid.

PubMed

Shahpiri, Azar; Talaei, Nasim; Finnie, Christine

2015-01-01

Cereal seed germination involves mobilization of storage reserves in the starchy endosperm to support seedling growth. In response to gibberellin produced by the embryo the aleurone layer synthesizes hydrolases that are secreted to the endosperm for degradation of storage products. In this study analysis of intracellular protein accumulation and release from barley aleurone layers is presented for the important enzymes in starch degradation: α-amylase and limit dextrinase (LD). Proteins were visualized by immunoblotting in aleurone layers and culture supernatants from dissected aleurone layers incubated up to 72 h with either gibberellic acid (GA), abscisic acid (ABA) or salicylic acid (SA). The results show that α-amylase is secreted from aleurone layer treated with GA soon after synthesis but the release of LD to culture supernatants was significantly delayed and coincided with a general loss of proteins from aleurone layers. Release of LD was found to differ from that of amylase and was suggested to depend on programmed cell death (PCD). Despite detection of intracellular amylase in untreated aleurone layers or aleurone layers treated with ABA or SA, α-amylase was not released from these samples. Nevertheless, the release of α-amylase was observed from aleurone layers treated with GA+ABA or GA+SA. © 2014 Society of Chemical Industry.

Patterns of cortisol and alpha-amylase reactivity to psychosocial stress in maltreated women.

PubMed

Mielock, Alyssa S; Morris, Matthew C; Rao, Uma

2017-02-01

Childhood maltreatment can trigger enduring changes in major stress response systems, particularly in the context of major depressive disorder (MDD). However, the relative impact of maltreatment versus MDD on hypothalamic-pituitary-adrenal axis and sympathetic-adrenal-medullary system stress reactivity is not well understood. This study examined salivary cortisol and alpha-amylase responses to the Trier Social Stress Test (TSST) in 26 maltreated (15 with current MDD) and 26 non-maltreated (17 with current MDD) women. Maltreated women showed greater anticipatory cortisol reactivity during the TSST protocol compared to non-maltreated women. Maltreated women also showed rapid deceleration in cortisol levels. Whereas non-maltreated women showed initial declines in alpha-amylase levels but rapidly increasing alpha-amylase levels during the TSST protocol, maltreated women did not exhibit changes in alpha-amylase levels during the TSST protocol. Contrary to expectation, MDD did not impact cortisol or alpha-amylase responses. The present study is limited by retrospective report of childhood maltreatment, cross-sectional design, and modest sample sizes. These findings suggest that childhood maltreatment plays a greater role driving alterations in cortisol and alpha-amylase stress reactivity than MDD. Understanding the biological embedding of maltreatment is critical for elucidating mechanisms linking these experiences to risk for negative mental and physical health outcomes. Copyright © 2016 Elsevier B.V. All rights reserved.

Immobilization of α-amylase onto a calix[4]arene derivative: Evaluation of its enzymatic activity.

PubMed

Veesar, Irshad Ali; Solangi, Imam Bakhsh; Memon, Shahabuddin

2015-06-01

In order to enhance the cost-effectiveness practicability of enzymes in many industries such as pharmaceutical, food, medical and some other technological processes, there is great need to immobilize them onto a solid supports. In this study, a new and efficient immobilization of α-amylase from Saccharomyces cerevisiae has been developed by using the surface functionalization of calix[4]arene as support. A glutaraldehyde-containing amino group functionalized calix[4]arene was used to immobilize α-amylase covalently. In this procedure, imide bonds are formed between amino groups on the protein and aldehyde groups on the calix[4]arene surface. The surface modified support was characterized using Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM). The effect of various preparation conditions on the immobilized α-amylase process such as immobilization time, enzyme concentration, temperature and pH were investigated. The influence of pH and temperature on the activity of free and immobilized α-amylase was also studied using starch as substrate. The optimum reaction temperature and pH value for the enzymatic conversion catalyzed by the immobilized α-amylase were 25°C and 7, respectively. Compared to the free enzyme, the immobilized α-amylase retained 85% of its original activity and exhibited significant thermal stability than the free one and excellent durability. Copyright © 2015 Elsevier Inc. All rights reserved.

Enzymatic activities in different strains isolated from healthy and brittle leaf disease affected date palm leaves: study of amylase production conditions.

PubMed

Mouna, Jrad; Imen, Fendri; Choba Ines, Ben; Nourredine, Drira; Adel, Kadri; Néji, Gharsallah

2015-02-01

The present study aimed to investigate and compare the enzymatic production of endophytic bacteria isolated from healthy and brittle leaf disease affected date palm leaves (pectinase, cellulase, lipase, and amylase). The findings revealed that the enzymatic products from the bacterial isolates of healthy date palm leaves were primarily 33% amylolytic enzyme, 33 % cellulase, 25 % pectinase, and 25 % lipase. The isolates from brittle leaf disease date palm leaves, on the other hand, were noted to produce 16 % amylolytic enzyme, 20 % cellulose, 50 % pectinase, and 50 % lipase. The effects of temperature and pH on amylase, pectinase, and cellulose activities were investigated. The Bacillus subtilis JN934392 strain isolated from healthy date palm leaves produced higher levels of amylase activity at pH 7. A Box Behnken Design (BBD) was employed to optimize amylase extraction. Maximal activity was observed at pH and temperature ranges of pH 6-6.5 and 37-39 °C, respectively. Under those conditions, amylase activity was noted to be attained 9.37 U/ml. The results showed that the enzyme was able to maintain more than 50 % of its activity over a temperature range of 50-80 °C, with an optimum at 70 °C. This bacterial amylase showed high activity compared to other bacteria, which provides support for its promising candidacy for future industrial application.

Encapsulation of alpha-amylase into starch-based biomaterials: an enzymatic approach to tailor their degradation rate.

PubMed

Azevedo, Helena S; Reis, Rui L

2009-10-01

This paper reports the effect of alpha-amylase encapsulation on the degradation rate of a starch-based biomaterial. The encapsulation method consisted in mixing a thermostable alpha-amylase with a blend of corn starch and polycaprolactone (SPCL), which were processed by compression moulding to produce circular disks. The presence of water was avoided to keep the water activity low and consequently to minimize the enzyme activity during the encapsulation process. No degradation of the starch matrix occurred during processing and storage (the encapsulated enzyme remained inactive due to the absence of water), since no significant amount of reducing sugars was detected in solution. After the encapsulation process, the released enzyme activity from the SPCL disks after 28days was found to be 40% comparatively to the free enzyme (unprocessed). Degradation studies on SPCL disks, with alpha-amylase encapsulated or free in solution, showed no significant differences on the degradation behaviour between both conditions. This indicates that alpha-amylase enzyme was successfully encapsulated with almost full retention of its enzymatic activity and the encapsulation of alpha-amylase clearly accelerates the degradation rate of the SPCL disks, when compared with the enzyme-free disks. The results obtained in this work show that degradation kinetics of the starch polymer can be controlled by the amount of encapsulated alpha-amylase into the matrix.

Human Parotid Gland Alpha-Amylase Secretion as a Function of Chronic Hyperbaric Exposure

DTIC Science & Technology

1979-01-01

parotid ...Pullman, WA 99163 Gilman, S. C, G. J. Fischer, R. J. Biersner, R. D. Thornton, and D. A. Miller. 1979. Human parotid gland alpha-amylase secretion...as a function of chronic hyperbaric exposure. Undersea Biomed. Res. 6(3):303-307.—Secretion of a-amylase by the human parotid gland increased

Mechanism of removal of undesirable residual amylase, insoluble starch, and select colorants from refinery streams by powdered activated carbons

USDA-ARS?s Scientific Manuscript database

There is a need in the world-wide sugar industry to find a practical and economical solution to remove or inactivate residual alpha-amylases that are high temperature stable from factory or refinery streams. A survey of refineries that used amylase and had activated carbon systems for decolorization...

General Subject 1. Report to ICUMSA on the determination of commercial alpha-amylase activity by a spectrophotometric method

USDA-ARS?s Scientific Manuscript database

A report is given on a new industrial method for the determination of the activity or strength of commercial alpha-amylase at a sugarcane factory or refinery, as well as a recommendation. At the present time, the activities or strengths of commercial alpha-amylases cannot be directly compared becau...

Influence of media composition on the production of alkaline α-amylase from Bacillus subtilis CB-18.

PubMed

Ogbonnaya, Nwokoro; Odiase, Anthonia

2012-01-01

Starch, a homopolysaccharide is an important and an abundant food reserve and energy source. Starches are processed to yield different products which find many industrial applications. Alpha-amylases hydrolyze starch by cleaving α-1,4-glucosidic bonds and have been used in food, textile and pharmaceutical industries [Sun et al. 2010]. Enzymatic conversion of starch with amylase presents an economically superior alternative to the conventional method of starch gelatinization. Alkaline α-amylase has an important position in the global enzyme market as a constituent of detergent. In this paper, we screened soil bacteria and an isolate, alkalophilic Bacillus subtilis CB-18 was found to produce an alkaline α-amylase in different media. MATERIAL AND METHODS. Screening of the isolates for amylolytic activity was carried out by growing bacteria isolated from the soil in starch agar plates and subsequently staining the plates with iodine solution to reveal zones of hydrolysis of starch. The selected isolate, Bacillus subtlis CB-18 was grown in different media at alkaline pH to evaluate the influence of media composition on alkaline α-amylase production. Enzyme assay was carried out by growing the culture in a broth medium and obtaining cell - free culture supernatant after centrifugation at 2515 × g for 15 minutes Amylase activity was determined by incubating 0.5 ml of crude enzyme solution in 0.1M Tris/HCl buffer (pH 8.5) with 0.5 ml of 1% soluble starch solution. The reaction was terminated by the addition of DNS reagent and reducing sugar produced from the amylolytic reaction was determined. Bacillus subtilis CB-18 used for this work was selected because it produced 7 mm zone diameter on starch agar plate. This organism was cultured in different alkaline broth media containing 2% soluble starch as inducer carbohydrate for α-amylase production. Among the carbon sources used for enzyme production, sorbitol was the best to stimulate enzyme production with α-amylase activity of 758 U/mL after 48 h. Peptone was the best nitrogen source for enzyme production with α-amylase activity of 680 U/mL after 48 h. Metal ions including Ca (2+), Mn(2+) and Mg(2+) stimulated enzyme production while Hg(2+) and Ag(+) repressed enzyme production. The best enzyme yields were observed in basal media containing agro-based substrates. This work reports the production of alkaline α-amylase by Bacillus subtlis CB-18 in different media. Enzyme production was highest when agro-based media were used to formulate the media.

Latent associations of low serum amylase with decreased plasma insulin levels and insulin resistance in asymptomatic middle-aged adults

PubMed Central

2012-01-01

Background Low serum amylase is likely to be associated with obesity and metabolic abnormalities, which are often accompanied by impaired insulin action. However, it is unclear whether low serum amylase is associated with impaired insulin action in clinical settings. Therefore, we investigated the associations of low serum amylase with plasma insulin levels, and obesity-related parameters, including leptin. Research design and methods We measured serum amylase, plasma insulin, obesity-related parameters such as leptin, cardiometabolic risk factors, and anthropometric parameters in a cross-sectional study of 54 asymptomatic subjects (mean age 48.6 ± 7.6 years) who were not being treated for diabetes. Results Body mass index (BMI) and plasma glucose at 120 min after a 75-g oral glucose tolerance test (OGTT) were significantly higher in subjects with low serum amylase (< 60 IU/l, n = 21) than in those with normal-to-high serum amylase (n = 33) (P = 0.04 and P = 0.004, respectively). In univariate correlation analysis, serum amylase was significantly correlated with BMI alone (r = –0.39, P = 0.004). By contrast, multivariate logistic analysis showed that each 1-SD increase in quantitative insulin sensitivity check index, and each 1-SD decrease in plasma insulin OGTT at 0 and 60 min, homeostasis model assessment of insulin resistance (HOMA)-R, and HOMA-β were significantly associated with low serum amylase, particularly after adjusting for BMI. When subjects were divided into three groups according to HOMA-R, serum amylase levels were significantly lower in subjects with HOMA-R > 2.5 (n = 23) compared with subjects with HOMA-R 1.6–2.5 (n = 10) (61.1 ± 13.6 U/ml versus 76.9 ± 20.5 U/ml, Bonferroni test, P = 0.02), but not compared with subjects with HOMA-R<1.6 (n = 21; 62.7 ± 17.6 U/ml). Similar trends were observed when subjects were divided according to plasma leptin and fasting plasma insulin levels. Conclusions These results suggest that after adjusting for BMI, low serum amylase is associated with decreased basal insulin levels and insulin secretion, as well as high insulin resistance. The nature of these associations remains to be elucidated in further studies. PMID:22748134

Chemical constituents of Swertia longifolia Boiss. with α-amylase inhibitory activity.

PubMed

Saeidnia, Soodabeh; Ara, Leila; Hajimehdipoor, Homa; Read, Roger W; Arshadi, Sattar; Nikan, Marjan

2016-01-01

α-Amylase inhibitors play a critical role in the control of diabetes and many of medicinal plants have been found to act as α-amylase inhibitors. Swertia genus, belonging to the family Gentianaceae, comprises different species most of which have been used in traditional medicine of several cultures as antidiabetic, anti-pyretic, analgesic, liver and gastrointestinal tonic. Swertia longifolia Boiss. is the only species of Swertia growing in Iran. In the present investigation, phytochemical study of S. longifolia was performed and α-amylase inhibitory effects of the plant fractions and purified compounds were determined. Aerial parts of the plant were extracted with hexane, chloroform, methanol and water, respectively. The components of the hexane and chloroform fractions were isolated by different chromatographic methods and their structures were determined by (1)H NMR and (13)C NMR data. α-Amylase inhibitory activity was determined by a colorimetric assay using 3,5-dinitro salysilic acid. During phytochemical examination, α-amyrin, β-amyrin and β-sitosterol were purified from the hexane fraction, while ursolic acid, daucosterol and swertiamarin were isolated from chloroform fraction. The results of the biochemical assay revealed α-amylase inhibitory activity of hexane, chloroform, methanol and water fractions, of which the chloroform and methanol fractions were more potent (IC50 16.8 and 18.1 mg/ml, respectively). Among examined compounds, daucosterol was found to be the most potent α-amylase inhibitor (57.5% in concentration 10 mg/ml). With regard to α-amylase inhibitory effects of the plant extracts, purified constituents, and antidiabetic application of the species of Swertia genus in traditional medicine of different countries, S. longifolia seems more appropriate species for further mechanistic antidiabetic evaluations.

Chemical constituents of Swertia longifolia Boiss. with α-amylase inhibitory activity

PubMed Central

Saeidnia, Soodabeh; Ara, Leila; Hajimehdipoor, Homa; Read, Roger W.; Arshadi, Sattar; Nikan, Marjan

2016-01-01

α-Amylase inhibitors play a critical role in the control of diabetes and many of medicinal plants have been found to act as α-amylase inhibitors. Swertia genus, belonging to the family Gentianaceae, comprises different species most of which have been used in traditional medicine of several cultures as antidiabetic, anti-pyretic, analgesic, liver and gastrointestinal tonic. Swertia longifolia Boiss. is the only species of Swertia growing in Iran. In the present investigation, phytochemical study of S. longifolia was performed and α-amylase inhibitory effects of the plant fractions and purified compounds were determined. Aerial parts of the plant were extracted with hexane, chloroform, methanol and water, respectively. The components of the hexane and chloroform fractions were isolated by different chromatographic methods and their structures were determined by 1H NMR and 13C NMR data. α-Amylase inhibitory activity was determined by a colorimetric assay using 3,5-dinitro salysilic acid. During phytochemical examination, α-amyrin, β-amyrin and β-sitosterol were purified from the hexane fraction, while ursolic acid, daucosterol and swertiamarin were isolated from chloroform fraction. The results of the biochemical assay revealed α-amylase inhibitory activity of hexane, chloroform, methanol and water fractions, of which the chloroform and methanol fractions were more potent (IC50 16.8 and 18.1 mg/ml, respectively). Among examined compounds, daucosterol was found to be the most potent α-amylase inhibitor (57.5% in concentration 10 mg/ml). With regard to α-amylase inhibitory effects of the plant extracts, purified constituents, and antidiabetic application of the species of Swertia genus in traditional medicine of different countries, S. longifolia seems more appropriate species for further mechanistic antidiabetic evaluations. PMID:27051429

Genomic and functional characterization of coleopteran insect-specific α-amylase inhibitor gene from Amaranthus species.

PubMed

Bhide, Amey J; Channale, Sonal M; Yadav, Yashpal; Bhattacharjee, Kabita; Pawar, Pankaj K; Maheshwari, V L; Gupta, Vidya S; Ramasamy, Sureshkumar; Giri, Ashok P

2017-06-01

The smallest 32 amino acid α-amylase inhibitor from Amaranthus hypochondriacus (AAI) is reported. The complete gene of pre-protein (AhAI) encoding a 26 amino acid (aa) signal peptide followed by the 43 aa region and the previously identified 32 aa peptide was cloned successfully. Three cysteine residues and one disulfide bond conserved within known α-amylase inhibitors were present in AhAI. Identical genomic and open reading frame was found to be present in close relatives of A. hypochondriacus namely Amaranthus paniculatus, Achyranthes aspera and Celosia argentea. Interestingly, the 3'UTR of AhAI varied in these species. The highest expression of AhAI was observed in A. hypochondriacus inflorescence; however, it was not detected in the seed. We hypothesized that the inhibitor expressed in leaves and inflorescence might be transported to the seeds. Sub-cellular localization studies clearly indicated the involvement of AhAI signal peptide in extracellular secretion. Full length rAhAI showed differential inhibition against α-amylases from human, insects, fungi and bacteria. Particularly, α-amylases from Helicoverpa armigera (Lepidoptera) were not inhibited by AhAI while Tribolium castaneum and Callosobruchus chinensis (Coleoptera) α-amylases were completely inhibited. Molecular docking of AhAI revealed tighter interactions with active site residues of T. castaneum α-amylase compared to C. chinensis α-amylase, which could be the rationale behind the disparity in their IC 50 . Normal growth, development and adult emergence of C. chinensis were hampered after feeding on rAhAI. Altogether, the ability of AhAI to affect the growth of C. chinensis demonstrated its potential as an efficient bio-control agent, especially against stored grain pests.

Arabidopsis thaliana AMY3 is a unique redox-regulated chloroplastic α-amylase.

PubMed

Seung, David; Thalmann, Matthias; Sparla, Francesca; Abou Hachem, Maher; Lee, Sang Kyu; Issakidis-Bourguet, Emmanuelle; Svensson, Birte; Zeeman, Samuel C; Santelia, Diana

2013-11-22

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 (Asp(666)), 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 Cys(499) and Cys(587) is central to this regulation. This work provides new insights into how α-amylase activity may be regulated in the chloroplast.

Bioassay-guided fractionation and identification of α-amylase inhibitors from Syzygium cumini leaves.

PubMed

Poongunran, Jeyakumaran; Perera, Handunge Kumudu Irani; Jayasinghe, Lalith; Fernando, Irushika Thushari; Sivakanesan, Ramaiah; Araya, Hiroshi; Fujimoto, Yoshinori

2017-12-01

Pancreatic α-amylase and α-glucosidase inhibitors serve as important strategies in the management of blood glucose. Even though Syzygium cumini (L.) Skeels (Myrtaceae) (SC) is used extensively to treat diabetes; scientific evidence on antidiabetic effects of SC leaves is scarce. SC leaf extract was investigated for α-amylase inhibitory effect and continued with isolation and identification of α-amylase inhibitors. Bioassay-guided fractionation was conducted using in vitro α-amylase inhibitory assay (with 20-1000 μg/mL test material) to isolate the inhibitory compounds from ethyl acetate extract of SC leaves. Structures of the isolated inhibitory compounds were elucidated using 1 H NMR and 13 C NMR spectroscopic analysis and direct TLC and HPLC comparison with authentic samples. Study period was from October 2013 to October 2015. An active fraction obtained with chromatographic separation of the extract inhibited porcine pancreatic α-amylase with an IC 50 of 39.9 μg/mL. Furthermore, it showed a strong inhibition on α-glucosidase with an IC 50 of 28.2 μg/mL. The active fraction was determined to be a 3:1 mixture of ursolic acid and oleanolic acid. Pure ursolic acid and oleanolic acid showed IC 50 values of 6.7 and 57.4 μg/mL, respectively, against α-amylase and 3.1 and 44.1 μg/mL respectively, against α-glucosidase. The present study revealed strong α-amylase and α-glucosidase inhibitory effects of ursolic acid and oleanolic acid isolated from SC leaves for the first time validating the use of SC leaves in antidiabetic therapy.

Amylase, lipase, and volume of drainage fluid in gastrectomy for the early detection of complications caused by pancreatic leakage.

PubMed

Seo, Kyung Won; Yoon, Ki Young; Lee, Sang Ho; Shin, Yeon Myung; Choi, Kyung Hyun; Hwang, Hyun Yong

2011-12-01

Pancreatic leakage is a serious complication of gastrectomy due to stomach cancer. Therefore, we analyzed amylase and lipase concentrations in blood and drainage fluid, and evaluated the volume of drainage fluid to discern their usefulness as markers for the early detection of serious pancreatic leakage requiring reoperation after gastrectomy. From January 2001 to December 2007, we retrospectively analyzed data from 24,072 patient samples. We divided patients into two groups; 1) complications with pancreatic leakage (CG), and 2) no complications associated with pancreatic leakage (NCG). Values of amylase and lipase in the blood and drainage fluid, volume of the drainage fluid, and relationships among the volumes, amylase values, and lipase values in the drainage fluid were evaluated, respectively in the two groups. The mean amylase values of CG were significantly higher than those of NCG in blood and drainage fluid (P < 0.05). For lipase, statistically significant differences were observed in drainage fluid (P < 0.05). The mean volume (standard deviation) of the drained fluid through the tube between CG (n = 22) and NCG (n = 236) on postoperative day 1 were 368.41 (266.25) and 299.26 (300.28), respectively. There were no statistically significant differences between the groups (P = 0.298). There was a correlation between the amylase and lipase values in the drainage fluid (r = 0.812, P = 0.000). Among postoperative amylase and lipase values in blood and drainage fluid, and the volume of drainage fluid, the amylase in drainage fluid was better differentiated between CG and NCG than other markers. The volume of the drainage fluid did not differ significantly between groups.

Diosgenin from Dioscorea bulbifera: novel hit for treatment of type II diabetes mellitus with inhibitory activity against α-amylase and α-glucosidase.

PubMed

Ghosh, Sougata; More, Piyush; Derle, Abhishek; Patil, Ajay B; Markad, Pramod; Asok, Adersh; Kumbhar, Navanath; Shaikh, Mahemud L; Ramanamurthy, Boppana; Shinde, Vaishali S; Dhavale, Dilip D; Chopade, Balu A

2014-01-01

Diabetes mellitus is a multifactorial metabolic disease characterized by post-prandial hyperglycemia (PPHG). α-amylase and α-glucosidase inhibitors aim to explore novel therapeutic agents. Herein we report the promises of Dioscorea bulbifera and its bioactive principle, diosgenin as novel α-amylase and α-glucosidase inhibitor. Among petroleum ether, ethyl acetate, methanol and 70% ethanol (v/v) extracts of bulbs of D. bulbifera, ethyl acetate extract showed highest inhibition upto 72.06 ± 0.51% and 82.64 ± 2.32% against α-amylase and α-glucosidase respectively. GC-TOF-MS analysis of ethyl acetate extract indicated presence of high diosgenin content. Diosgenin was isolated and identified by FTIR, 1H NMR and 13C NMR and confirmed by HPLC which showed an α-amylase and α-glucosidase inhibition upto 70.94 ± 1.24% and 81.71 ± 3.39%, respectively. Kinetic studies confirmed the uncompetitive mode of binding of diosgenin to α-amylase indicated by lowering of both Km and Vm. Interaction studies revealed the quenching of intrinsic fluorescence of α-amylase in presence of diosgenin. Similarly, circular dichroism spectrometry showed diminished negative humped peaks at 208 nm and 222 nm. Molecular docking indicated hydrogen bonding between carboxyl group of Asp300, while hydrophobic interactions between Tyr62, Trp58, Trp59, Val163, His305 and Gln63 residues of α-amylase. Diosgenin interacted with two catalytic residues (Asp352 and Glu411) from α-glucosidase. This is the first report of its kind that provides an intense scientific rationale for use of diosgenin as novel drug candidate for type II diabetes mellitus.

Mammalian mucosal α-glucosidases coordinate with α-amylase in the initial starch hydrolysis stage to have a role in starch digestion beyond glucogenesis.

PubMed

Dhital, Sushil; Lin, Amy Hui-Mei; Hamaker, Bruce R; Gidley, Michael J; Muniandy, Anbuhkani

2013-01-01

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.

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

PubMed Central

Dhital, Sushil; Lin, Amy Hui-Mei; Hamaker, Bruce R.; Gidley, Michael J.; Muniandy, Anbuhkani

2013-01-01

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. PMID:23638112

Structural elucidation and molecular characterization of Marinobacter sp. α-amylase.

PubMed

Kumar, Sumit; Khan, Rizwan Hasan; Khare, S K

2016-01-01

Halophiles have been perceived as potential source of novel enzymes in recent years. The interest emanates from their ability to catalyze efficiently under high salt and organic solvents. Marinobacter sp. EMB8 α-amylase was found to be active and stable in salt and organic solvents. A study was carried out using circular dichroism (CD), fluorescence spectroscopy, and bioinformatics analysis of similar protein sequence to ascertain molecular basis of salt and solvent adaptability of α-amylase. Structural changes recorded in the presence of varying amounts of NaCl exhibited an increase in negative ellipticity as a function of salt, confirming that salt stabilizes the protein and increases the secondary structure, making it catalytically functional. The data of intrinsic and extrinsic fluorescence (using 1-anilinonaphthalene 8-sulfonate [ANS] as probe) further confirmed the role of salt. The α-amylase was active in the presence of nonpolar solvents, namely, hexane and decane, but inactivated by ethanol. The decrease in the activity was correlated with the loss of tertiary structure in the presence of ethanol. Guanidine hydrochloride and pH denaturation indicated the molten globule state at pH 4.0. Partial N-terminal amino acid sequence of the purified α-amylase revealed the relatedness to Pseudoalteromonas sp. α-amylase. "FVHLFEW" was found as the N-terminal signature sequence. Bioinformatics analysis was done using M. algicola α-amylase protein having the same N-terminal signature sequence. The three-dimensional structure of Marinobacter α-amylase was deduced using the I-TASSER server, which reflected the enrichment of acidic amino acids on the surface, imparting the stability in the presence of salt. Our study clearly indicate that salt is necessary for maintaining the secondary and tertiary structure of halophilic protein, which is a necessary prerequisite for catalysis.

Dynamics of the Streptococcus gordonii Transcriptome in Response to Medium, Salivary α-Amylase, and Starch

PubMed Central

Haase, Elaine M.; Feng, Xianghui; Pan, Jiachuan; Miecznikowski, Jeffrey C.

2015-01-01

Streptococcus gordonii, a primary colonizer of the tooth surface, interacts with salivary α-amylase via amylase-binding protein A (AbpA). This enzyme hydrolyzes starch to glucose, maltose, and maltodextrins that can be utilized by various oral bacteria for nutrition. Microarray studies demonstrated that AbpA modulates gene expression in response to amylase, suggesting that the amylase-streptococcal interaction may function in ways other than nutrition. The goal of this study was to explore the role of AbpA in gene regulation through comparative transcriptional profiling of wild-type KS1 and AbpA− mutant KS1ΩabpA under various environmental conditions. A portion of the total RNA isolated from mid-log-phase cells grown in 5% CO2 in (i) complex medium with or without amylase, (ii) defined medium (DM) containing 0.8% glucose with/without amylase, and (iii) DM containing 0.2% glucose and amylase with or without starch was reverse transcribed to cDNA and the rest used for RNA sequencing. Changes in the expression of selected genes were validated by quantitative reverse transcription-PCR. Maltodextrin-associated genes, fatty acid synthesis genes and competence genes were differentially expressed in a medium-dependent manner. Genes in another cluster containing a putative histidine kinase/response regulator, peptide methionine sulfoxide reductase, thioredoxin protein, lipoprotein, and cytochrome c-type protein were downregulated in KS1ΩabpA under all of the environmental conditions tested. Thus, AbpA appears to modulate genes associated with maltodextrin utilization/transport and fatty acid synthesis. Importantly, in all growth conditions AbpA was associated with increased expression of a potential two-component signaling system associated with genes involved in reducing oxidative stress, suggesting a role in signal transduction and stress tolerance. PMID:26025889

Correlation between salivary alpha-amylase and stress-related anxiety.

PubMed

Rashkova, Maya R; Ribagin, Lora S; Toneva, Nina G

2012-01-01

Salivary alpha-amylase is a useful biomarker that can be used in assessing human psychobiological and social behavioural processes. Studying it opens up possibilities for the creation of novel concepts concerning the interaction of biological and social processes and their impact on health and behaviour. The levels of salivary alpha-amylase and situation anxiety self-assessment using Spielberger test were measured twice in 30 individuals aged 21.37 +/- 0.96 yrs (18 females and 12 males): once during stressful situation (prior to examination) and, again a month later, in stress-free environment (during a training session). Salivary alpha-amylase was measured using kinetic reaction kit Salimetrics LLC--USA. The mean level of salivary alpha-amylase measured during the first measurement 156.0 +/- 93.33 U/ml. During the second measurement in the absence of intense stress, the levels were two times lower - 74.03 +/- 58.06 U/ml and the differences were statistically significant (P < 0.001). We found a statistically significant correlation between the levels of salivary alpha-amylase in both measurements (P < 0.01). The correlation coefficient was r = 0.472 (P < 0.01). The adapted version of the State-Trait Anxiety Inventory score (STAI) created by Spielberger is appropriate for assessment of stress-related anxiety in young individuals. Salivary alpha-amylase may be used as a biomarker for objective evaluation of the psychosomatic state of individuals in a stressful environment. The combination of psychological test and objective indicator such as salivary alpha-amylase is an excellent tool for objective evaluation of individual's state in stressful environment. Similar tests may be used in assessment of patients' behaviours at dental treatment that may be considered a stressor in most patients.

Amylase activity is associated with AMY2B copy numbers in dog: implications for dog domestication, diet and diabetes.

PubMed

Arendt, Maja; Fall, Tove; Lindblad-Toh, Kerstin; Axelsson, Erik

2014-10-01

High amylase activity in dogs is associated with a drastic increase in copy numbers of the gene coding for pancreatic amylase, AMY2B, that likely allowed dogs to thrive on a relatively starch-rich diet during early dog domestication. Although most dogs thus probably digest starch more efficiently than do wolves, AMY2B copy numbers vary widely within the dog population, and it is not clear how this variation affects the individual ability to handle starch nor how it affects dog health. In humans, copy numbers of the gene coding for salivary amylase, AMY1, correlate with both salivary amylase levels and enzyme activity, and high amylase activity is related to improved glycemic homeostasis and lower frequencies of metabolic syndrome. Here, we investigate the relationship between AMY2B copy numbers and serum amylase activity in dogs and show that amylase activity correlates with AMY2B copy numbers. We then describe how AMY2B copy numbers vary in individuals from 20 dog breeds and find strong breed-dependent patterns, indicating that the ability to digest starch varies both at the breed and individual level. Finally, to test whether AMY2B copy number is strongly associated with the risk of developing diabetes mellitus, we compare copy numbers in cases and controls as well as in breeds with varying diabetes susceptibility. Although we see no such association here, future studies using larger cohorts are needed before excluding a possible link between AMY2B and diabetes mellitus. © 2014 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics.

Comparison of salivary levels of mucin and amylase and their relation with clinical parameters obtained from patients with aggressive and chronic periodontal disease

PubMed Central

ACQUIER, Andrea Beatriz; PITA, Alejandra Karina De Couto; BUSCH, Lucila; SÁNCHEZ, Gabriel Antonio

2015-01-01

Objective Salivary mucin and amylase levels are increased in patients with chronic periodontitis (CP). Due to the fact that aggressive periodontitis (AgP) not only differs from chronic periodontitis in terms of its clinical manifestation, the aim of this study was to compare salivary mucin and amylase levels and their relation to the clinical parameters of patients with aggressive periodontitis with that of patients with chronic periodontitis. Material and Methods Eighty subjects were divided into two groups: 20 patients with AgP and their 20 matched controls and 20 patients with CP and their 20 matched controls, based on clinical attachment loss (CAL), probing pocket depth (PPD) and bleeding on probing (BOP). Whole unstimulated saliva was obtained and mucin, amylase and protein were determined by colorimetric methods. Pearson’s correlation analysis was used to determine the relationship between salivary mucin, amylase and protein levels and the clinical parameters. Results Salivary mucin, amylase and protein levels were increased in patients with AgP and CP but there were no differences between them or between control groups. Pearson’s correlation analysis, determined in the entire subjects studied, showed a positive and significant correlation of mucin, amylase and proteins with CAL and PPD and a negative correlation with the flow rate. When Pearson’s correlation analysis was carried out in each group separately, Fisher’s z transformation showed no significant difference between both groups. Conclusion Comparison of the salivary levels of mucin, amylase and protein and their relationship with clinical parameters of AgP patients with that of CP patients revealed no differences between both groups. PMID:26221923

Comparative genomics and evolution of the amylase-binding proteins of oral streptococci.

PubMed

Haase, Elaine M; Kou, Yurong; Sabharwal, Amarpreet; Liao, Yu-Chieh; Lan, Tianying; Lindqvist, Charlotte; Scannapieco, Frank A

2017-04-20

Successful commensal bacteria have evolved to maintain colonization in challenging environments. The oral viridans streptococci are pioneer colonizers of dental plaque biofilm. Some of these bacteria have adapted to life in the oral cavity by binding salivary α-amylase, which hydrolyzes dietary starch, thus providing a source of nutrition. Oral streptococcal species bind α-amylase by expressing a variety of amylase-binding proteins (ABPs). Here we determine the genotypic basis of amylase binding where proteins of diverse size and function share a common phenotype. ABPs were detected in culture supernatants of 27 of 59 strains representing 13 oral Streptococcus species screened using the amylase-ligand binding assay. N-terminal sequences from ABPs of diverse size were obtained from 18 strains representing six oral streptococcal species. Genome sequencing and BLAST searches using N-terminal sequences, protein size, and key words identified the gene associated with each ABP. Among the sequenced ABPs, 14 matched amylase-binding protein A (AbpA), 6 matched amylase-binding protein B (AbpB), and 11 unique ABPs were identified as peptidoglycan-binding, glutamine ABC-type transporter, hypothetical, or choline-binding proteins. Alignment and phylogenetic analyses performed to ascertain evolutionary relationships revealed that ABPs cluster into at least six distinct, unrelated families (AbpA, AbpB, and four novel ABPs) with no phylogenetic evidence that one group evolved from another, and no single ancestral gene found within each group. AbpA-like sequences can be divided into five subgroups based on the N-terminal sequences. Comparative genomics focusing on the abpA gene locus provides evidence of horizontal gene transfer. The acquisition of an ABP by oral streptococci provides an interesting example of adaptive evolution.

Amylase addition increases starch ruminal digestion in first-lactation cows fed high and low starch diets.

PubMed

Nozière, P; Steinberg, W; Silberberg, M; Morgavi, D P

2014-01-01

The objective of this study was to evaluate the effect of an exogenous amylase preparation on digestion of low- and high-starch diets in dairy cattle. Rumen and total-tract nutrient digestibility were measured in a 4×4 Latin square design with 28-d periods using 4 first-lactation cows cannulated at the rumen and duodenum. Corn silage-based diets had 20 or 30% starch, attained by changing the composition of concentrate, with or without addition of an exogenous amylase preparation. Effects of the enzyme additive were observed on ruminal digestibility but not at the total-tract level. Ruminal digestibility of starch increased from 75% in control to 81% with amylase supplementation. This difference in ruminal starch digestion was compensated postruminally, so that the total-tract digestibility of starch was almost complete and did not differ between treatments. The amylase supplement also increased the true ruminal digestibility of organic matter but did not affect microbial N flow to the duodenum. Amylase supplement reduced the proportion of acetate and butyrate and increased that of propionate, particularly in the high-starch diet, where it tended to increase the concentration of total volatile fatty acids in the rumen. Other effects were a higher amylase activity in the solid-associated microbial community and a tendency for lower numbers of protozoa. In contrast, we observed no changes in intake, production, dry matter and fiber (neutral detergent fiber and acid detergent fiber) digestibility, or ruminal digestion, and no or small changes on selected fibrolytic and amylolytic bacteria and on the microbial community in general. We conclude that the exogenous amylase improved starch digestion in the rumen in first-lactation cows with moderate intake and production levels. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Diosgenin from Dioscorea bulbifera: Novel Hit for Treatment of Type II Diabetes Mellitus with Inhibitory Activity against α-Amylase and α-Glucosidase

PubMed Central

Ghosh, Sougata; More, Piyush; Derle, Abhishek; Patil, Ajay B.; Markad, Pramod; Asok, Adersh; Kumbhar, Navanath; Shaikh, Mahemud L.; Ramanamurthy, Boppana; Shinde, Vaishali S.; Dhavale, Dilip D.; Chopade, Balu A.

2014-01-01

Diabetes mellitus is a multifactorial metabolic disease characterized by post-prandial hyperglycemia (PPHG). α-amylase and α-glucosidase inhibitors aim to explore novel therapeutic agents. Herein we report the promises of Dioscorea bulbifera and its bioactive principle, diosgenin as novel α-amylase and α-glucosidase inhibitor. Among petroleum ether, ethyl acetate, methanol and 70% ethanol (v/v) extracts of bulbs of D. bulbifera, ethyl acetate extract showed highest inhibition upto 72.06 ± 0.51% and 82.64 ± 2.32% against α-amylase and α-glucosidase respectively. GC-TOF-MS analysis of ethyl acetate extract indicated presence of high diosgenin content. Diosgenin was isolated and identified by FTIR, 1H NMR and 13C NMR and confirmed by HPLC which showed an α-amylase and α-glucosidase inhibition upto 70.94 ± 1.24% and 81.71 ± 3.39%, respectively. Kinetic studies confirmed the uncompetitive mode of binding of diosgenin to α-amylase indicated by lowering of both Km and Vm. Interaction studies revealed the quenching of intrinsic fluorescence of α-amylase in presence of diosgenin. Similarly, circular dichroism spectrometry showed diminished negative humped peaks at 208 nm and 222 nm. Molecular docking indicated hydrogen bonding between carboxyl group of Asp300, while hydrophobic interactions between Tyr62, Trp58, Trp59, Val163, His305 and Gln63 residues of α-amylase. Diosgenin interacted with two catalytic residues (Asp352 and Glu411) from α-glucosidase. This is the first report of its kind that provides an intense scientific rationale for use of diosgenin as novel drug candidate for type II diabetes mellitus. PMID:25216353

Leucine Affects α-Amylase Synthesis through PI3K/Akt-mTOR Signaling Pathways in Pancreatic Acinar Cells of Dairy Calves.

PubMed

Guo, Long; Liang, Ziqi; Zheng, Chen; Liu, Baolong; Yin, Qingyan; Cao, Yangchun; Yao, Junhu

2018-05-23

Dietary nutrient utilization, particularly starch, is potentially limited by digestion in dairy cow small intestine because of shortage of α-amylase. Leucine acts as an effective signal molecular in the mTOR signaling pathway, which regulates a series of biological processes, especially protein synthesis. It has been reported that leucine could affect α-amylase synthesis and secretion in ruminant pancreas, but mechanisms have not been elaborated. In this study, pancreatic acinar (PA) cells were used as a model to determine the cellular signal of leucine influence on α-amylase synthesis. PA cells were isolated from newborn Holstein dairy bull calves and cultured in Dulbecco's modifed Eagle's medium/nutrient mixture F12 liquid media containing four leucine treatments (0, 0.23, 0.45, and 0.90 mM, respectively), following α-amylase activity, zymogen granule, and signal pathway factor expression detection. Rapamycin, a specific inhibitor of mTOR, was also applied to PA cells. Results showed that leucine increased ( p < 0.05) synthesis of α-amylase as well as phosphorylation of PI3K, Akt, mTOR, and S6K1 while reduced ( p < 0.05) GCN2 expression. Inhibition of mTOR signaling downregulated the α-amylase synthesis. In addition, the extracellular leucine dosage significantly influenced intracellular metabolism of isoleucine ( p < 0.05). Overall, leucine regulates α-amylase synthesis through promoting the PI3K/Akt-mTOR pathway and reducing the GCN2 pathway in PA cells of dairy calves. These pathways form the signaling network that controls the protein synthesis and metabolism. It would be of great interest in future studies to explore the function of leucine in ruminant nutrition.

Purification of an alpha amylase from Aspergillus flavus NSH9 and molecular characterization of its nucleotide gene sequence.

PubMed

Karim, Kazi Muhammad Rezaul; Husaini, Ahmad; Sing, Ngieng Ngui; Sinang, Fazia Mohd; Roslan, Hairul Azman; Hussain, Hasnain

2018-04-01

In this study, an alpha-amylase enzyme from a locally isolated Aspergillus flavus NSH9 was purified and characterized. The extracellular α-amylase was purified by ammonium sulfate precipitation and anion-exchange chromatography at a final yield of 2.55-fold and recovery of 11.73%. The molecular mass of the purified α-amylase was estimated to be 54 kDa using SDS-PAGE and the enzyme exhibited optimal catalytic activity at pH 5.0 and temperature of 50 °C. The enzyme was also thermally stable at 50 °C, with 87% residual activity after 60 min. As a metalloenzymes containing calcium, the purified α-amylase showed significantly increased enzyme activity in the presence of Ca 2+ ions. Further gene isolation and characterization shows that the α-amylase gene of A. flavus NSH9 contained eight introns and an open reading frame that encodes for 499 amino acids with the first 21 amino acids presumed to be a signal peptide. Analysis of the deduced peptide sequence showed the presence of three conserved catalytic residues of α-amylase, two Ca 2+ -binding sites, seven conserved peptide sequences, and several other properties that indicates the protein belongs to glycosyl hydrolase family 13 capable of acting on α-1,4-bonds only. Based on sequence similarity, the deduced peptide sequence of A. flavus NSH9 α-amylase was also found to carry two potential surface/secondary-binding site (SBS) residues (Trp 237 and Tyr 409) that might be playing crucial roles in both the enzyme activity and also the binding of starch granules.

Assessment of salivary amylase as a stress biomarker in pregnant patients.

PubMed

Guglielminotti, J; Dehoux, M; Mentré, F; Bedairia, E; Montravers, P; Desmonts, J-M; Longrois, D

2012-01-01

Chronic stress during pregnancy has been associated with worsened maternal and fetal outcomes. Acute stress immediately before spinal anaesthesia for caesarean section may contribute to hypotension. Therefore objective measures of acute stress may help identify women at risk of adverse outcomes. Salivary alpha-amylase is a stress biomarker that has so far been poorly investigated during pregnancy. The reference change value is the difference between two sequential results that must be exceeded for a change to be considered clinically relevant. Our first aim was to determine if salivary alpha-amylase increased in pregnant patients when subjected to the stress of transfer to the operating room. Our second aim was to determine if changes in salivary alpha-amylase were likely to be clinically significant by measuring reference change value in healthy volunteers. In 15 pregnant patients undergoing planned caesarean section under spinal anaesthesia, salivary alpha-amylase, systolic blood pressure, heart rate, and immediate anxiety were measured on the morning of surgery on the ward and again in the operating room. The reference change value was calculated from 18 healthy volunteers. A median 220% increase in salivary alpha-amylase activity (P=0.0015) and a 17% increase in systolic blood pressure (P=0.0006) were observed between the ward and operating room. No changes of immediate anxiety or heart rate were observed. Reference change value was ±76% in volunteers and 13 of the 15 pregnant patients had a salivary alpha-amylase increase greater than the reference change value. When pregnant women are taken to the operating room, a clinically and statistically significant increase in salivary alpha-amylase was observed. Further studies are required to define its clinical usefulness. Copyright © 2011 Elsevier Ltd. All rights reserved.

α-Amylase in Vaginal Fluid: Association With Conditions Favorable to Dominance of Lactobacillus.

PubMed

Nasioudis, Dimitrios; Beghini, Joziani; Bongiovanni, Ann Marie; Giraldo, Paulo C; Linhares, Iara M; Witkin, Steven S

2015-11-01

Vaginal glycogen is degraded by host α-amylase and then converted to lactic acid by Lactobacilli. This maintains the vaginal pH at ≤4.5 and prevents growth of other bacteria. Therefore, host α-amylase activity may promote dominance of Lactobacilli. We evaluated whether the α-amylase level in vaginal fluid is altered in women with bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC) and whether its concentration was associated with levels of lactic acid isomers and host mediators. Vaginal fluid was obtained from 43 women with BV, 50 women with VVC, and 62 women with no vulvovaginal disorders. Vaginal fluid concentrations of α-amylase, secretory leukocyte protease inhibitor (SLPI), hyaluronan, hyaluronidase-1, β-defensin, and elafin were measured by enzyme-linked immunosorbent assay (ELISA). Vaginal concentrations of neutrophil gelatinase-associated lipocalin (NGAL), matrix metalloproteinase (MMP) 8, and d- and l-lactic acid levels in these patients were previously reported. The median vaginal fluid α-amylase level was 1.83 mU/mL in control women, 1.45 mU/mL in women with VVC, and 1.07 mU/mL in women with BV. Vaginal levels of α-amylase were correlated with d-lactic acid (P = .003) but not with l-lactic acid (P > .05) and with SLPI (P < .001), hyaluronidase-1 (P < .001), NGAL (P = .001), and MMP-8 (P = .005). The exfoliation of glycogen-rich epithelial cells into the vaginal lumen by hyaluronidase-1 and MMP-8 may increase glycogen availability and promote α-amylase activity. The subsequent enhanced availability of glycogen breakdown products would favor proliferation of Lactobacilli, the primary producers of d-lactic acid in the vagina. Concomitant production of NGAL and SLPI would retard growth of BV-related bacteria. © The Author(s) 2015.

Salivary amylase and stress during stressful environment: three Mars analog mission crews study.

PubMed

Rai, Balwant; Kaur, Jasdeep; Foing, Bernard H

2012-06-14

After the establishment of the space age physicians, human factors engineers, neurologist and psychologists and their special attention to work on people's capability to meet up the physical, psychological, neuroscience and interpersonal strains of working in space, it has been regarded as an issue that seeks urgent consideration. Not study was conducted on effect of simulated Mars analog environment on stress and salivary amylase. So, this study aimed to confirm whether salivary amylase is act as stress biomarker in crew members who took part in Mars analog mission in an isolated and stressful environment. The 18 crew members were selected who took part in Mars Analog Research Station, Utah. Salivary amylase was measured using a biosensor of salivary amylase monitor and State-Trait Anxiety Inventory score at pre-extravehicular activity, post-extravehicular activity and on before mission. The state and trait anxiety scores at pre-extravehicular activity for each commander were elevated as compared to after extravehicular activity. There were significant differences in the state and trait anxiety scores between before extravehicular activity and after extravehicular activity of Commander and other members, also there were significant differences in values of before-extravehicular activity between commanders and other members. There were significant differences in values of salivary amylase at before extravehicular activity and after extravehicular activity between commander group and other members. There was significant correlation between salivary amylase and state and trait anxiety scores in all groups. Measuring salivary amylase level could be useful for stress assessment of crew members and population working in a stressful and isolated environment. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Aspergillus 6V4, a Strain Isolated from Manipueira, Produces High Amylases Levels by Using Wheat Bran as a Substrate

PubMed Central

Celestino, Jessyca dos Reis; Duarte, Ana Caroline; Silva, Cláudia Maria de Melo; Sena, Hellen Holanda; Ferreira, Maria do Perpétuo Socorro Borges Carriço; Mallmann, Neila Hiraishi; Lima, Natacha Pinheiro Costa; Tavares, Chanderlei de Castro; de Souza, Rodrigo Otávio Silva; Souza, Érica Simplício; Souza, João Vicente Braga

2014-01-01

The aim of this study was screening fungi strains, isolated from manipueira (a liquid subproduct obtained from the flour production of Manihot esculenta), for amylases production and investigating production of these enzymes by the strain Aspergillus 6V4. The fungi isolated from manipueira belonged to Ascomycota phylum. The strain Aspergillus 6V4 was the best amylase producer in the screening assay of starch hydrolysis in petri dishes (ASHPD) and in the assay in submerged fermentation (ASbF). The strain Aspergillus 6V4 produced high amylase levels (335 UI/L) using wheat bran infusion as the exclusive substrate and the supplementation of this substrate with peptone decreased the production of this enzyme. The moisture content of 70% was the best condition for the production of Aspergillus 6V4 amylases (385 IU/g) in solid state fermentation (SSF). PMID:24724017

A multidisciplinary approach to an unusual cause of hyperamylasaemia

PubMed Central

Logie, James John; Cox, Mary; Sharkey, Joanne; Williams, Alistair

2015-01-01

Clinical features together with elevation of pancreatic enzymes are the key diagnostic indicators of acute pancreatitis. We report a case of a woman in her 50s who presented with abdominal distension and serum amylase raised to more than 30 times the upper limit of normal. She was initially treated for acute pancreatitis, however, she was not symptomatic of this and the pancreas appeared to be normal on CT scan. Further investigations revealed the patient had a high-grade serous ovarian carcinoma with nodal metastatic spread. An amylase-secreting ovarian tumour was suspected, which was supported by elevated salivary-amylase isoenzymes, consistent with previous reports in the literature. The patient was treated with chemotherapy and surgery, during which her serum amylase and CA-125 initially fell significantly, but eventually both increased, reflecting disease progression. This case serves as an important reminder to consider non-pancreatic causes of raised serum amylase, to avoid misdiagnosis. PMID:26150631

The 53-kDa proteolytic product of precursor starch-hydrolyzing enzyme of Aspergillus niger has Taka-amylase-like activity.

PubMed

Ravi-Kumar, K; Venkatesh, K S; Umesh-Kumar, S

2007-04-01

The 53-kDa amylase secreted by Aspergillus niger due to proteolytic processing of the precursor starch-hydrolyzing enzyme was resistant to acarbose, a potent alpha-glucosidase inhibitor. The enzyme production was induced when A. niger was grown in starch medium containing the inhibitor. Antibodies against the precursor enzyme cross-reacted with the 54-kDa Taka-amylase protein of A. oryzae. It resembled Taka-amylase in most of its properties and also hydrolyzed starch to maltose of alpha-anomeric configuration. However, it did not degrade maltotriose formed during the reaction and was not inhibited by zinc ions.

Experimental and computational approaches to reveal the potential of Ficus deltoidea leaves extract as α-amylase inhibitor.

PubMed

Abu Bakar, Amirul Ridzuan; Manaharan, Thamilvaani; Merican, Amir Feisal; Mohamad, Saharuddin Bin

2018-02-01

Ficus deltoidea leaves extract are known to have good therapeutic properties such as antioxidant, anti-inflammatory and anti-diabetic. We showed that 50% ethanol-water extract of F. deltoidea leaves and its pungent compounds vitexin and isovitexin exhibited significant (p < 0.05) α-amylase inhibition with IC 50 (vitexin: 4.6 μM [0.02 μg/mL]; isovitexin: 0.06 μg/mL [13.8 μM] and DPPH scavenging with IC 50 (vitexin: 92.5 μM [0.4 μg/mL]; isovitexin: 0.5 μg/mL [115.4 μM]). Additionally, molecular docking analysis confirmed that vitexin has a higher binding affinity (-7.54 kcal/mol) towards α-amylase compared to isovitexin (-5.61 kcal/mol). On the other hand, the molecular dynamics findings showed that vitexin-α-amylase complex is more stable during the simulation of 20 ns when compared to the isovitexin-α-amylase complex. Our results suggest that vitexin is more potent and stable against α-amylase enzyme, thus it could develop as a therapeutic drug for the treatment of diabetes.

HPLC method for measurement of human salivary α-amylase inhibition by aqueous plant extracts.

PubMed

Takács, István; Takács, Ákos; Pósa, Anikó; Gyémánt, Gyöngyi

2017-06-01

Control of hyperglycemia is an important treatment in metabolic disorders such as type II diabetes and obesity. α-Amylase, as the first enzyme of glucose release from dietary polysaccharides, is a potential target to identify new sources of novel anti-obesity and anti-diabetic drugs. In this work, different herbal extracts as α-amylase inhibitors were studied by measuring the rate of the cleavage of a maltooligomer substrate 2-chloro-4-nitrophenyl-β-D-maltoheptoside. Measurement of chromophore containing products after reversed phase HPLC separation was used for α-amylase activity measurement. Rates of hydrolysis catalysed by human salivary α-amylase were determined in the presence and absence of lyophilised water extracts of eleven herbs. Remarkable bioactivities were found for extracts of Cinnamomum zeylanicum Blume (bark), Camellia sinensis L. (leaf), Ribes nigrum L. (leaf), Laurus nobilis L. (leaf), Vaccinium macrocarpon Aiton (fruit) and Syzygium aromaticum L. (bud). Determined IC 50 values were in 0.017-41 μg/ml range for these six selected plant extracts. Our results confirm the applicability of this HPLC-based method for the quick and reliable comparison of plants as α-amylase inhibitors.

Alpha-amylase from the Hyperthermophilic Archaeon Thermococcus thioreducens

NASA Technical Reports Server (NTRS)

Bernhardsdotter, E. C. M. J.; Pusey, M. L.; Ng, M. L.; Garriott, O. K.

2003-01-01

Extremophiles are microorganisms that thrive in, from an anthropocentric view, extreme environments such as hot springs. The ability of survival at extreme conditions has rendered enzymes from extremophiles to be of interest in industrial applications. One approach to producing these extremozymes entails the expression of the enzyme-encoding gene in a mesophilic host such as E.coli. This method has been employed in the effort to produce an alpha-amylase from a hyperthermophile (an organism that displays optimal growth above 80 C) isolated from a hydrothermal vent at the Rainbow vent site in the Atlantic Ocean. alpha-amylases catalyze the hydrolysis of starch to produce smaller sugars and constitute a class of industrial enzymes having approximately 25% of the enzyme market. One application for thermostable alpha-amylases is the starch liquefaction process in which starch is converted into fructose and glucose syrups. The a-amylase encoding gene from the hyperthermophile Thermococcus thioreducens was cloned and sequenced, revealing high similarity with other archaeal hyperthermophilic a-amylases. The gene encoding the mature protein was expressed in E.coli. Initial characterization of this enzyme has revealed an optimal amylolytic activity between 85-90 C and around pH 5.3-6.0.

High Temperature-Induced Expression of Rice α-Amylases in Developing Endosperm Produces Chalky Grains.

PubMed

Nakata, Masaru; Fukamatsu, Yosuke; Miyashita, Tomomi; Hakata, Makoto; Kimura, Rieko; Nakata, Yuriko; Kuroda, Masaharu; Yamaguchi, Takeshi; Yamakawa, Hiromoto

2017-01-01

Global warming impairs grain filling in rice and reduces starch accumulation in the endosperm, leading to chalky-appearing grains, which damages their market value. We found previously that high temperature-induced expression of starch-lytic α-amylases during ripening is crucial for grain chalkiness. Because the rice genome carries at least eight functional α-amylase genes, identification of the α-amylase(s) that contribute most strongly to the production of chalky grains could accelerate efficient breeding. To identify α-amylase genes responsible for the production of chalky grains, we characterized the histological expression pattern of eight α-amylase genes and the influences of their overexpression on grain appearance and carbohydrate components through a series of experiments with transgenic rice plants. The promoter activity of most α - amylase genes was elevated to various extents at high temperature. Among them, the expression of Amy1A and Amy3C was induced in the internal, especially basal to dorsal, region of developing endosperm, whereas that of Amy3D was confined near the ventral aleurone. These regions coincided with the site of occurrence of chalkiness, which was in clear contrast to conventionally known expression patterns of the enzyme in the scutellum and aleurone during seed germination. Furthermore, overexpression of α-amylase genes, except for Amy3E , in developing endosperm produced various degrees of chalky grains without heat exposure, whereas that of Amy3E yielded normal translucent grains, as was the case in the vector control, even though Amy3E -overexpressing grains contained enhanced α-amylase activities. The weight of the chalky grains was decreased due to reduced amounts of starch, and microscopic observation of the chalky part of these grains revealed that their endosperm consisted of loosely packed round starch granules that had numerous pits on their surface, confirming the hydrolysis of the starch reserve by α-amylases. Moreover, the chalky grains contained increased amounts of soluble sugars including maltooligosaccharides at the expense of starch. The integrated analyses proposed that expression of Amy1A, Amy3C , and Amy3D at the specific regions of the developing endosperm could generate the chalkiness. This finding provides the fundamental knowledge to narrow down the targets for the development of high temperature-tolerant premium rice.

High Temperature-Induced Expression of Rice α-Amylases in Developing Endosperm Produces Chalky Grains

PubMed Central

Nakata, Masaru; Fukamatsu, Yosuke; Miyashita, Tomomi; Hakata, Makoto; Kimura, Rieko; Nakata, Yuriko; Kuroda, Masaharu; Yamaguchi, Takeshi; Yamakawa, Hiromoto

2017-01-01

Global warming impairs grain filling in rice and reduces starch accumulation in the endosperm, leading to chalky-appearing grains, which damages their market value. We found previously that high temperature-induced expression of starch-lytic α-amylases during ripening is crucial for grain chalkiness. Because the rice genome carries at least eight functional α-amylase genes, identification of the α-amylase(s) that contribute most strongly to the production of chalky grains could accelerate efficient breeding. To identify α-amylase genes responsible for the production of chalky grains, we characterized the histological expression pattern of eight α-amylase genes and the influences of their overexpression on grain appearance and carbohydrate components through a series of experiments with transgenic rice plants. The promoter activity of most α-amylase genes was elevated to various extents at high temperature. Among them, the expression of Amy1A and Amy3C was induced in the internal, especially basal to dorsal, region of developing endosperm, whereas that of Amy3D was confined near the ventral aleurone. These regions coincided with the site of occurrence of chalkiness, which was in clear contrast to conventionally known expression patterns of the enzyme in the scutellum and aleurone during seed germination. Furthermore, overexpression of α-amylase genes, except for Amy3E, in developing endosperm produced various degrees of chalky grains without heat exposure, whereas that of Amy3E yielded normal translucent grains, as was the case in the vector control, even though Amy3E-overexpressing grains contained enhanced α-amylase activities. The weight of the chalky grains was decreased due to reduced amounts of starch, and microscopic observation of the chalky part of these grains revealed that their endosperm consisted of loosely packed round starch granules that had numerous pits on their surface, confirming the hydrolysis of the starch reserve by α-amylases. Moreover, the chalky grains contained increased amounts of soluble sugars including maltooligosaccharides at the expense of starch. The integrated analyses proposed that expression of Amy1A, Amy3C, and Amy3D at the specific regions of the developing endosperm could generate the chalkiness. This finding provides the fundamental knowledge to narrow down the targets for the development of high temperature-tolerant premium rice. PMID:29270189

Exploring the effect of bisphenol S on sludge hydrolysis and mechanism of the interaction between bisphenol S and α-Amylase through spectrophotometric methods.

PubMed

Yang, Hang; Hou, Guangying; Zhang, Li; Ju, Lei; Liu, Chunguang

2017-02-01

Sewage sludge, as a very significant sources of BPS (up to 523mg/kg dw) introduction into the environment, must be handled properly. Therefore, it is important to access BPS removal and its effect on sludge treatment with the biological treatment. However, it is unclear for its effect on the hydrolysis of sludge. In this research, impact of BPS on sludge hydrolysis by α-Amylase is studied from the respect of component of soluble organic matter in sludge using three-dimensional fluorescence spectra. Enzyme activity assay suggests that sludge hydrolysis is inhibited due to the denaturation of α-Amylase with BPS exposure. In order to illuminate the interaction mechanism between BPS and α-Amylase, UV-vis, steady-state fluorescence, circular dichroism, synchronous fluorescence, light scattering spectra, enzyme activity assay and molecule docking techniques are applied. Results show that BPS interacts with α-Amylase by hydrophobic bond in the activity region of α-Amylase. This interaction not only causes an unfolding skeleton structure of α-Amylase and a less hydrophobic microenvironment of tyrosine and tryptophan residues, but also leads to a specific fluorophore quenching involving static and dynamic type. This work provides direct evidence about enzyme toxicity of BPS and establishes a new strategy to investigate the interaction between protein and BPS at a molecular level, which is helpful for clarifying the bioactivities of BPS. Copyright © 2016 Elsevier B.V. All rights reserved.

Multi-spectral and thermodynamic analysis of the interaction mechanism between Cu2+ and α-amylase and impact on sludge hydrolysis.

PubMed

Zhou, Ruiqi; Liu, Hong; Hou, Guangying; Ju, Lei; Liu, Chunguang

2017-04-01

An increasing amount of heavy metals (e.g., Cu 2+ ) is being discharged into sewage treatment plants and is accumulating in sludge, which is toxic to the enzyme in sludge or soil when the sludge is used as fertilizer, resulting in unfavorable effect on the biological treatment of sludge and the circulation and conversion of materials in soil. In this research, effect of Cu 2+ on sludge hydrolysis by α-amylase is studied from the respect of concentration and components of soluble organic matter in sludge, using three-dimensional fluorescence spectra. Results show that Cu 2+ exposure not only inhibits the hydrolysis of sludge due to the denaturation of α-amylase but also affects the components of soluble organic matter in sludge. In order to illuminate the interaction mechanism between Cu 2+ and α-amylase (a model of hydrolase in sludge), multi-spectra and isothermal titration microcalorimetry techniques are applied. Results show that the secondary structure of α-amylase is changed as that the α-helical content increases and the structure loosens. The microenvironment of amino acid residue in α-amylase is changed that the hydrophobicity decreases and the polarity increases with Cu 2+ exposure. Isothermal titration calorimetry results show that Van der Waals force and hydrogen bond exist in the interaction between Cu 2+ and α-amylase. Results from this research would favor the development of advanced process for the biological treatment of sludge containing heavy metals.

Elevated bile amylase level without pancreaticobiliary maljunction is a risk factor for gallbladder carcinoma.

PubMed

Fujimoto, Takaaki; Ohtsuka, Takao; Nakashima, Yohei; Gotoh, Yoshitaka; Date, Kenjiro; Mori, Yasuhisa; Sadakari, Yoshihiko; Takahata, Shunichi; Oda, Yoshinao; Nakamura, Masafumi

2017-02-01

Elevated bile amylase level in patients with pancreaticobiliary maljunction (PBM) or high confluence of pancreaticobiliary ducts (HCPBD) is well known as a risk factor for gallbladder carcinoma (GBC) development. However, the effects of occult pancreaticobiliary reflux (OPR), a condition characterized by high bile amylase level in the presence of an anatomically normal pancreaticobiliary junction, on GBC development remain unclear. The aim of this study was to assess the relationship between OPR and GBC. Clinicopathological data of 52 patients who were preoperatively diagnosed with gallbladder (GB) tumor (22 malignant, 30 benign) were retrospectively reviewed. All of the patients underwent preoperative endoscopic retrograde cholangiopancreatography to evaluate pancreaticobiliary junction morphology and bile amylase level. The relationship between the histological diagnosis of GB lesions, and pancreaticobiliary junction morphology and bile amylase level were investigated. Pancreaticobiliary maljunction, HCPBD, and normal pancreaticobiliary junction (NPJ) were identified in 12, nine, and 31 patients, respectively. The rates of GBC in patients with PBM, HCPBD, and NPJ were 58% (7/12), 67% (6/9), and 29% (9/31), respectively. Of the 31 patients with NPJ, 22 had OPR and nine of these had GBC. None of the patients with NPJ and normal bile amylase level had GBC. Additionally, among patients with NPJ, bile amylase level was significantly higher in patients with GBC than in patients with benign tumors. Occult pancreaticobiliary reflux, like PBM and HCPBD, is a risk factor for GBC development. © 2017 Japanese Society of Hepato-Biliary-Pancreatic Surgery.

Salivary α-amylase reflects change in attentional demands during postural control: comparison with probe reaction time.

PubMed

Akizuki, Kazunori; Ohashi, Yukari

2014-12-01

The influence of attention on postural control and the relationship between attention and falling has been reported in previous studies. Although a dual-task procedure is commonly used to measure attentional demand, such procedures are affected by allocation policy, which is a mental strategy to divide attention between simultaneous tasks. Therefore, we examined the effectiveness of salivary α-amylase, which is a physiological method for measuring attentional demand during postural control. Sixteen healthy participants performed a postural-control task using the Balance System, which is a device that can be calibrated to a specific stability level ("Level 1 = least stable" to "Level 8 = most stable"). Levels 1, 2, and 3 were used for this study. Dependent variables measured were overall stability index, which represents the variance of platform displacement in degrees from a horizontal plane; probe reaction time, which was measured using a sound stimulator and recorder; and salivary α-amylase, which was measured using a portable salivary amylase analyzer. As stability level of the test task decreased, both stability index and probe reaction time significantly increased. In addition, we identified a positive moderate correlation between probe reaction time and salivary α-amylase. Our results suggest that salivary α-amylase and probe reaction time reflect the change in attentional demands during a postural-control task and that salivary α-amylase may be an effective tool for evaluating attentional demands during postural control because it is noninvasive and simple to perform.

Screening alpha-glucosidase and alpha-amylase inhibitors from natural compounds by molecular docking in silico.

PubMed

Jhong, Chien-Hung; Riyaphan, Jirawat; Lin, Shih-Hung; Chia, Yi-Chen; Weng, Ching-Feng

2015-01-01

The alpha-glucosidase inhibitor is a common oral anti-diabetic drug used for controlling carbohydrates normally converted into simple sugars and absorbed by the intestines. However, some adverse clinical effects have been observed. The present study seeks an alternative drug that can regulate the hyperglycemia by down-regulating alpha-glucosidase and alpha-amylase activity by molecular docking approach to screen the hyperglycemia antagonist against alpha-glucosidase and alpha-amylase activities from the 47 natural compounds. The docking data showed that Curcumin, 16-hydroxy-cleroda-3,13-dine-16,15-olide (16-H), Docosanol, Tetracosanol, Antroquinonol, Berberine, Catechin, Quercetin, Actinodaphnine, and Rutin from 47 natural compounds had binding ability towards alpha-amylase and alpha-glucosidase as well. Curcumin had a better biding ability of alpha-amylase than the other natural compounds. Analyzed alpha-glucosidase activity reveals natural compound inhibitors (below 0.5 mM) are Curcumin, Actinodaphnine, 16-H, Quercetin, Berberine, and Catechin when compared to the commercial drug Acarbose (3 mM). A natural compound with alpha-amylase inhibitors (below 0.5 mM) includes Curcumin, Berberine, Docosanol, 16-H, Actinodaphnine/Tetracosanol, Catechin, and Quercetin when compared to Acarbose (1 mM). When taken together, the implication is that molecular docking is a fast and effective way to screen alpha-glucosidase and alpha-amylase inhibitors as lead compounds of natural sources isolated from medicinal plants. © 2015 International Union of Biochemistry and Molecular Biology.

Polymeric amylase nanoparticles as a new semi-synthetic enzyme system for hydrolysis of starch.

PubMed

Say, R; Şenay, R Hilal; Biçen, Özlem; Ersöz, Arzu; Şişman Yılmaz, Filiz; Akgöl, Sinan; Denizli, Adil

2013-05-01

α-Amylase (EC 3.2.1.1; α-D-1,4,glucan glucanohydrolase) catalyzes the hydrolysis of α-D-(1,4)-glucosidic linkages in starch, glycogen, and various malto-oligosaccharides, by releasing α-anomeric products. In this study, a novel method has been developed to prepare nanoprotein particles that carry α-amylase as a monomer by using a photosensitive microemulsion polymerization process. The nanostructured α-amylase with photosensitive features have been characterized by fluorescence spectroscopy, transmission electron microscopy (TEM) and Zeta Sizer. The fluorescence intensity of amylase nanoparticles was determined to be 658 a.u. at 610 nm and the average particle size of nanoamylase was found to be about 71.8 nm. Both free α-amylase and nanoparticles were used in the hydrolysis of starch under varying reaction conditions such as pH and temperature that affect enzyme activity and the results were compared to each other. Km values were 0.26 and 0.87 mM and Vmax values were 0.36 IU mg(-1) and 22.32 IU mg(-1) for nanoenzyme and free enzyme, respectively. Then, thermal stability, storage stability and reusability were investigated and according to the results, activity was preserved 60% at 60 °C; 20% at 70-80 °C temperature values and 80% after 105 days storage. Finally after 10 cycles, the activity was preserved 90% and this novel enzymatic polymeric amylase nanoparticle has showed considerable potential as reusable catalyst. Copyright © 2012 Elsevier B.V. All rights reserved.

Evaluation of amylase and lipase levels in blunt trauma abdomen patients.

PubMed

Kumar, Subodh; Sagar, Sushma; Subramanian, Arulselvi; Albert, Venencia; Pandey, Ravindra Mohan; Kapoor, Nitika

2012-04-01

There are studies to prove the role of amylase and lipase estimation as a screening diagnostic tool to detect diseases apart from acute pancreatitis. However, there is sparse literature on the role of serum and urine amylase, lipase levels, etc to help predict the specific intra-abdominal injury after blunt trauma abdomen (BTA). To elucidate the significance of elevation in the levels of amylase and lipase in serum and urine samples as reliable parameters for accurate diagnosis and management of blunt trauma to the abdomen. A prospective analysis was done on the trauma patients admitted in Jai Prakash Narayan Apex Trauma Center, AIIMS, with blunt abdomen trauma injuries over a period of six months. Blood and urine samples were collected on days 1, 3, and 5 of admission for the estimation of amylase and lipase, liver function tests, serum bicarbonates, urine routine microscopy for red blood cells, and complete hemogram. Clinical details such as time elapsed from injury to admission, type of injury, trauma score, and hypotension were noted. Patients were divided into groups according to the single or multiple organs injured and according to their hospital outcome (dead/discharged). Wilcoxon's Rank sum or Kruskal-Wallis tests were used to compare median values in two/three groups. Data analysis was performed using STATA 11.0 statistical software. A total of 55 patients with median age 26 (range, 6-80) years, were enrolled in the study. Of these, 80% were males. Surgery was required for 20% of the patients. Out of 55 patients, 42 had isolated single organ injury [liver or spleen or gastrointestinal tract (GIT) or kidney]. Patients with pancreatic injury were excluded. In patients who suffered liver injuries, urine lipase levels on day 1, urine lipase/amylase ratio along with aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) on days 1, 3, and 5, were found to be significant. Day 1 serum amylase, AST, ALT, hemoglobin, and hematocrit levels were found significant in patients who had spleen injury. Serum amylase levels on day 5 and ALP on day 3 were significant in patients who had GIT injury. Urine amylase levels on day 5 were found to be statistically significant in patients who had kidney injury. In patients with isolated organ injury to the liver or spleen, the levels of urine amylase were elevated on day 1 and gradually decreased on days 3 and 5, whereas in patients with injury to GIT, the urine amylase levels were observed to gradually increase on days 3 and 5. Although amylase and lipase levels in the serum and urine are not cost-effective clinical tools for routine diagnosis of extra-pancreatic abdominal injuries in BTA, but when coupled with other laboratory tests such as liver enzymes, they may be significant in predicting specific intra-abdominal injury.

Evaluation of amylase and lipase levels in blunt trauma abdomen patients

PubMed Central

Kumar, Subodh; Sagar, Sushma; Subramanian, Arulselvi; Albert, Venencia; Pandey, Ravindra Mohan; Kapoor, Nitika

2012-01-01

Background: There are studies to prove the role of amylase and lipase estimation as a screening diagnostic tool to detect diseases apart from acute pancreatitis. However, there is sparse literature on the role of serum and urine amylase, lipase levels, etc to help predict the specific intra-abdominal injury after blunt trauma abdomen (BTA). Aim: To elucidate the significance of elevation in the levels of amylase and lipase in serum and urine samples as reliable parameters for accurate diagnosis and management of blunt trauma to the abdomen. Materials and Methods: A prospective analysis was done on the trauma patients admitted in Jai Prakash Narayan Apex Trauma Center, AIIMS, with blunt abdomen trauma injuries over a period of six months. Blood and urine samples were collected on days 1, 3, and 5 of admission for the estimation of amylase and lipase, liver function tests, serum bicarbonates, urine routine microscopy for red blood cells, and complete hemogram. Clinical details such as time elapsed from injury to admission, type of injury, trauma score, and hypotension were noted. Patients were divided into groups according to the single or multiple organs injured and according to their hospital outcome (dead/discharged). Wilcoxon's Rank sum or Kruskal–Wallis tests were used to compare median values in two/three groups. Data analysis was performed using STATA 11.0 statistical software. Results: A total of 55 patients with median age 26 (range, 6-80) years, were enrolled in the study. Of these, 80% were males. Surgery was required for 20% of the patients. Out of 55 patients, 42 had isolated single organ injury [liver or spleen or gastrointestinal tract (GIT) or kidney]. Patients with pancreatic injury were excluded. In patients who suffered liver injuries, urine lipase levels on day 1, urine lipase/amylase ratio along with aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) on days 1, 3, and 5, were found to be significant. Day 1 serum amylase, AST, ALT, hemoglobin, and hematocrit levels were found significant in patients who had spleen injury. Serum amylase levels on day 5 and ALP on day 3 were significant in patients who had GIT injury. Urine amylase levels on day 5 were found to be statistically significant in patients who had kidney injury. In patients with isolated organ injury to the liver or spleen, the levels of urine amylase were elevated on day 1 and gradually decreased on days 3 and 5, whereas in patients with injury to GIT, the urine amylase levels were observed to gradually increase on days 3 and 5. Conclusion: Although amylase and lipase levels in the serum and urine are not cost-effective clinical tools for routine diagnosis of extra-pancreatic abdominal injuries in BTA, but when coupled with other laboratory tests such as liver enzymes, they may be significant in predicting specific intra-abdominal injury. PMID:22787343

Comparisons of amylolytic enzyme activities and ß-amylases with differing Bmy1 intron III alleles to sugar production during congress mashing with North American barley cultivars

USDA-ARS?s Scientific Manuscript database

This study was conducted to determine the relationships between patterns of activity development of malt amylolytic enzymes (a-amylase, ß-amylase, and limit dextrinase) and sugar production in two- and six-row North American cultivars during the course of Congress mashing and to test two hypotheses:...

Occupation of low-affinity cholecystokinin (CCK) receptors by CCK activates signal transduction and stimulates amylase secretion in pancreatic acinar cells.

PubMed

Vinayek, R; Patto, R J; Menozzi, D; Gregory, J; Mrozinski, J E; Jensen, R T; Gardner, J D

1993-03-10

Based on the effects of monensin on binding of 125I-CCK-8 and its lack of effect on CCK-8-stimulated amylase secretion we previously proposed that pancreatic acinar cells possess three classes of CCK receptors: high-affinity receptors, low-affinity receptors and very low-affinity receptors [1]. In the present study we treated pancreatic acini with carbachol to induce a complete loss of high-affinity CCK receptors and then examined the action of CCK-8 on inositol trisphosphate IP3(1,4,5), cytosolic calcium and amylase secretion in an effort to confirm and extend our previous hypothesis. We found that first incubating pancreatic acini with 10 mM carbachol decreased binding of 125I-CCK-8 measured during a second incubation by causing a complete loss of high-affinity CCK receptors with no change in the low-affinity CCK receptors. Carbachol treatment of acini, however, did not alter the action of CCK-8 on IP3(1,4,5), cytosolic calcium or amylase secretion or the action of CCK-JMV-180 on amylase secretion or on the supramaximal inhibition of amylase secretion caused by CCK-8. The present findings support our previous hypothesis that pancreatic acinar cells possess three classes of CCK receptors and suggest that high-affinity CCK receptors do not mediate the action of CCK-8 on enzyme secretion, that low-affinity CCK receptors may mediate the action of CCK on cytosolic calcium that does not involve IP3(1,4,5) and produce the upstroke of the dose-response curve for CCK-8-stimulated amylase secretion and that very low-affinity CCK receptors mediate the actions of CCK on IP3(1,4,5) and cytosolic calcium and produce the downstroke of the dose-response curve for CCK-8-stimulated amylase secretion. Moreover, CCK-JMV-180 is a full agonist for stimulating amylase secretion by acting at low-affinity CCK receptors and is an antagonist at very low-affinity CCK receptors.

Starch digestibility, energy utilization, and growth performance of broilers fed corn-soybean basal diets supplemented with enzymes.

PubMed

Stefanello, C; Vieira, S L; Santiago, G O; Kindlein, L; Sorbara, J O B; Cowieson, A J

2015-10-01

A study was conducted to evaluate the effects of dietary α-amylase and β-xylanase supplementation of corn-soy diets, formulated with or without supplemental phytase, on growth performance, energy utilization, and starch digestibility in broiler chickens. A total of 336 slow-feathering, Cobb × Cobb 500 male broilers were randomly distributed to 6 treatments having 8 replicates of 7 birds each. Birds were fed a common starter diet to d 14 post-hatch (3,050 kcal/kg AMEn, 21.7% CP, 1.05% Ca, and 0.53% nPP). The experimental diets were provided afterwards until d 25. A 2 × 3 factorial arrangement of 2 control diets (basal = corn-soy diet without added phytase or PHY = corn-soy diet formulated with 1,000 phytase units/kg) and 3 carbohydrase supplementations (0, 80 kilo-Novo α-amylase units/kg, or 80 kilo-Novo α-amylase units/kg + 100 fungal β-xylanase units/kg) was used from d 14 to 25. Excreta were collected from 21 to 24 d and all birds were euthanized at 25 d for jejunum and ileum content collection. Samples of feed, excreta, and jejunal and ileal digesta were analyzed for determination of total tract retention and ileal apparent digestibility. No interactions between diet and carbohydrase were observed. Broilers fed diets formulated with phytase or supplemented with amylase + xylanase had higher BW gain (BWG) and lower FCR (P < 0.05) when compared with birds fed diets without carbohydrases. Relative to the basal diet, AMEn was increased (P < 0.01) by 70 kcal/kg and 99 kcal/kg when birds were fed the diet supplemented with amylase and amylase + xylanase, respectively. Starch digestibility in the jejunum and ileum was increased (P < 0.05) by 3.5% and 2.4%, respectively, when birds were fed the diet supplemented with amylase + xylanase. Results from this experiment show that corn-soy diets having phytase and supplemented with amylase and xylanase led to increased growth performance, AMEn, and starch digestibility in broilers. Furthermore, the efficacy of exogenous amylase and xylanase was independent of the presence of microbial phytase. © 2015 Poultry Science Association Inc.

[POLYMORPHISM OF ALFA-AMYLASE AND CONJUGATION IN COMMON WHEAT ENZYME TYPES WITH QUANTITATIVE TRAITS OF PLANTS].

PubMed

Netsvetaev, V P; Bondarenko, L S; Motorina, I P

2015-01-01

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.

Preparation and characterization of a dextran-amylase conjugate.

PubMed

Marshall, J J

1976-07-01

Bacillus amyloliquefaciens alpha-amylase was attached to dextran after activation of the polysaccharide by using a modification of the cyanogen bromide method. The soluble dextran-amylase conjugate was purified by molecular-sieve chromatography. The conjugated enzyme has greater stability than the unmodified enzyme at low pH values, during heat treatment, and on removal of calcium ions with a chelating agent. Attachment of dextran to alpha-amylase did not alter the Michaelis constant of the enzyme acting on starch. The polysaccharide-enzyme conjugate probably consists of a cross-linked aggregate of many dextran and many enzyme molecules, in which a proportion of the enzyme molecules, although not inactivated, are unable to express their activity, except after dextranase treatment.

Bacillus licheniformis BT5.9 Isolated from Changar Hot Spring, Malang, Indonesia, as a Potential Producer of Thermostable α-amylase

PubMed Central

Ibrahim, Darah; Zhu, Han Li; Yusof, Nuraqilah; Isnaeni; Hong, Lim Sheh

2013-01-01

A total of 34 bacterial isolates were obtained from soil samples collected from Changar Hot Spring, Malang, Indonesia. Of these, 13 isolates produced a zone of hydrolysis in starch-nutrient agar medium and generated various amylases in liquid medium. One isolate was selected as the best amylase producer and was identified as Bacillus licheniformis BT5.9. The improvement of culture conditions (initial medium pH of 5.0, cultivation temperature of 50°C, agitation speed of 100 rpm and inoculum size of 1.7 × 109 cells/ml) provided the highest amylase production (0.327 U/ml). PMID:24575243

Baker's asthma in a child.

PubMed

Alonso, E; Ausín, A; Elices, A; Moreno-Escobosa, M; Ibáñez, M; Laso, M

2001-01-01

baker's asthma is a well-known occupational lung disease which usually develops in adults. We report the case of a two years old boy who suffered from asthma, urticaria and atopic dermatitis for twelve months, whose symptoms were associated to visits to his grandfather's bakery. skin prick tests (SPT) were made to dust mites, moulds, flours, alfa-amylase and egg. It was also determined total IgE and specific IgE antibodies to alfa-amylase and flours. Subsequently, a challenge test was carried out with wheat flour. The SPTs were positive to flours, alfa-amylase and egg. The determination of specific IgE antibodies showed 2.64 kU/L to wheat, 0.79 kU/L to glyadin and 2.98 kU/L to alfa-amylase. The patient developed asthma and rhinitis after manipulating wheat flour for 10 min. we demonstrated a type I hypersensitivity to wheat flour and alfa-amylase in a two years old child by SPT, specific IgE antibodies and challenge test. This case in the childhood equivalent of occupational baker's asthma.

Alternative method for quantification of alfa-amylase activity.

PubMed

Farias, D F; Carvalho, A F U; Oliveira, C C; Sousa, N M; Rocha-Bezerrra, L C B; Ferreira, P M P; Lima, G P G; Hissa, D C

2010-05-01

A modification of the sensitive agar diffusion method was developed for macro-scale determination of alfa-amylase. The proposed modifications lower costs with the utilisation of starch as substrate and agar as supporting medium. Thus, a standard curve was built using alfa-amylase solution from Aspergillus oryzae, with concentrations ranging from 2.4 to 7,500 U.mL-1. Clear radial diffusion zones were measured after 4 hours of incubation at 20 A degrees C. A linear relationship between the logarithm of enzyme activities and the area of clear zones was obtained. The method was validated by testing alpha-amylase from barley at the concentrations of 2.4; 60; 300 and 1,500 U.mL-1. The proposed method turned out to be simpler, faster, less expensive and able to determine on a macro-scale alpha-amylase over a wide range (2.4 to 7,500 U.mL-1) in scientific investigation as well as in teaching laboratory activities.

SusG: A Unique Cell-Membrane-Associated [alpha]-Amylase from a Prominent Human Gut Symbiont Targets Complex Starch Molecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koropatkin, Nicole M.; Smith, Thomas J.

SusG is an {alpha}-amylase and part of a large protein complex on the outer surface of the bacterial cell and plays a major role in carbohydrate acquisition by the animal gut microbiota. Presented here, the atomic structure of SusG has an unusual extended, bilobed structure composed of amylase at one end and an unprecedented internal carbohydrate-binding motif at the other. Structural studies further demonstrate that the carbohydrate-binding motif binds maltooligosaccharide distal to, and on the opposite side of, the amylase catalytic site. SusG has an additional starch-binding site on the amylase domain immediately adjacent to the active cleft. Mutagenesis analysismore » demonstrates that these two additional starch-binding sites appear to play a role in catabolism of insoluble starch. However, elimination of these sites has only a limited effect, suggesting that they may have a more important role in product exchange with other Sus components.« less

Cloning, molecular characterization and heterologous expression of AMY1, an alpha-amylase gene from Cryptococcus flavus.

PubMed

Galdino, Alexsandro S; Ulhoa, Cirano J; Moraes, Lídia Maria P; Prates, Maura V; Bloch, Carlos; Torres, Fernando A G

2008-03-01

A Cryptococcus flavus gene (AMY1) encoding an extracellular alpha-amylase has been cloned. The nucleotide sequence of the cDNA revealed an ORF of 1896 bp encoding for a 631 amino acid polypeptide with high sequence identity with a homologous protein isolated from Cryptococcus sp. S-2. The presence of four conserved signature regions, (I) (144)DVVVNH(149), (II) (235)GLRIDSLQQ(243), (III) (263)GEVFN(267), (IV) (327)FLENQD(332), placed the enzyme in the GH13 alpha-amylase family. Furthermore, sequence comparison suggests that the C. flavusalpha-amylase has a C-terminal starch-binding domain characteristic of the CBM20 family. AMY1 was successfully expressed in Saccharomyces cerevisiae. The time course of amylase secretion in S. cerevisiae resulted in a maximal extracellular amylolytic activity (3.93 U mL(-1)) at 60 h of incubation. The recombinant protein had an apparent molecular mass similar to the native enzyme (c. 67 kDa), part of which was due to N-glycosylation.

Cloning and Expression Analysis of the Bombyx mori α-amylase Gene (Amy) from the Indigenous Thai Silkworm Strain, Nanglai

PubMed Central

Ngernyuang, Nipaporn; Kobayashi, Isao; Promboon, Amornrat; Ratanapo, Sunanta; Tamura, Toshiki; Ngernsiri, Lertluk

2011-01-01

α-Amylase is a common enzyme for hydrolyzing starch. In the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), α-amylase is found in both digestive fluid and hemolymph. Here, the complete genomic sequence of the Amy gene encoding α-amylase from a local Thai silkworm, the Nanglai strain, was obtained. This gene was 7981 bp long with 9 exons. The full length Amy cDNA sequence was 1749 bp containing a 1503 bp open reading frame. The ORF encoded 500 amino acid residues. The deduced protein showed 81–54% identity to other insect α-amylases and more than 50% identity to mammalian enzymes. Southern blot analysis revealed that in the Nanglai strain Amy is a single-copy gene. RT- PCR showed that Amy was transcribed only in the foregut. Transgenic B. mori also showed that the Amy promoter activates expression of the transgene only in the foregut. PMID:21529256

The bean. alpha. -amylase inhibitor is encoded by a lectin gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moreno, J.; Altabella, T.; Chrispeels, M.J.

The common bean, Phaseolus vulgaris, contains an inhibitor of insect and mammalian {alpha}-amylases that does not inhibit plant {alpha}-amylase. This inhibitor functions as an anti-feedant or seed-defense protein. We purified this inhibitor by affinity chromatography and found that it consists of a series of glycoforms of two polypeptides (Mr 14,000-19,000). Partial amino acid sequencing was carried out, and the sequences obtained are identical with portions of the derived amino acid sequence of a lectin-like gene. This lectin gene encodes a polypeptide of MW 28,000, and the primary in vitro translation product identified by antibodies to the {alpha}-amylase inhibitor has themore » same size. Co- and posttranslational processing of this polypeptide results in glycosylated polypeptides of 14-19 kDa. Our interpretation of these results is that the bean lectins constitute a gene family that encodes diverse plant defense proteins, including phytohemagglutinin, arcelin and {alpha}-amylase inhibitor.« less

Pancreatitis with normal lipase and amylase in setting of end-stage renal disease.

PubMed

Sharma, Anuj; Masood, Umair; Khan, Babar; Chawla, Kunal; Manocha, Divey

2017-09-01

Pancreatitis with normal lipase and amylase level is a rare phenomenon. This is especially true in patient with end-stage renal disease as lipase and amylase are renally excreted. Literature review reveals previous case report of pancreatitis with normal lipase and amylase level, however, none of them occurred in the setting of end-stage renal disease. Our case is the first such reported case of pancreatitis in such setting. Here we report a 30year old male with past medical history of end-stage renal disease who presented in emergency department with acute abdominal pain. Laboratory work up revealed normal lipase and amylase level. However, radiological work up was consistent with pancreatitis. This case report highlight the importance of taking the overall clinical picture rather than laboratory work up to rule in or rule out the diagnosis of pancreatitis. Furthermore, this should also serve an important reminder for clinicians to further investigate where clinical suspicion for pancreatitis is high. Copyright © 2017 Elsevier Inc. All rights reserved.

Astilbe thunbergii reduces postprandial hyperglycemia in a type 2 diabetes rat model via pancreatic alpha-amylase inhibition by highly condensed procyanidins.

PubMed

Kato, Eisuke; Kushibiki, Natsuka; Inagaki, Yosuke; Kurokawa, Mihoko; Kawabata, Jun

2017-09-01

Type 2 diabetes mellitus (T2DM) is a common global health problem. Prevention of this disease is an important task, and functional food supplements are considered an effective method. We found potent pancreatic α-amylase inhibition in Astilbe thunbergii root extract (AT) and confirmed that AT treatment in a T2DM rat model reduces post-starch administration blood glucose levels. Activity-guided isolation revealed procyanidin (AT-P) as the α-amylase inhibitory component with IC 50  = 1.7 μg/mL against porcine pancreatic α-amylase. Structure analysis of AT-P revealed it is a B-type procyanidin comprised of four types of flavan-3-ols, some with a galloyl group, and catechin attached as the terminal unit. The abundant AT-P content and its comparable α-amylase inhibition to acarbose, the anti-diabetic medicine, suggest that AT is a promising food supplement for diabetes prevention.

Phytonutrients for controlling starch digestion: evaluation of grape skin extract.

PubMed

Miao, Ming; Jiang, Huan; Jiang, Bo; Zhang, Tao; Cui, Steve W; Jin, Zhengyu

2014-02-15

The objective of this work was to evaluate the structure-function relationship between grape skin extract and human α-amylase. The grape skin extract was characterised as resveratrol-3-O-glucoside by RP-HPLC-ESI-MS, which showed strong inhibition towards α-amylase and the IC50 value was 1.35 mg/ml. The kinetic results demonstrated grape skin extract obeyed the non-competitive mode against amylase. Fluorescence data revealed the ability of grape skin binding to amylase belonged to static quenching mechanism with a complex formation and there was only one binding site in α-amylase for grape skin extract. Docking study showed a best pose with total energy value of -118.3 kJ/mol and grape skin extract interacted with side chain of Asp300 with hydrogen bonds and Van der Waals forces. This preliminary observation provides the basis for further evaluation of the suitability of grape skin extract as natural inhibitor with potential health benefits. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

In vitro investigations of α-amylase mediated hydrolysis of cyclodextrins in the presence of ibuprofen, flurbiprofen, or benzo[a]pyrene.

PubMed

Lumholdt, Ludmilla Riisager; Holm, René; Jørgensen, Erling Bonne; Larsen, Kim Lambertsen

2012-11-15

In vitro studies of α-amylase degradation of α-, β- and γ-cyclodextrins and 2-hydroxypropyl-β- and -γ-cyclodextrins were investigated spectrophotometrically by measuring the formation of reducing sugars, the reaction products of α-amylase degradation. This was done to evaluate potential degradation and thereby biological conversion of the cyclodextrins if dosed orally, as the intestinal tract contains α-amylase for digestive purposes. The results demonstrated that only γ- and 2-hydroxypropyl-γ-cyclodextrins can be degraded by α-amylase to a relevant extent, that is, γ- and 2-hydroxypropyl-γ-cyclodextrins have different biopharmaceutical behaviours than the other evaluated cyclodextrins. The rate of degradation was affected by the addition of the inclusion complex forming additives flurbiprofen, ibuprofen and benzo[a]pyrene. This effect between the degradation dynamics and the included additives was caused by a correlation between solubility of the additives and the stability of the complex. Copyright © 2012 Elsevier Ltd. All rights reserved.

Molecular improvements in microbial α-amylases for enhanced stability and catalytic efficiency.

PubMed

Sindhu, Raveendran; Binod, Parameswaran; Madhavan, Aravind; Beevi, Ummalyma Sabeela; Mathew, Anil Kuruvilla; Abraham, Amith; Pandey, Ashok; Kumar, Vinod

2017-12-01

α-Amylases is one of the most important industrial enzyme which contributes to 25% of the industrial enzyme market. Though it is produced by plant, animals and microbial source, those from microbial source seems to have potential applications due to their stability and economic viability. However a large number of α-amylases from different sources have been detailed in the literature, only few numbers of them could withstand the harsh industrial conditions. Thermo-stability, pH tolerance, calcium independency and oxidant stability and starch hydrolyzing efficiency are the crucial qualities for α-amylase in starch based industries. Microbes can be genetically modified and fine tuning can be done for the production of enzymes with desired characteristics for specific applications. This review focuses on the native and recombinant α-amylases from microorganisms, their heterologous production and the recent molecular strategies which help to improve the properties of this industrial enzyme. Copyright © 2017 Elsevier Ltd. All rights reserved.

Solid State Fermentation of a Raw Starch Digesting Alkaline Alpha-Amylase from Bacillus licheniformis RT7PE1 and Its Characteristics.

PubMed

Tabassum, Romana; Khaliq, Shazia; Rajoka, Muhammad Ibrahim; Agblevor, Foster

2014-01-01

The thermodynamic and kinetic properties of solids state raw starch digesting alpha amylase from newly isolated Bacillus licheniformis RT7PE1 strain were studied. The kinetic values Q p , Y p/s , Y p/X , and q p were proved to be best with 15% wheat bran. The molecular weight of purified enzyme was 112 kDa. The apparent K m and V max values for starch were 3.4 mg mL(-1) and 19.5 IU mg(-1) protein, respectively. The optimum temperature and pH for α -amylase were 55°C, 9.8. The half-life of enzyme at 95°C was 17h. The activation and denaturation activation energies were 45.2 and 41.2 kJ mol(-1), respectively. Both enthalpies (ΔH (∗)) and entropies of activation (ΔS (∗)) for denaturation of α -amylase were lower than those reported for other thermostable α -amylases.

Solid State Fermentation of a Raw Starch Digesting Alkaline Alpha-Amylase from Bacillus licheniformis RT7PE1 and Its Characteristics

PubMed Central

Tabassum, Romana; Khaliq, Shazia; Rajoka, Muhammad Ibrahim; Agblevor, Foster

2014-01-01

The thermodynamic and kinetic properties of solids state raw starch digesting alpha amylase from newly isolated Bacillus licheniformis RT7PE1 strain were studied. The kinetic values Q p, Y p/s, Y p/X, and q p were proved to be best with 15% wheat bran. The molecular weight of purified enzyme was 112 kDa. The apparent K m and V max values for starch were 3.4 mg mL−1 and 19.5 IU mg−1 protein, respectively. The optimum temperature and pH for α-amylase were 55°C, 9.8. The half-life of enzyme at 95°C was 17h. The activation and denaturation activation energies were 45.2 and 41.2 kJ mol−1, respectively. Both enthalpies (ΔH ∗) and entropies of activation (ΔS ∗) for denaturation of α-amylase were lower than those reported for other thermostable α-amylases. PMID:24587909

Zinc oxide nanoparticles as novel alpha-amylase inhibitors

NASA Astrophysics Data System (ADS)

Dhobale, Sandip; Thite, Trupti; Laware, S. L.; Rode, C. V.; Koppikar, Soumya J.; Ghanekar, Ruchika-Kaul; Kale, S. N.

2008-11-01

Amylase inhibitors, also known as starch blockers, contain substances that prevent dietary starches from being absorbed by the body via inhibiting breakdown of complex sugars to simpler ones. In this sense, these materials are projected as having potential applications in diabetes control. In this context, we report on zinc oxide nanoparticles as possible alpha-amylase inhibitors. Zinc oxide nanoparticles have been synthesized using soft-chemistry approach and 1-thioglycerol was used as a surfactant to yield polycrystalline nanoparticles of size ˜18 nm, stabilized in wurtzite structure. Conjugation study and structural characterization have been done using x-ray diffraction technique, Fourier transform infrared spectroscopy, UV-visible spectroscopy, and transmission electron microscopy. Cytotoxicity studies on human fibrosarcoma (HT-1080) and skin carcinoma (A-431) cell lines as well as mouse primary fibroblast cells demonstrate that up to a dose of 20 μg/ml, ZnO nanoparticles are nontoxic to the cells. We report for the first time the alpha-amylase inhibitory activity of ZnO nanoparticles wherein an optimum dose of 20 μg/ml was sufficient to exhibit 49% glucose inhibition at neutral pH and 35 °C temperature. This inhibitory activity was similar to that obtained with acarbose (a standard alpha-amylase inhibitor), thereby projecting ZnO nanoparticles as novel alpha-amylase inhibitors.

A hyper-thermostable α-amylase from Pyrococcus furiosus accumulates in Nicotiana tabacum as functional aggregates.

PubMed

Zhu, Hong; Reynolds, L Bruce; Menassa, Rima

2017-06-19

Alpha amylase hydrolyzes α-bonds of polysaccharides such as starch and produces malto-oligosaccharides. Its starch saccharification applications make it an essential enzyme in the textile, food and brewing industries. Commercially available α-amylase is mostly produced from Bacillus or Aspergillus. A hyper-thermostable and Ca 2++ independent α-amylase from Pyrococcus furiosus (PFA) expressed in E.coli forms insoluble inclusion bodies and thus is not feasible for industrial applications. We expressed PFA in Nicotiana tabacum and found that plant-produced PFA forms functional aggregates with an accumulation level up to 3.4 g/kg FW (fresh weight) in field conditions. The aggregates are functional without requiring refolding and therefore have potential to be applied as homogenized plant tissue without extraction or purification. PFA can also be extracted from plant tissue upon dissolution in a mild reducing buffer containing SDS. Like the enzyme produced in P. furiosus and in E. coli, plant produced PFA preserves hyper-thermophilicity and hyper-thermostability and has a long shelf life when stored in lyophilized leaf tissue. With tobacco's large biomass and high yield, hyper-thermostable α-amylase was produced at a scale of 42 kg per hectare. Tobacco may be a suitable bioreactor for industrial production of active hyperthermostable alpha amylase.

Enhanced Production and Characterization of a Solvent Stable Amylase from Solvent Tolerant Bacillus tequilensis RG-01: Thermostable and Surfactant Resistant

PubMed Central

Tiwari, Soni; Shukla, Neha; Mishra, Pooja; Gaur, Rajeeva

2014-01-01

Ten bacterial strains isolated from the soil samples in the presence of cyclohexane were screened for amylase production. Among them, culture RG-01 was adjudged as the best amylase producer and was identified as Bacillus tequilensis from MTCC, Chandigarh. The isolate showed maximum amylase production (8100 U/mL) in the presence of starch, peptone, and Ca2+ ions at 55°C pH 7.0 within 24 h of incubation. The enzyme was stable in the presence of n-dodecane, isooctane, n-decane, xylene, toluene, n-hexane, n-butanol, and cyclohexane, respectively. The presence of benzene, methanol, and ethanol marginally reduced the amylase stability, respectively. The enzyme was showed it 100% activity at 55°C and pH 7.0 with 119% and 127% stability at 55°C and pH 7.0, respectively. The enzyme was also stable in the presence of SDS, Tween-40, Tween-60, and Tween-80 (1%) and was found stimulatory effect, respectively. Only Triton-X-100 showed a moderate inhibitory effect (5%) on amylase activity. This isolate (Bacillus tequilensis RG-01) may be useful in several industrial applications owing to its thermotolerant and organic solvents and surfactants resistance characteristics. PMID:25401163

Salivary Alpha-Amylase Correlates with Subjective Heat Pain Perception.

PubMed

Wittwer, Amrei; Krummenacher, Peter; La Marca, Roberto; Ehlert, Ulrike; Folkers, Gerd

2016-06-01

Self-reports of pain are important for an adequate therapy. This is a problem with patients and infants who are restricted in providing an accurate verbal estimation of their pain. Reliable, real-time, economical, and non-invasive physiological correlates might contribute to a more comprehensive description of pain. Salivary alpha-amylase constitutes one candidate biomarker, which reflects predominantly sympathetic nervous system alterations under stressful conditions and can be measured non-invasively. The current study investigated the effects of acute heat pain on salivary alpha-amylase activity. Heat pain tolerance was measured on the non-dominant forearm. Participants completed visual analog scales on pain intensity and unpleasantness. Saliva samples were collected directly after pain induction. Twenty-seven healthy volunteers were recruited for this study. While salivary alpha-amylase levels correlated positively with intensity and unpleasantness ratings in response to acute heat pain stimuli, there was no corresponding association with pain tolerance. Salivary alpha-amylase is suggested to be an indirect physiologic correlate of subjective heat pain perception. Future studies should address the role of salivary alpha-amylase depending on the origin of pain, the concerned tissue, and other pain assessment methods. © 2016 American Academy of Pain Medicine. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Isolation and characterization of a cold-active, alkaline, detergent stable α-amylase from a novel bacterium Bacillus subtilis N8.

PubMed

Arabacı, Nihan; Arıkan, Burhan

2018-05-28

A cold-active alkaline amylase producer Bacillus subtilis N8 was isolated from soil samples. Amylase synthesis optimally occurred at 15°C and pH 10.0 on agar plates containing starch. The molecular weight of the enzyme was found to be 205 kDa by performing SDS-PAGE. While the enzyme exhibited the highest activity at 25°C and pH 8.0, it was highly stable in alkaline media (pH 8.0-12.0) and retained 96% of its original activity at low temperatures (10-40°C) for 24 hr. While the amylase activity increased in the presence of β-mercaptoethanol (103%); Ba 2+ , Ca 2+ , Na + , Zn 2+ , Mn 2+ , H 2 O 2 , and Triton X-100 slightly inhibited the activity. The enzyme showed resistance to some denaturants: such as SDS, EDTA, and urea (52, 65, and 42%, respectively). N8 α-amylase displayed the maximum remaining activity of 56% with 3% NaCl. The major final products of starch were glucose, maltose, and maltose-derived oligosaccharides. This novel cold-active α-amylase has the potential to be used in the industries of detergent and food, bioremediation process and production of prebiotics.

Inhibition of α-amylase and α-glucosidase activities by ethanolic extract of Telfairia occidentalis (fluted pumpkin) leaf

PubMed Central

Oboh, G; Akinyemi, AJ; Ademiluyi, AO

2012-01-01

Objective To investigate the inhibitory effect of Telfairia occidentalis Hook f. (Curcubitaceae) (T. occidentalis) leaf on key enzyme linked to type-2 diabetes (α - amylase and α - glucosidase) as well as assess the effect of blanching (a commonly practiced food processing technique) of the vegetable on these key enzymes. Methods Fresh leaves of T. occidentalis were blanched in hot water for 10 minutes, and the extracts of both the fresh and blanched vegetables were prepared and used for subsequent analysis. The inhibitory effect of the extract on α - amylase and α - glucosidase activities as well as some antioxidant parameter was determined in vitro. Results The result revealed that unprocessed T. occidentalis leaf reduce Fe3+ to Fe2+ and also inhibited α - amylase and α - glucosidase activities in a dose dependent manner. However, blanching of the leafy vegetables caused a significant (P<0.05) increase in the antioxidant properties but decrease their ability to inhibit α - amylase and α - glucosidase activities. Conclusions This antioxidant properties and enzyme inhibition could be part of the mechanism by which they are used in the treatment/prevention of type-2 diabetes. However, the blanched vegetable reduces their ability to inhibit both α - amylase and α - glucosidase activity in vitro. PMID:23570004

Induction of a Mitosis Delay and Cell Lysis by High-Level Secretion of Mouse α-Amylase from Saccharomyces cerevisiae

PubMed Central

Wang, Bi-Dar; Kuo, Tsong-Teh

2001-01-01

Some foreign proteins are produced in yeast in a cell cycle-dependent manner, but the cause of the cell cycle dependency is unknown. In this study, we found that Saccharomyces cerevisiae cells secreting high levels of mouse α-amylase have elongated buds and are delayed in cell cycle completion in mitosis. The delayed cell mitosis suggests that critical events during exit from mitosis might be disturbed. We found that the activities of PP2A (protein phosphatase 2A) and MPF (maturation-promoting factor) were reduced in α-amylase-oversecreting cells and that these cells showed a reduced level of assembly checkpoint protein Cdc55, compared to the accumulation in wild-type cells. MPF inactivation is due to inhibitory phosphorylation on Cdc28, as a cdc28 mutant which lacks an inhibitory phosphorylation site on Cdc28 prevents MPF inactivation and prevents the defective bud morphology induced by overproduction of α-amylase. Our data also suggest that high levels of α-amylase may downregulate PPH22, leading to cell lysis. In conclusion, overproduction of heterologous α-amylase in S. cerevisiae results in a negative regulation of PP2A, which causes mitotic delay and leads to cell lysis. PMID:11472949

The enhanced inhibition of water extract of black tea under baking treatment on α-amylase and α-glucosidase.

PubMed

Tong, Da-Peng; Zhu, Ke-Xue; Guo, Xiao-Na; Peng, Wei; Zhou, Hui-Ming

2018-02-01

This paper studied the inhibition of water extract of natural or baked black tea on the activity of α-amylase and α- glucosidase. Baking treatment was found to be one effective way to enhance the inhibition of black tea on both α-amylase and α- glucosidase, and IC 50 of water extract of baked black tea (BBTWE) were 1.213mg/mL and 4.190mg/mL, respectively, while IC 50 of water extract of black tea (BTWE) were 1.723mg/mL and 6.056mg/mL, respectively. This study further studied the mechanism of the effect of water extract on α-amylase and α- glucosidase using HPLC, circular dichroism, and synchronous fluorescence. HPLC analysis of tea polyphenols showed that the content of tea polyphenols with low polarity increased after baking. In addition, BBTWE had higer abilty on decreasing the hydrophobicity of tryptophan residues than BTWE for both α-amylase and α- glucosidase.The increase of α-helix proportion of α-amylase when treated with BBTWE was more obvious than that when treated with BTWE. In a word, thermal process of baked foods may be beneficial for tea polyphenols to reduce the rate of starch digestion. Copyright © 2017 Elsevier B.V. All rights reserved.

Β-Amylase from Starchless Seeds of Trigonella Foenum-Graecum and Its Localization in Germinating Seeds

PubMed Central

Srivastava, Garima; Kayastha, Arvind M.

2014-01-01

Fenugreek (Trigonella foenum-graecum) seeds do not contain starch as carbohydrate reserve. Synthesis of starch is initiated after germination. A β-amylase from ungerminated fenugreek seeds was purified to apparent electrophoretic homogeneity. The enzyme was purified 210 fold with specific activity of 732.59 units/mg. Mr of the denatured enzyme as determined from SDS-PAGE was 58 kD while that of native enzyme calculated from size exclusion chromatography was 56 kD. Furthermore, its identity was confirmed to be β-amylase from MALDI-TOF analysis. The optimum pH and temperature was found to be 5.0 and 50°C, respectively. Starch was hydrolyzed at highest rate and enzyme showed a Km of 1.58 mg/mL with it. Antibodies against purified Fenugreek β-amylase were generated in rabbits. These antibodies were used for localization of enzyme in the cotyledon during different stages of germination using fluorescence and confocal microscopy. Fenugreek β-amylase was found to be the major starch degrading enzyme depending on the high amount of enzyme present as compared to α-amylase and also its localization at the periphery of amyloplasts. A new finding in terms of its association with protophloem was observed. Thus, this enzyme appears to be important for germination of seeds. PMID:24551136

Characterization of a Novel Maltose-Forming α-Amylase from Lactobacillus plantarum subsp. plantarum ST-III.

PubMed

Jeon, Hye-Yeon; Kim, Na-Ri; Lee, Hye-Won; Choi, Hye-Jeong; Choung, Woo-Jae; Koo, Ye-Seul; Ko, Dam-Seul; Shim, Jae-Hoon

2016-03-23

A novel maltose (G2)-forming α-amylase from Lactobacillus plantarum subsp. plantarum ST-III was expressed in Escherichia coli and characterized. Analysis of conserved amino acid sequence alignments showed that L. plantarum maltose-producing α-amylase (LpMA) belongs to glycoside hydrolase family 13. The recombinant enzyme (LpMA) was a novel G2-producing α-amylase. The properties of purified LpMA were investigated following enzyme purification. LpMA exhibited optimal activity at 30 °C and pH 3.0. It produced only G2 from the hydrolysis of various substrates, including maltotriose (G3), maltopentaose (G5), maltosyl β-cyclodextrin (G2-β-CD), amylose, amylopectin, and starch. However, LpMA was unable to hydrolyze cyclodextrins. Reaction pattern analysis using 4-nitrophenyl-α-d-maltopentaoside (pNPG5) demonstrated that LpMA hydrolyzed pNPG5 from the nonreducing end, indicating that LpMA is an exotype α-amylase. Kinetic analysis revealed that LpMA had the highest catalytic efficiency (kcat/Km ratio) toward G2-β-CD. Compared with β-amylase, a well-known G2-producing enzyme, LpMA produced G2 more efficiently from liquefied corn starch due to its ability to hydrolyze G3.

Purification and characterization of midgut α-amylase in a predatory bug, Andralus spinidens.

PubMed

Sorkhabi-Abdolmaleki, Sahar; Zibaee, Arash; Hoda, Hassan; Fazeli-Dinan, Mahmoud

2014-05-13

α-Amylases are widespread enzymes that catalyze endohydrolysis of long α-1,4-glucan chains such as starch and glycogen. The highest amylolytic activity was found in 5th instar nymphs and midgut of the predatory bug, Andrallus spinidens F. (Hemiptera: Pentatomidae). The α-amylase was purified following a three-step procedure. The purified α-amylase had a specific activity of 13.46 U/mg protein, recovery of 4.21, purification fold of 13.87, and molecular weight of 21.3 kDa. The enzyme had optimal pH and temperature of 7 and 45°C, respectively. Na+, Mn+, Mg2+, and Zn2+ significantly decreased activity of the purified α-amylase, but some concentrations of K+, Ca2+, and Cu2+ had the opposite effect. EDTA, EGTA, and DTC significantly decreased enzymatic activity, showing the presence of metal ions in the catalytic site of the enzyme. Kinetic parameters of the purified α-amylase showed a Km of 3.71% in starch and 4.96% for glycogen, suggesting that the enzyme had a higher affinity for starch. This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits unrestricted use, provided that the paper is properly attributed.

Effects of 50 Hz magnetic field exposure on the stress marker α-amylase in the rat mammary gland.

PubMed

Fedrowitz, Maren; Hass, Ralf; Löscher, Wolfgang

2012-07-01

Concerns about adverse health effects of environmental exposure to 50/60 Hz magnetic fields (MF) have initiated numerous studies on laboratory animals with varying outcomes. Previously, we reported that rat strains responded differently to MF regarding mammary cell proliferation and tumor development indicating that (epi)genetic factors might influence MF effects in the breast tissue, yet without any identified mechanism. In the present study, α-amylase, recently introduced as a stress marker in humans, was investigated in the mammary gland of Fischer 344 (F344) and Lewis rats, two strains with distinct stress sensitivity. F344 rats were sham- and MF-exposed (50 Hz, 100 μT) for different time periods, Lewis rats for two weeks. For comparison, diethylstilbestrol was administered at single or repeated doses. α-Amylase activity was significantly enhanced in the F344 mammary glands after 2 and 4 weeks of MF, whereas no reproducible effects were observed in Lewis rats. Diethylstilbestrol increased the α-amylase after repeated dosing. Although α-amylase represents a difficult parameter in animal studies because of its stress sensitivity, it should be considered for investigations in humans and cell cultures as a biomarker for MF susceptibility and a target to examine possible MF mechanisms since α-amylase affects cell growth.

Characterizing Novel Thermophilic Amylase Producing Bacteria From Taptapani Hot Spring, Odisha, India

PubMed Central

Sen, Sudip Kumar; Raut, Sangeeta; Satpathy, Soumya; Rout, Prangya Ranjan; Bandyopadhyay, Bidyut; Das Mohapatra, Pradeep Kumar

2014-01-01

Background: Amylases play a vital role in biotechnological studies and rank an important position in the world enzyme market (25% to 33%). Bioprocess method of amylase production is more effective than the other sources, since the technique is easy, cost effective, fast, and the enzymes of required properties can be procured. Objectives: The current study aimed to report the characteristics of novel amylase producing bacterial strains isolated from Taptapani hot spring, Odisha, India. Materials and Methods: Bacterial strains were isolated by dilution plating method from the water samples collected from Taptapani Hot Spring, Odisha and screened for amylase production through starch hydrolysis. The bacterial isolates were identified morphologically, biochemically, and finally by 16S rDNA profiling. Results: Based on the morphological, physiological, biochemical characteristics and the molecular characterization, the isolates SS1, SS2, and SS3 were identified as Bacillus barbaricus, Aeromonas veroni, and Stenotrophomonas maltophilia, respectively. The approximate molecular weight of enzymes from SS1, SS2, and SS3 strains were 19 kDa, 56 kDa and 49 kDa, respectively. Conclusions: The current report isolates, characterizes, and demonstrates the novel heat-adapted amylase-producing bacteria SS1, SS2 and SS3 from Taptapani hot spring, indicating its potentiality and stability under acidic conditions. PMID:25741425

Efficient Expression of Maltohexaose-Forming α-Amylase from Bacillus stearothermophilus in Brevibacillus choshinensis SP3 and Its Use in Maltose Production

PubMed Central

Duan, Xuguo

2017-01-01

The maltohexaose-forming, Ca2+-independent α-amylase gene from Bacillus stearothermophilus (AmyMH) was efficiently expressed in Brevibacillus choshinensis SP3. To improve the production of AmyMH in B. choshinensis SP3, the temperature and initial pH of culture medium were optimized. In addition, single-factor and response surface methodologies were pursued to optimize culture medium. Addition of proline to the culture medium significantly improved the production of recombinant α-amylase in B. choshinensis SP3. This improvement may result from improved cellular integrity of recombinant B. choshinensis SP3 in existence of proline. Culture medium optimization resulted in an 8-fold improvement in α-amylase yield, which reached 1.72 × 104 U·mL−1. The recombinant α-amylase was applied to the production of maltose on a laboratory scale. A maltose content of 90.72%, which could be classified as an extremely high maltose syrup, could be achieved using 15% (m/v) corn starch as the substrate. This study demonstrated that the B. choshinensis SP3 expression system was able to produce substantial quantities of recombinant α-amylase that has potential application in the starch industry. PMID:29250543

Determining the binding affinities of phenolic compounds to proteins by quenching of the intrinsic tryptophan fluorescence.

PubMed

Rawel, Harshadrai M; Frey, Simone K; Meidtner, Karina; Kroll, Jürgen; Schweigert, Florian J

2006-08-01

The noncovalent binding of selected phenolic compounds (chlorogenic-, ferulic-, gallic acid, quercetin, rutin, and isoquercetin) to proteins (HSA, BSA, soy glycinin, and lysozyme) was studied by an indirect method applying the quenching of intrinsic tryptophan fluorescence. From the data obtained, the binding constants were calculated by nonlinear regression (one site binding; y = Bx/k + x). It has been reported that tannins inhibit human salivary amylase and that these complexes may reduce the development of cariogenic plaques. Further, amylase contains two tryptophan residues in its active site. Therefore, in a second part of the study involving 31 human subjects, evidence was sought for noncovalent interactions between the phenols of green tea and saliva proteins as measured by the fluorescence intensity. Amylase activity was determined before and after the addition of green tea to saliva of 31 subjects. Forty percent of the subjects showed an increase in amylase activity contrary to studies reporting only a decrease in activity. The interactions of tannin with amylase result in a decrease of its activity. It still remains to be elucidated why amylase does not react uniformly under conditions of applying green tea to saliva. Further, in terms of using phenols as caries inhibitors this finding should be of importance.

Branch pattern of starch internal structure influences the glucogenesis by mucosal Nt-maltase-glucoamylase.

PubMed

Lin, Amy Hui-Mei; Ao, Zihua; Quezada-Calvillo, Roberto; Nichols, Buford L; Lin, Chi-Tien; Hamaker, Bruce R

2014-10-13

To produce sufficient amounts of glucose from food starch, both α-amylase and mucosal α-glucosidases are required. We found previously that the digestion rate of starch is influenced by its susceptibility to mucosal α-glucosidases. In the present study, six starches and one glycogen were pre-hydrolyzed by α-amylase for various time periods, and then further hydrolyzed with the mucosal α-glucosidase, the N-terminal subunit of maltase-glucoamylase (Nt-MGAM), to generate free glucose. Results showed that α-amylase amplified the Nt-MGAM glucogenesis, and that the amplifications differed in various substrates. The amount of branches within α-amylase hydrolysate substrates was highly related to the rate of Nt-MGAM glucogenesis. After de-branching, the hydrolysates showed three fractions, Fraction 1, 2, and 3, in size exclusion chromatographs. We found that the α-amylase hydrolysates with higher quantity of the Fraction 3 (molecules with relatively short chain-length) and shorter average chain-length of this fraction had lower rates of Nt-MGAM glucogenesis. This study revealed that the branch pattern of α-amylase hydrolysates modulates glucose release by Nt-MGAM. It further supported the hypothesis that the internal structure of starch affects its digestibility at the mucosal α-glucosidase level. Published by Elsevier Ltd.

Salivary cortisol and α-amylase: subclinical indicators of stress as cardiometabolic risk.

PubMed

Cozma, S; Dima-Cozma, L C; Ghiciuc, C M; Pasquali, V; Saponaro, A; Patacchioli, F R

2017-02-06

Currently, the potential for cardiovascular (CV) stress-induced risk is primarily based on the theoretical (obvious) side effects of stress on the CV system. Salivary cortisol and α-amylase, produced respectively by the hypothalamus-pituitary-adrenal (HPA) axis and the sympathetic-adrenomedullary (SAM) system during stress response, are still not included in the routine evaluation of CV risk and require additional and definitive validation. Therefore, this article overviews studies published between 2010 and 2015, in which salivary cortisol and α-amylase were measured as stress biomarkers to examine their associations with CV/CMR (cardiometabolic risk) clinical and subclinical indicators. A comprehensive search of PubMed, Web of Science and Scopus electronic databases was performed, and 54 key articles related to the use of salivary cortisol and α-amylase as subclinical indicators of stress and CV/CMR factors, including studies that emphasized methodological biases that could influence the accuracy of study outcomes, were ultimately identified. Overall, the biological impact of stress measured by salivary cortisol and α-amylase was associated with CV/CMR factors. Results supported the use of salivary cortisol and α-amylase as potential diagnostic tools for detecting stress-induced cardiac diseases and especially to describe the mechanisms by which stress potentially contributes to the pathogenesis and outcomes of CV diseases.

Effects of Abscisic Acid and Ethylene on the Gibberellic Acid-Induced Synthesis of α-Amylase by Isolated Wheat Aleurone Layers 1

PubMed Central

Varty, Keith; Arreguín, Barbarín L.; Gómez, Miguel T.; López, Pablo Jaime T.; Gómez, Miguel Angel L.

1983-01-01

Gibberellic acid-induced α-amylase synthesis in wheat aleurone layers (Triticum aestivum L. var Potam S-70) escaped from transcriptional control 30 h after addition of the hormone, as evidenced by the tissue's loss of susceptibility to cordycepin. Abscisic acid inhibited the accumulation of α-amylase activity when added to the tissue during this cordycepin-insensitive phase of enzyme induction. α-Amylase synthesis was not restored by the addition of cordycepin, indicating that the response to abscisic acid was not dependent upon the continuous synthesis of a short lived RNA. When ethylene was added simultaneously or some time after abscisic acid, the accumulation of α-amylase activity was sustained or quickly restored. The loss of susceptibility to cordycepin was completely prevented when aleurone layers were incubated with a combination of gibberellic and abscisic acids from the start of the induction period. This effect of abscisic acid was not reversed by ethylene. On the basis of these observations, it is suggested that abscisic acid inhibits both the transcription and translation of α-amylase mRNA, and that only the latter site of action is susceptible to reversal by ethylene. The rate of incorporation of [methyl-14C]choline into phospholipids was also inhibited by abscisic acid. Ethylene reversed this effect. The effects of abscisic acid and ethylene on phospholipid synthesis were not dependent upon the presence of gibberellic acid. No direct relationship was found between the control of α-amylase synthesis and membrane formation by abscisic acid and ethylene. PMID:16663284

Abdominal Drainage and Amylase Measurement for Detection of Leakage After Gastrectomy for Gastric Cancer.

PubMed

Schots, Judith P M; Luyer, Misha D P; Nieuwenhuijzen, Grard A P

2018-05-07

To investigate the value of daily measurement of drain amylase for detecting leakage in gastric cancer surgery. This was a retrospective analysis including all patients who underwent a gastrectomy for gastric cancer. From January 2013 until December 2015, an intra-abdominal drain was routinely placed. Drain amylase was measured daily. Receiver operator characteristic curves were created to assess the ability of amylase to predict leakage. Sensitivity, specificity, and negative and positive predictive value of amylase in drain fluid were determined. Leakage of the gastrojejunostomy or esophagojejunostomy, enteroenterostomy, duodenal stump, or pancreas was diagnosed by CT scan, endoscopy, or during re-operation. From January 2016 until April 2017, no drain was inserted. Surgical outcome and postoperative complications were compared between both groups. Median drain amylase concentrations were higher for each postoperative day in patients with leakage. The optimal cutoff value was 1000 IU/L (sensitivity 77.8%, specificity 98.2%, negative predictive value 96.6%). Sixty-seven consecutive procedures were performed with a drain and 40 procedures without. No differences in group characteristics were observed except for gender. Fourteen patients (13.1%) had a leakage. The incidence and severity of leakage were not different between the patients with and without a drain. There was no significant difference in time to diagnosis (1 vs. 0 days; p 0.34), mortality rate (7.5 vs. 2.5%; p 0.41), and median length of hospital stay (9 days in both groups; p 0.46). Daily amylase measurement in drain fluid does not influence the early recognition and management of leakage in gastric cancer surgery.

Reflection on design and testing of pancreatic alpha-amylase inhibitors: an in silico comparison between rat and rabbit enzyme models

PubMed Central

2012-01-01

Background Inhibitors of pancreatic alpha-amylase are potential drugs to treat diabetes and obesity. In order to find compounds that would be effective amylase inhibitors, in vitro and in vivo models are usually used. The accuracy of models is limited, but these tools are nonetheless valuable. In vitro models could be used in large screenings involving thousands of chemicals that are tested to find potential lead compounds. In vivo models are still used as preliminary mean of testing compounds behavior in the whole organism. In the case of alpha-amylase inhibitors, both rats and rabbits could be chosen as in vivo models. The question was which animal could present more accuracy with regard to its pancreatic alpha-amylase. Results As there is no crystal structure of these enzymes, a molecular modeling study was done in order to compare the rabbit and rat enzymes with the human one. The overall result is that rabbit enzyme could probably be a better choice in this regard, but in the case of large ligands, which could make putative interactions with the −4 subsite of pancreatic alpha-amylase, interpretation of results should be made cautiously. Conclusion Molecular modeling tools could be used to choose the most suitable model enzyme that would help to identify new enzyme inhibitors. In the case of alpha-amylase, three-dimensional structures of animal enzymes show differences with the human one which should be taken into account when testing potential new drugs. PMID:23352052

FGF7 and cell density are required for final differentiation of pancreatic amylase-positive cells from human ES cells.

PubMed

Takizawa-Shirasawa, Sakiko; Yoshie, Susumu; Yue, Fengming; Mogi, Akimi; Yokoyama, Tadayuki; Tomotsune, Daihachiro; Sasaki, Katsunori

2013-12-01

The major molecular signals of pancreatic exocrine development are largely unknown. We examine the role of fibroblast growth factor 7 (FGF7) in the final induction of pancreatic amylase-containing exocrine cells from induced-pancreatic progenitor cells derived from human embryonic stem (hES) cells. Our protocol consisted in three steps: Step I, differentiation of definitive endoderm (DE) by activin A treatment of hES cell colonies; Step II, differentiation of pancreatic progenitor cells by re-plating of the cells of Step I onto 24-well plates at high density and stimulation with all-trans retinoic acid; Step III, differentiation of pancreatic exocrine cells with a combination of FGF7, glucagon-like peptide 1 and nicotinamide. The expression levels of pancreatic endodermal markers such as Foxa2, Sox17 and gut tube endoderm marker HNF1β were up-regulated in both Step I and II. Moreover, in Step III, the induced cells expressed pancreatic markers such as amylase, carboxypeptidase A and chymotrypsinogen B, which were similar to those in normal human pancreas. From day 8 in Step III, cells immunohistochemically positive for amylase and for carboxypeptidase A, a pancreatic exocrine cell product, were induced by FGF7. Pancreatic progenitor Pdx1-positive cells were localized in proximity to the amylase-positive cells. In the absence of FGF7, few amylase-positive cells were identified. Thus, our three-step culture protocol for human ES cells effectively induces the differentiation of amylase- and carboxypeptidase-A-containing pancreatic exocrine cells.

Close relationship of a novel Flavobacteriaceae α-amylase with archaeal α-amylases and good potentials for industrial applications

PubMed Central

2014-01-01

Background Bioethanol production from various starchy materials has received much attention in recent years. α-Amylases are key enzymes in the bioconversion process of starchy biomass to biofuels, food or other products. The properties of thermostability, pH stability, and Ca-independency are important in the development of such fermentation process. Results A novel Flavobacteriaceae Sinomicrobium α-amylase (FSA) was identified and characterized from genomic analysis of a novel Flavobacteriaceae species. It is closely related with archaeal α-amylases in the GH13_7 subfamily, but is evolutionary distant with other bacterial α-amylases. Based on the conserved sequence alignment and homology modeling, with minor variation, the Zn2+- and Ca2+-binding sites of FSA were predicated to be the same as those of the archaeal thermophilic α-amylases. The recombinant α-amylase was highly expressed and biochemically characterized. It showed optimum activity at pH 6.0, high enzyme stability at pH 6.0 to 11.0, but weak thermostability. A disulfide bond was introduced by site-directed mutagenesis in domain C and resulted in the apparent improvement of the enzyme activity at high temperature and broad pH range. Moreover, about 50% of the enzyme activity was detected under 100°C condition, whereas no activity was observed for the wild type enzyme. Its thermostability was also enhanced to some extent, with the half-life time increasing from 25 to 55 minutes at 50°C. In addition, after the introduction of the disulfide bond, the protein became a Ca-independent enzyme. Conclusions The improved stability of FSA suggested that the domain C contributes to the overall stability of the enzyme under extreme conditions. In addition, successfully directed modification and special evolutionary status of FSA imply its directional reconstruction potentials for bioethanol production, as well as for other industrial applications. PMID:24485248

Dynamics of the Streptococcus gordonii Transcriptome in Response to Medium, Salivary α-Amylase, and Starch.

PubMed

Haase, Elaine M; Feng, Xianghui; Pan, Jiachuan; Miecznikowski, Jeffrey C; Scannapieco, Frank A

2015-08-15

Streptococcus gordonii, a primary colonizer of the tooth surface, interacts with salivary α-amylase via amylase-binding protein A (AbpA). This enzyme hydrolyzes starch to glucose, maltose, and maltodextrins that can be utilized by various oral bacteria for nutrition. Microarray studies demonstrated that AbpA modulates gene expression in response to amylase, suggesting that the amylase-streptococcal interaction may function in ways other than nutrition. The goal of this study was to explore the role of AbpA in gene regulation through comparative transcriptional profiling of wild-type KS1 and AbpA(-) mutant KS1ΩabpA under various environmental conditions. A portion of the total RNA isolated from mid-log-phase cells grown in 5% CO2 in (i) complex medium with or without amylase, (ii) defined medium (DM) containing 0.8% glucose with/without amylase, and (iii) DM containing 0.2% glucose and amylase with or without starch was reverse transcribed to cDNA and the rest used for RNA sequencing. Changes in the expression of selected genes were validated by quantitative reverse transcription-PCR. Maltodextrin-associated genes, fatty acid synthesis genes and competence genes were differentially expressed in a medium-dependent manner. Genes in another cluster containing a putative histidine kinase/response regulator, peptide methionine sulfoxide reductase, thioredoxin protein, lipoprotein, and cytochrome c-type protein were downregulated in KS1ΩabpA under all of the environmental conditions tested. Thus, AbpA appears to modulate genes associated with maltodextrin utilization/transport and fatty acid synthesis. Importantly, in all growth conditions AbpA was associated with increased expression of a potential two-component signaling system associated with genes involved in reducing oxidative stress, suggesting a role in signal transduction and stress tolerance. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Characterization of a Digestive α-Amylase in the Midgut of Pieris brassicae L. (Lepidoptera: Pieridae)

PubMed Central

Sharifloo, Ali; Zibaee, Arash; Sendi, Jalal J.; Jahroumi, Khalil Talebi

2016-01-01

The current study deals with a digestive α-amylase in the larvae of Pieris brassicae L. through purification, enzymatic characterization, gene expression, and in vivo effect of a specific inhibitor, Acarbose. Although α-amylase activity was the highest in the whole gut homogenate of larvae but compartmentalization of amylolytic activity showed an equal activity in posterior midgut (PM) and anterior midgut (AM). A three step purification using ammonium sulfate, Sepharyl G-100 and DEAE-Cellulose Fast flow revealed an enzyme with a specific activity of 5.18 U/mg, recovery of 13.20, purification fold of 19.25 and molecular weight of 88 kDa. The purified α-amylase had the highest activity at optimal pH and temperature of 8 and 35°C. Also, the enzyme had Vmax values of 4.64 and 3.02 U/mg protein and Km values of 1.37 and 1.74% using starch and glycogen as substrates, respectively. Different concentrations of acarbose, ethylenediamine tetraacetic acid, and ethylene glycol-bis (β-aminoethylether) N, N, N′, N′-tetraacetic acid significantly decreased activity of the purified α-amylase. The 4th instar larvae of P. brassicae were fed on the treated leaves of Raphanus sativus L. with 0.22 mM of Acarbose to find in vivo effects on nutritional indices, α-amylase activity, and gene expression. The significant differences were only found in conversion efficiency of digested food, relative growth rate, and metabolic cost of control and fed larvae on Acarbose. Also, amylolytic activity significantly decreased in the treated larvae by both biochemical and native-PAGE experiments. Results of RT-PCR revealed a gene with 621 bp length responsible for α-amylase expression that had 75% identity with Papilio xuthus and P. polytes. Finally, qRT-PCR revealed higher expression of α-amylase in control larvae compared to acarbose-fed ones. PMID:27014094

Close relationship of a novel Flavobacteriaceae α-amylase with archaeal α-amylases and good potentials for industrial applications.

PubMed

Li, Chunfang; Du, Miaofen; Cheng, Bin; Wang, Lushan; Liu, Xinqiang; Ma, Cuiqing; Yang, Chunyu; Xu, Ping

2014-01-31

Bioethanol production from various starchy materials has received much attention in recent years. α-Amylases are key enzymes in the bioconversion process of starchy biomass to biofuels, food or other products. The properties of thermostability, pH stability, and Ca-independency are important in the development of such fermentation process. A novel Flavobacteriaceae Sinomicrobium α-amylase (FSA) was identified and characterized from genomic analysis of a novel Flavobacteriaceae species. It is closely related with archaeal α-amylases in the GH13_7 subfamily, but is evolutionary distant with other bacterial α-amylases. Based on the conserved sequence alignment and homology modeling, with minor variation, the Zn2+- and Ca2+-binding sites of FSA were predicated to be the same as those of the archaeal thermophilic α-amylases. The recombinant α-amylase was highly expressed and biochemically characterized. It showed optimum activity at pH 6.0, high enzyme stability at pH 6.0 to 11.0, but weak thermostability. A disulfide bond was introduced by site-directed mutagenesis in domain C and resulted in the apparent improvement of the enzyme activity at high temperature and broad pH range. Moreover, about 50% of the enzyme activity was detected under 100°C condition, whereas no activity was observed for the wild type enzyme. Its thermostability was also enhanced to some extent, with the half-life time increasing from 25 to 55 minutes at 50°C. In addition, after the introduction of the disulfide bond, the protein became a Ca-independent enzyme. The improved stability of FSA suggested that the domain C contributes to the overall stability of the enzyme under extreme conditions. In addition, successfully directed modification and special evolutionary status of FSA imply its directional reconstruction potentials for bioethanol production, as well as for other industrial applications.

Antidiabetic potential: in vitro inhibition effects of some natural phenolic compounds on α-glycosidase and α-amylase enzymes.

PubMed

Taslimi, Parham; Gulçin, İlhami

2017-10-01

α-Glycosidase is a catalytic enzyme and it destroys the complex carbohydrates into simple absorbable sugar units. The natural phenolic compounds were tested for their antidiabetic properties as α-glycosidase and α-amylase inhibitors. The phenolic compounds investigated in this study have been used as antidiabetic common medicines. This paper aimed to consider their capability to inhibit α-amylase and α-glycosidase, two significant enzymes defined in serum glucose adjustment. These examination recorded impressive inhibition profiles with IC 50 values in the range of 137.36-737.23 nM against α-amylase and 29.01-157.96 nM against α-glycosidase. © 2017 Wiley Periodicals, Inc.

Exposure to inhalable dust, wheat flour and alpha-amylase allergens in industrial and traditional bakeries.

PubMed

Bulat, Petar; Myny, Katrien; Braeckman, Lutgart; van Sprundel, Marc; Kusters, Edouard; Doekes, Gert; Pössel, Kerstin; Droste, Jos; Vanhoorne, Michel

2004-01-01

This study was designed to characterize exposure to inhalable dust, wheat flour and alpha-amylase allergens in industrial and traditional bakeries. The study included 70 bakeries from the northern part of Belgium. Based on the degree of automation and a clear division of individual job tasks, four bakeries were identified as industrial and the remaining 66 were identified as traditional ones. Personal, as well as stationary, samples of inhalable dust were collected during full shift periods, usually 5-7 h. The portable pumps aspirated 2 l/min through Teflon personal dust samplers (Millipore, pore size 1.0 microm) mounted in PAS-6 sampling heads. In the collected samples the inhalable dust, wheat flour and alpha-amylase allergens were determined. Wheat flour allergens were measured using enzyme-linked immunosorbent assay inhibition and an antiwheat IgG4 serum pool. The alpha-amylase allergens were measured using a sandwich enzyme immunoassay with affinity-purified polyclonal rabbit IgG antibodies. In total, 440 samples (300 personal and 140 stationary) were processed. The highest inhalable dust exposure was observed in traditional bakeries among bread [geometric mean (GM) 2.10 mg/m3] and bread and pastry workers (GM 1.80 mg/m3). In industrial bakeries the highest dust exposure was measured in bread-producing workers (GM 1.06 mg/m3). Similar relations were observed for wheat flour and alpha-amylase allergens. Bread baking workers in traditional bakeries had the highest exposure to both allergens (wheat flour GM 22.33 microg/m(3), alpha-amylase GM 0.61 ng/m3). The exposure to wheat flour and alpha-amylase allergens in industrial bakeries was higher in bread baking workers (wheat flour GM 6.15 microg/m3, alpha-amylase GM 0.47 ng/m3) than in bread packing workers (wheat flour GM 2.79 microg/m3, alpha-amylase GM 0.15 ng/m3). The data presented suggest that, on average, exposure in the Belgium bakeries studied-industrial as well as traditional-is lower than or similar to bakeries in The Netherlands, Canada, Sweden, the United Kingdom and Finland. Furthermore, the exposure levels in traditional bakeries seem to be higher than in industrial bakeries.

Caffeic and chlorogenic acids inhibit key enzymes linked to type 2 diabetes (in vitro): a comparative study.

PubMed

Oboh, Ganiyu; Agunloye, Odunayo M; Adefegha, Stephen A; Akinyemi, Ayodele J; Ademiluyi, Adedayo O

2015-03-01

Chlorogenic acid is a major phenolic compound that forms a substantial part of plant foods and is an ester of caffeic acid and quinic acid. However, the effect of the structures of both chlorogenic and caffeic acids on their antioxidant and antidiabetic potentials have not been fully understood. Thus, this study sought to investigate and compare the interaction of caffeic acid and chlorogenic acid with α-amylase and α-glucosidase (key enzymes linked to type 2 diabetes) activities in vitro. The inhibitory effect of the phenolic acids on α-amylase and α-glucosidase activities was evaluated. Thereafter, their antioxidant activities as typified by their 1,1-diphenyl-2 picrylhydrazyl radical scavenging ability and ferric reducing antioxidant properties were determined. The results revealed that both phenolic acids inhibited α-amylase and α-glucosidase activities in a dose-dependent manner (2-8 μg/mL). However, caffeic acid had a significantly (p<0.05) higher inhibitory effect on α-amylase [IC50 (concentration of sample causing 50% enzyme inhibition)=3.68 μg/mL] and α-glucosidase (IC50=4.98 μg/mL) activities than chlorogenic acid (α-amylase IC50=9.10 μg/mL and α-glucosidase IC50=9.24 μg/mL). Furthermore, both phenolic acids exhibited high antioxidant properties, with caffeic acid showing higher effects. The esterification of caffeic acid with quinic acid, producing chlorogenic acid, reduces their ability to inhibit α-amylase and α-glucosidase activities. Thus, the inhibition of α-amylase and α-glucosidase activities by the phenolic acids could be part of the possible mechanism by which the phenolic acids exert their antidiabetic effects.

Biochemical, structural and functional diversity between two digestive α-amylases from Helicoverpa armigera.

PubMed

Bhide, Amey J; Channale, Sonal M; Patil, Sucheta S; Gupta, Vidya S; Ramasamy, Sureshkumar; Giri, Ashok P

2015-09-01

Helicoverpa armigera (Lepidoptera) feeds on various plants using diverse digestive enzymes as one of the survival tool-kit. The aim of the present study was to understand biochemical properties of recombinant α-amylases of H. armigera viz., HaAmy1 and HaAmy2. The open reading frames of HaAmy1 and HaAmy2 were cloned in Pichia pastoris and expressed heterologously. Purified recombinant enzymes were characterized for their biochemical and biophysical attributes using established methods. Sequence alignment and homology modeling showed that HaAmy1 and HaAmy2 were conserved in their amino acid sequences and structures. HaAmy1 and HaAmy2 showed optimum activity at 60°C; however, they differed in their optimum pH. Furthermore, HaAmy2 showed higher affinity for starch and amylopectin whereas HaAmy1 had higher catalytic efficiency. HaAmy1 and HaAmy2 were inhibited to the same magnitude by a synthetic amylase inhibitor (acarbose) while wheat amylase inhibitor showed about 2-fold higher inhibition of HaAmy1 than HaAmy2 at pH7 while 6-fold difference at pH11. Interactions of HaAmy1 and HaAmy2 with wheat amylase inhibitor revealed 2:1 stoichiometric ratio and much more complex interaction with HaAmy1. The diversity of amylases in perspective of their biochemical and biophysical properties, and their differential interactions with amylase inhibitors signify the potential role of these enzymes in adaptation of H. armigera on diverse plant diets. Characterization of digestive enzymes of H. armigera provides the molecular basis for the polyphagous nature and thus could assist in designing future strategies for the insect control. Copyright © 2015 Elsevier B.V. All rights reserved.

Alpha-amylase inhibitor, CS-1036 binds to serum amylase in a concentration-dependent and saturable manner.

PubMed

Honda, Tomohiro; Kaneno-Urasaki, Yoko; Ito, Takashi; Kimura, Takako; Matsushima, Nobuko; Okabe, Hiromi; Yamasaki, Atsushi; Izumi, Takashi

2014-03-01

(2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxy-β-D-glucopyranosyl)-α-D-glucopyranoside (CS-1036), which is an α-amylase inhibitor, exhibited biphasic and sustained elimination with a long t1/2 (18.4-30.0 hours) in rats and monkeys, but exhibited a short t1/2 (3.7-7.9 hours) in humans. To clarify the species differences in the t1/2, the plasma protein binding of CS-1036 was evaluated by ultrafiltration. A concentration-dependent and saturable plasma protein binding of CS-1036 was observed in rats and monkeys with the dissociation rate constant (KD) of 8.95 and 27.2 nM, and maximal binding capacity (Bmax) of 52.8 and 22.1 nM, respectively. By the assessments of the recombinant amylase and immunoprecipitation, the major binding protein of CS-1036 in rats was identified as salivary amylase (KD 5.64 nM). CS-1036 also showed concentration-dependent and saturable binding to human salivary and pancreatic amylase, with similar binding affinity in rats. However, the protein binding of CS-1036 was constant in human plasma (≤10.2%) due to the lower serum amylase level compared with rats and monkeys. From the calculation of the unbound fraction (fu) in plasma based on in vitro KD and Bmax, the dose-dependent increase in fu after oral administration is speculated to lead to a dose-dependent increase in total body clearance and a high area under the curve/dose at lower doses, such as 0.3 mg/kg in rats.

Obesity, starch digestion and amylase: association between copy number variants at human salivary (AMY1) and pancreatic (AMY2) amylase genes

PubMed Central

Carpenter, Danielle; Dhar, Sugandha; Mitchell, Laura M.; Fu, Beiyuan; Tyson, Jess; Shwan, Nzar A.A.; Yang, Fengtang; Thomas, Mark G.; Armour, John A.L.

2015-01-01

The human salivary amylase genes display extensive copy number variation (CNV), and recent work has implicated this variation in adaptation to starch-rich diets, and in association with body mass index. In this work, we use paralogue ratio tests, microsatellite analysis, read depth and fibre-FISH to demonstrate that human amylase CNV is not a smooth continuum, but is instead partitioned into distinct haplotype classes. There is a fundamental structural distinction between haplotypes containing odd or even numbers of AMY1 gene units, in turn coupled to CNV in pancreatic amylase genes AMY2A and AMY2B. Most haplotypes have one copy each of AMY2A and AMY2B and contain an odd number of copies of AMY1; consequently, most individuals have an even total number of AMY1. In contrast, haplotypes carrying an even number of AMY1 genes have rearrangements leading to CNVs of AMY2A/AMY2B. Read-depth and experimental data show that different populations harbour different proportions of these basic haplotype classes. In Europeans, the copy numbers of AMY1 and AMY2A are correlated, so that phenotypic associations caused by variation in pancreatic amylase copy number could be detected indirectly as weak association with AMY1 copy number. We show that the quantitative polymerase chain reaction (qPCR) assay previously applied to the high-throughput measurement of AMY1 copy number is less accurate than the measures we use and that qPCR data in other studies have been further compromised by systematic miscalibration. Our results uncover new patterns in human amylase variation and imply a potential role for AMY2 CNV in functional associations. PMID:25788522

Obesity, starch digestion and amylase: association between copy number variants at human salivary (AMY1) and pancreatic (AMY2) amylase genes.

PubMed

Carpenter, Danielle; Dhar, Sugandha; Mitchell, Laura M; Fu, Beiyuan; Tyson, Jess; Shwan, Nzar A A; Yang, Fengtang; Thomas, Mark G; Armour, John A L

2015-06-15

The human salivary amylase genes display extensive copy number variation (CNV), and recent work has implicated this variation in adaptation to starch-rich diets, and in association with body mass index. In this work, we use paralogue ratio tests, microsatellite analysis, read depth and fibre-FISH to demonstrate that human amylase CNV is not a smooth continuum, but is instead partitioned into distinct haplotype classes. There is a fundamental structural distinction between haplotypes containing odd or even numbers of AMY1 gene units, in turn coupled to CNV in pancreatic amylase genes AMY2A and AMY2B. Most haplotypes have one copy each of AMY2A and AMY2B and contain an odd number of copies of AMY1; consequently, most individuals have an even total number of AMY1. In contrast, haplotypes carrying an even number of AMY1 genes have rearrangements leading to CNVs of AMY2A/AMY2B. Read-depth and experimental data show that different populations harbour different proportions of these basic haplotype classes. In Europeans, the copy numbers of AMY1 and AMY2A are correlated, so that phenotypic associations caused by variation in pancreatic amylase copy number could be detected indirectly as weak association with AMY1 copy number. We show that the quantitative polymerase chain reaction (qPCR) assay previously applied to the high-throughput measurement of AMY1 copy number is less accurate than the measures we use and that qPCR data in other studies have been further compromised by systematic miscalibration. Our results uncover new patterns in human amylase variation and imply a potential role for AMY2 CNV in functional associations. © The Author 2015. Published by Oxford University Press.

Noncovalent PEGylation through Protein-Polyelectrolyte Interaction: Kinetic Experiment and Molecular Dynamics Simulation.

PubMed

Kurinomaru, Takaaki; Kuwada, Kengo; Tomita, Shunsuke; Kameda, Tomoshi; Shiraki, Kentaro

2017-07-20

Noncovalent binding of polyethylene glycol (PEG) to a protein surface is a unique protein handling technique to control protein function and stability. A diblock copolymer containing PEG and polyelectrolyte chains (PEGylated polyelectrolyte) is a promising candidate for noncovalent attachment of PEG to a protein surface because of the binding through multiple electrostatic interactions without protein denaturation. To obtain a deeper understanding of protein-polyelectrolyte interaction at the molecular level, we investigated the manner in which cationic PEGylated polyelectrolyte binds to anionic α-amylase in enzyme kinetic experiments and molecular dynamics (MD) simulations. Cationic PEG-block-poly(N,N-dimethylaminoethyl) (PEG-b-PAMA) inhibited the enzyme activity of anionic α-amylase due to binding of PAMA chains. Enzyme kinetics revealed that the inhibition of α-amylase activity by PEG-b-PAMA is noncompetitive inhibition manner. In MD simulations, the PEG-b-PAMA molecule was initially located at six different placements of the x-, y-, and z-axis ±20 Å from the center of α-amylase, which showed that the PEG-b-PAMA nonspecifically bound to the α-amylase surface, corresponding to the noncompetitive inhibition manner that stems from the polymer binding to an enzyme surface other than the active site. In addition, the enzyme activity of α-amylase in the presence of PEG-b-PAMA was not inhibited by increasing the ionic strength, consistent with the MD simulation; i.e., PEG-b-PAMA did not interact with α-amylase in high ionic strength conditions. The results reported in this paper suggest that enzyme inhibition by PEGylated polyelectrolyte can be attributed to the random electrostatic interaction between protein and polyelectrolyte.

Performance Evaluation of a Salivary Amylase Biosensor for Stress Assessment in Military Field Research.

PubMed

Peng, Henry T; Savage, Erin; Vartanian, Oshin; Smith, Shane; Rhind, Shawn G; Tenn, Catherine; Bjamason, Stephen

2016-05-01

A convenient biosensor for real-time measurement of biomarkers for in-field psychophysiological stress research and military operations is desirable. We evaluated a hand-held device for measuring salivary amylase as a stress marker in medical technicians undergoing combat casualty care training using two different modalities in operating room and field settings. Salivary amylase activity was measured by two biosensor methods: directly sampling saliva with a test strip placed under the tongue or pipetting a fixed volume of precollected saliva onto the test strip, followed by analyzing the sample on the strip using a biosensor. The two methods were compared for their accuracy and sensitivity to detect the stress response using an enzyme assay method as a standard. The measurements from the under-the-tongue method were not as consistent with those from the standard assay method as the values obtained from the pipetting method. The under-the-tongue method did not detect any significant increase in the amylase activity due to stress in the operating room (P > 0.1), in contrast to the significant increases observed using the pipetting method and assay method with a significance level less than 0.05 and 0.1, respectively. Furthermore, the under-the-tongue method showed no increased amylase activity in the field testing, while both the pipetting method and assay method showed increased amylase activity in the same group (P < 0.1). The accuracy and consistency of the biosensors need to be improved when used to directly measure salivary amylase activity under the tongue for stress assessment in military medical training. © 2015 Her Majesty the Queen in Right of Canada. Journal of Clinical Laboratory Analysis published by Wiley Periodicals, Inc. Reproduced with the permission DRDC Editorial Board.

The baseline serum value of α-amylase is a significant predictor of distance running performance.

PubMed

Lippi, Giuseppe; Salvagno, Gian Luca; Danese, Elisa; Tarperi, Cantor; La Torre, Antonio; Guidi, Gian Cesare; Schena, Federico

2015-02-01

This study was planned to investigate whether serum α-amylase concentration may be associated with running performance, physiological characteristics and other clinical chemistry analytes in a large sample of recreational athletes undergoing distance running. Forty-three amateur runners successfully concluded a 21.1 km half-marathon at 75%-85% of their maximal oxygen uptake (VO2max). Blood was drawn during warm up and 15 min after conclusion of the run. After correction for body weight change, significant post-run increases were observed for serum values of alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, bilirubin, creatine kinase (CK), iron, lactate dehydrogenase (LDH), triglycerides, urea and uric acid, whereas the values of body weight, glomerular filtration rate, total and low density lipoprotein-cholesterol were significantly decreased. The concentration of serum α-amylase was unchanged. In univariate analysis, significant associations with running performance were found for gender, VO2max, training regimen and pre-run serum values of α-amylase, CK, glucose, high density lipoprotein-cholesterol, LDH, urea and uric acid. In multivariate analysis, only VO2max (p=0.042) and baseline α-amylase (p=0.021) remained significant predictors of running performance. The combination of these two variables predicted 71% of variance in running performance. The baseline concentration of serum α-amylase was positively correlated with variation of serum glucose during the trial (r=0.345; p=0.025) and negatively with capillary blood lactate at the end of the run (r=-0.352; p=0.021). We showed that the baseline serum α-amylase concentration significantly and independently predicts distance running performance in recreational runners.

Kinetics of α-amylase and α-glucosidase inhibitory potential of Zea mays Linnaeus (Poaceae), Stigma maydis aqueous extract: An in vitro assessment.

PubMed

Sabiu, S; O'Neill, F H; Ashafa, A O T

2016-05-13

Corn silk (Zea mays L., Stigma maydis) is an important herb used traditionally in many parts of the world to treat array of diseases including diabetes mellitus. Inhibitors of α-amylase and α-glucosidase offer an effective strategy to modulate levels of post prandial hyperglycaemia via control of starch metabolism. This study evaluated α-amylase and α-glucosidase inhibitory potentials of corn silk aqueous extract. Active principles and antioxidant attributes of the extract were also analysed. The α-amylase inhibitory potential of the extract was investigated by reacting its different concentrations with α-amylase and starch solution, while α-glucosidase inhibition was determined by pre-incubating α-glucosidase with different concentrations of the extract followed by addition of p-nitrophenylglucopyranoside. The mode(s) of inhibition of the enzymes were determined using Lineweaver-Burke plot. In vitro analysis of the extract showed that it exhibited potent and moderate inhibitory potential against α-amylase and α-glucosidase, respectively. The inhibition was concentration-dependent with respective half-maximal inhibitory concentration (IC50) values of 5.89 and 0.93mg/mL. Phytochemical analyses revealed the presence of alkaloids, flavonoids, phenols, saponins, tannins and phytosterols as probable inhibitory constituents. Furthermore, the extract remarkably scavenges reactive oxygen species like DPPH and nitric oxide radicals, elicited good reducing power and a significant metal chelating attributes. Overall, the non-competitive and uncompetitive mechanism of action of corn silk extract is due to its inhibitory effects on α-amylase and α-glucosidase, respectively. Consequently, this will reduce the rate of starch hydrolysis, enhance palliated glucose levels, and thus, lending credence to hypoglycaemic candidature of corn silk. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Amelioration in secretion of hyperthermostable and Ca2+ -independent alpha-amylase of Geobacillus thermoleovorans by some polyamines and their biosynthesis inhibitor methylglyoxal-bis-guanylhydrazone.

PubMed

Uma Maheswar Rao, J L; Satyanarayana, T

2004-01-01

Effect of polyamines and their biosynthesis inhibitors on the production of hyperthermostable and Ca2+ -independent alpha-amylase by Geobacillus thermoleovorans MTCC 4220. The alpha-amylase was produced in starch-yeast extract-tryptone (SYT) broth with different polyamines (PA) and polyamine biosynthesis inhibitors, methylglyoxal-bis-guanylhydrazone (MGBG) and cyclohexylammonium sulphate (CHA) at 70 degrees C. The bacterial pellets were obtained after growing G. thermoleovorans at different temperatures, and used in determining total PA. The cell-free culture filtrates were used in alpha-amylase assays. During growth, total polyamines in biomass increased till 2 h, and thereafter, decreased gradually. The total polyamine content was very high in the biomass cultivated at 55 degrees C when compared with that of higher temperatures. Enzyme titre enhanced up to 70 degrees C, and thereafter declined. Extracellular enzyme and protein levels declined in the presence of exogenously added PA. The intracellular enzyme titres, however, were higher in putrescine (put) and spermidine (spd) than in spermine (spm). Polyamine biosynthesis inhibitor, MGBG enhanced secretion of alpha-amylase in a laboratory fermentor as well as shake flasks, although CHA did not affect it. The intracellular accumulation of put in the presence of MGBG appeared to enhance synthesis and secretion of alpha-amylase. Extracellular enzyme and protein levels were low in the presence of exogenously added PA, but their intracellular levels, however, were higher in put and spd than in spm. A substantial increase in the synthesis and secretion of alpha-amylase was attained in G. thermoleovorans in the presence of polyamine biosynthesis inhibitor MGBG.

A bifunctional α-amylase/trypsin inhibitor from pigeonpea seeds: Purification, biochemical characterization and its bio-efficacy against Helicoverpa armigera.

PubMed

Gadge, Prafull P; Wagh, Sandip K; Shaikh, Faiyaz K; Tak, Rajesh D; Padul, Manohar V; Kachole, Manvendra S

2015-11-01

This paper evaluates α-amylase inhibitor (α-AI) mediated defense of pigeonpea against Helicoverpa armigera. A bifunctional α-amylase/trypsin inhibitor was purified from the seeds of pigeonpea by native liquid phase isoelectric focusing (N-LP-IEF), affinity chromatography and preparative electrophoresis. Its in-vivo and in-vitro interaction with midgut amylases of H. armigera was studied along with growth inhibitory activity. One and two dimensional (2D) zymographic analyses revealed that the purified inhibitor is dimeric glycoprotein (60.2kDa and 56kDa) exist in a multi-isomeric form with five pI variants (pI 5.5 to 6.3). It was found to be heat labile with complete inactivation up to 80°C and stable over a wide range of pH (4-11). The slow binding and competitive type of α-amylase inhibition was observed with 0.08μM of dissociation constant (Ki) for the enzyme-inhibitor complex (EI). The internal protein sequence of two subunits obtained by mass spectrometry matched with cereal-type α-AI, a conserved domain from AAI_LTSS superfamily and sialyltransferase-like protein respectively. In-vivo studies indicated up-regulation of total midgut α-amylase activity with negative effect on growth rate of H. armigera suggesting its suitability for pest control. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterisation and inhibition studies of Helicoverpa armigera (Hübner) gut α-amylase.

PubMed

Kaur, Rimaljeet; Gupta, Anil K; Taggar, Gaurav K

2015-09-01

The survival of a devastating pest, Helicoverpa armigera, is mainly dependent on the availability of α-amylase. Therefore, characterising H. armigera α-amylase and targeting it with effective inhibitors could aid in reducing its damaging effects. H. armigera gut possessed four isozymes of α-amylase. The molecular weight of the major purified isozyme ranged from 79 to 81 kDa. The purified enzyme was identified to be α-amylase on the basis of products formed from starch. The optimum pH and temperature were 10.0 and 50 °C respectively. The activation energy was 5.7 kcal mol(-1) . The enzyme showed high activity with starch and amylopectin, whereas dextrins were poor substrates. The Michaelis constant Km with starch, amylose and amylopectin was 0.45, 1.23 and 0.11 mg mL(-1) respectively. ZnSO4 , FeSO4 , CuSO4 , citric acid, oxalic acid and salicylic acid were potent inhibitors. ZnSO4 , salicylic acid and pigeonpea α-amylase inhibitor (∼21.0 kDa) acted primarily as competitive inhibitors, FeSO4 and citric acid displayed mainly anticompetitive behaviour, while CuSO4 and oxalic acid behaved mainly as non-competitive inhibitors. The identification of effective ecofriendly inhibitors could help in managing H. armigera infestation. © 2014 Society of Chemical Industry. © 2014 Society of Chemical Industry.

Aqueous extracts of Roselle (Hibiscus sabdariffa Linn.) varieties inhibit α-amylase and α-glucosidase activities in vitro.

PubMed

Ademiluyi, Adedayo O; Oboh, Ganiyu

2013-01-01

This study sought to investigate the inhibitory effect of aqueous extracts of two varieties (red and white) of Hibiscus sabdariffa (Roselle) calyces on carbohydrate hydrolyzing enzymes (α-amylase and α-glucosidase), with the aim of providing the possible mechanism for their antidiabetes properties. Aqueous extracts were prepared (1:100 w/v) and the supernatant used for the analysis. The extracts caused inhibition of α-amylase and α-glucosidase activities in vitro.The IC(50) revealed that the red variety (25.2 μg/mL) exhibited higher α-glucosidase inhibitory activity than the white variety (47.4 μg/mL), while the white variety (90.5 μg/mL) exhibited higher α-amylase inhibitory activity than the red variety (187.9 μg/mL). However, the α-glucosidase inhibitory activities of both calyces were higher than that of their α-amylase. In addition, the red variety possessed higher antioxidant capacity as exemplified by the (•)OH scavenging abilities, Fe(2+) chelating ability, and inhibition of Fe(2+)-induced pancreatic lipid peroxidation in vitro. The enzyme inhibitory activities and antioxidant properties of the roselle extracts agreed with their phenolic content. Hence, inhibition of α-amylase and α-glucosidase, coupled with strong antioxidant properties could be the possible underlying mechanism for the antidiabetes properties of H. sabdariffa calyces; however, the red variety appeared to be more potent.

Inhibitory activity of cinnamon bark species and their combination effect with acarbose against intestinal α-glucosidase and pancreatic α-amylase.

PubMed

Adisakwattana, Sirichai; Lerdsuwankij, Orathai; Poputtachai, Ubonwan; Minipun, Aukkrapon; Suparpprom, Chaturong

2011-06-01

Inhibition of α-glucosidase and pancreatic α-amylase is one of the therapeutic approaches for delaying carbohydrate digestion, resulting in reduced postprandial glucose. The aim of this study was to evaluate the phytochemical analysis and the inhibitory effect of various cinnamon bark species against intestinal α-glucosidase and pancreatic α-amylase. The results showed that the content of total phenolic, flavonoid, and condensed tannin ranged from 0.17 to 0.21 g gallic acid equivalent/g extract, from 48.85 to 65.52 mg quercetin equivalent/g extract, and from 0.12 to 0.15 g catechin equivalent/g extract, respectively. The HPLC fingerprints of each cinnamon species were established. Among cinnamon species, Thai cinnamon extract was the most potent inhibitor against the intestinal maltase with the IC(50) values of 0.58 ± 0.01 mg/ml. The findings also showed that Ceylon cinnamon was the most effective intestinal sucrase and pancreatic α-amylase inhibitor with the IC(50) values of 0.42 ± 0.02 and 1.23 ± 0.02 mg/ml, respectively. In addition, cinnamon extracts produced additive inhibition against intestinal α-glucosidase and pancreatic α-amylase when combined with acarbose. These results suggest that cinnamon bark extracts may be potentially useful for the control of postprandial glucose in diabetic patients through inhibition of intestinal α-glucosidase and pancreatic α-amylase.

Purification and Characterization of a Highly Efficient Calcium-Independent α-Amylase from Talaromyces pinophilus 1-95

PubMed Central

Xian, Liang; Wang, Fei; Luo, Xiang; Feng, Yu-Liang; Feng, Jia-Xun

2015-01-01

Alpha-amylase is a very important enzyme in the starch conversion process. Most of the α-amylases are calcium-dependent and exhibit poor performance in the simultaneous saccharification and fermentation process of industrial bioethanol production that uses starch as feedstock. In this study, an extracellular amylolytic enzyme was purified from the culture broth of newly isolated Talaromyces pinophilus strain 1-95. The purified amylolytic enzyme, with an apparent molecular weight of 58 kDa on SDS-PAGE, hydrolyzed maltopentaose, maltohexaose, and maltoheptaose into mainly maltose and maltotriose and minor amount of glucose, confirming the endo-acting mode of the enzyme, and hence, was named Talaromyces pinophilus α-amylase (TpAA). TpAA was most active at pH 4.0–5.0 (with the temperature held at 37°C) and 55°C (at pH 5.0), and stable within the pH range of 5.0–9.5 (at 4°C) and below 45°C (at pH 5.0). Interestingly, the Ca2+ did not improve its enzymatic activity, optimal temperature, or thermostability of the enzyme, indicating that the TpAA was Ca2+-independent. TpAA displayed higher enzyme activity toward malto-oligosaccharides and dextrin than other previously reported α-amylases. This highly active Ca2+-independent α-amylase may have potential applications in starch-to-ethanol conversion process. PMID:25811759

Starch Digestion-Related Amylase Genetic Variant Affects 2-Year Changes in Adiposity in Response to Weight-Loss Diets: The POUNDS Lost Trial.

PubMed

Heianza, Yoriko; Sun, Dianjianyi; Wang, Tiange; Huang, Tao; Bray, George A; Sacks, Frank M; Qi, Lu

2017-09-01

Salivary and pancreatic amylases (encoded by AMY1 and AMY2 genes, respectively) are responsible for digesting starchy foods. AMY1 and AMY2 show copy number variations that affect differences in amylase amount and activity, and AMY1 copies have been associated with adiposity. We investigated whether genetic variants determining amylase gene copies are associated with 2-year changes in adiposity among 692 overweight and obese individuals who were randomly assigned to diets varying in macronutrient content. We found that changes in body weight (BW) and waist circumference (WC) were significantly different according to the AMY1-AMY2 rs11185098 genotype. Individuals carrying the A allele (indicating higher amylase amount and activity) showed a greater reduction in BW and WC at 6, 12, 18, and 24 months than those without the A allele ( P < 0.05 for all). The association was stronger for long-term changes compared with short-term changes of these outcomes. The genetic effects on these outcomes did not significantly differ across diet groups. In conclusion, the genetic variant determining starch metabolism influences the response to weight-loss dietary intervention. Overweight and obese individuals carrying the AMY1-AMY2 rs11185098 genotype associated with higher amylase activity may have greater loss of adiposity during low-calorie diet interventions. © 2017 by the American Diabetes Association.

Close evolutionary relatedness among functionally distantly related members of the (alpha/beta)8-barrel glycosyl hydrolases suggested by the similarity of their fifth conserved sequence region.

PubMed

Janecek, S

1995-12-11

A short conserved sequence equivalent to the fifth conserved sequence region of alpha-amylases (173_LPDLD, Aspergillus oryzae alpha-amylase) comprising the calcium-ligand aspartate, Asp-175, was identified in the amino acid sequences of several members of the family of (alpha/beta)8-barrel glycosyl hydrolases. Despite the fact that the aspartate is not invariantly conserved, the stretch can be easily recognised in all sequences to be positioned 26-28 amino acid residues in front of the well-known catalytic aspartate (Asp-206, A. oryzae alpha-amylase) located in the beta 4-strand of the barrel. The identification of this region revealed remarkable similarities between some alpha-amylases (those from Bacillus megaterium, Bacillus subtilis and Dictyoglomus thermophilum) on the one hand and several different enzyme specificities (such as oligo-1,6-glucosidase, amylomaltase and neopullulanase, respectively) on the other hand. The most interesting example was offered by B. subtilis alpha-amylase and potato amylomaltase with the regions LYDWN and LYDWK, respectively. These observations support the idea that all members of the family of glycosyl hydrolases adopting the structure of the alpha-amylase-type (alpha/beta)8-barrel are mutually closely related and the strict evolutionary borders separating the individual enzyme specificities can be hardly defined.

Purification, Characterization, and Potential of Saline Waste Water Remediation of a Polyextremophilic α-Amylase from an Obligate Halophilic Aspergillus gracilis

PubMed Central

Ali, Imran; Akbar, Ali; Yanwisetpakdee, Benjawan; Prasongsuk, Sehanat; Lotrakul, Pongtharin; Punnapayak, Hunsa

2014-01-01

An obligate halophilic Aspergillus gracilis which was isolated from a hypersaline man-made saltern from Thailand was screened for its potential of producing extracellular α-amylase in the previous studies. In this study the α-amylase was extracted and purified by the help of column chromatography using Sephadex G-100 column. Presence of amylase was verified by SDS-PAGE analysis, showing a single band of approximately 35 kDa. The specific activity of the enzyme was found to be 131.02 U/mg. The Lineweaver-Burk plot showed the V max and K m values of 8.36 U/mg and 6.33 mg/mL, respectively. The enzyme was found to have the best activity at 5 pH, 60°C, and 30% of NaCl concentration, showing its polyextremophilic nature. The use of various additives did not show much variation in the activity of enzyme, showing its resilience against inhibitors. The enzyme, when tested for its use for synthetic waste water remediation by comparing its activity with commercial amylase in different salt concentrations showed that the α-amylase from A. gracilis was having better performance at increasing salt concentrations than the commercial one. This shows its potential to be applied in saline waste water and other low water activity effluents for bioremediation. PMID:24949415

Grape seed and tea extracts and catechin 3-gallates are potent inhibitors of α-amylase and α-glucosidase activity.

PubMed

Yilmazer-Musa, Meltem; Griffith, Anneke M; Michels, Alexander J; Schneider, Erik; Frei, Balz

2012-09-12

This study evaluated the inhibitory effects of plant-based extracts (grape seed, green tea, and white tea) and their constituent flavan-3-ol monomers (catechins) on α-amylase and α-glucosidase activity, two key glucosidases required for starch digestion in humans. To evaluate the relative potency of extracts and catechins, their concentrations required for 50 and 90% inhibition of enzyme activity were determined and compared to the widely used pharmacological glucosidase inhibitor, acarbose. Maximum enzyme inhibition was used to assess relative inhibitory efficacy. Results showed that grape seed extract strongly inhibited both α-amylase and α-glucosidase activity, with equal and much higher potency, respectively, than acarbose. Whereas tea extracts and catechin 3-gallates were less effective inhibitors of α-amylase, they were potent inhibitors of α-glucosidase. Nongallated catechins were ineffective. The data show that plant extracts containing catechin 3-gallates, in particular epigallocatechin gallate, are potent inhibitors of α-glucosidase activity and suggest that procyanidins in grape seed extract strongly inhibit α-amylase activity.

Characterisation of three starch degrading enzymes: thermostable β-amylase, maltotetraogenic and maltogenic α-amylases.

PubMed

Derde, L J; Gomand, S V; Courtin, C M; Delcour, J A

2012-11-15

Maltogenic α-amylase from Bacillus stearothermophilus (BStA) is widely used as bread crumb anti-firming enzyme. A maltotetraose-forming α-amylase from Pseudomonas saccharophila (PSA) was recently proposed as alternative, hence the need to compare both exo-acting enzymes with some endo-action component. A purely exo-acting thermostable β-amylase from Clostridium thermosulfurogenes (CTB) was included for reference purposes. Under the experimental conditions used, temperature optima of the enzymes are rather similar (60-65 °C), but temperature stability decreased in the order BStA, PSA and CTB. The action of the enzymes on different substrates and their impact on the rheological behaviour of maize starch suspensions demonstrated that, while CTB acts exclusively through an exo-action mechanism, BStA displayed limited endo-action which became more pronounced at higher temperatures. PSA has more substantial endo-action than BStA, which is rather temperature independent. This is important for their impact in processes such as breadmaking, where temperature is gradually increased. Copyright © 2012 Elsevier Ltd. All rights reserved.

Extraction and identification of α-amylase inhibitor peptides from Nephelium lappacheum and Nephelium mutabile seed protein using gastro-digestive enzymes.

PubMed

Evaristus, Natashya Anak; Wan Abdullah, Wan Nadiah; Gan, Chee-Yuen

2018-04-01

The potential of N. lappacheum and N. mutabile seed as a source of α-amylase inhibitor peptides was explored based on the local traditional practice of using the seed. Different gastro-digestive enzymes (i.e. pepsin or chymotrypsin) or a sequential digestion were used to extract the peptides. The effects of digestion time and enzyme to substrate (E:S) ratio on the α-amylase inhibitory activity were investigated. Results showed that chymotrypsin was effective in producing the inhibitor peptides from rambutan seed protein at E:S ratio 1:20 for 1 h, whereas pepsin was more effective for pulasan seed protein under the same condition. A total of 20 and 31 novel inhibitor peptides were identified, respectively. These peptides could bind with the subsites of α-amylase (i.e. Trp58, Trp59, Tyr62, Asp96, Arg195, Asp197, Glu233, His299, Asp300, and His305) and formed a sliding barrier that preventing the formation of enzyme/substrate intermediate leading to lower α-amylase activity. Copyright © 2018 Elsevier Inc. All rights reserved.

Influence of hydration and starch digestion on the transient rheology of an aqueous suspension of comminuted potato snack food.

PubMed

Boehm, Michael W; Warren, Frederick J; Moore, Jackson E; Baier, Stefan K; Gidley, Michael J; Stokes, Jason R

2014-11-01

Oral processing of most foods is inherently destructive: solids are broken into particles before reassembly into a hydrated bolus while salivary enzymes degrade food components. In order to investigate the underlying physics driving changes during oral processing, we capture the transient rheological behaviour of a simulated potato chip bolus during hydration by a buffer with or without α-amylase. In the absence of amylase and for all oil contents and solids weight fractions tested, we find a collapse of the transient data when graphed according to simple Fickian diffusion. In the presence of amylase, we find effects on the transient and pseudo steady state bolus rheology. Within the first minute of mixing, the amylase degrades only ≈6% of the starch but that leads to an order of magnitude reduction in the bolus elasticity, as compared to the case without amylase. Thus, for an in vitro bolus, only a small amount of starch needs to be digested to have a large impact on the bolus rheology very soon after mixing.

Application of a statistical design to the optimization of parameters and culture medium for alpha-amylase production by Aspergillus oryzae CBS 819.72 grown on gruel (wheat grinding by-product).

PubMed

Kammoun, Radhouane; Naili, Belgacem; Bejar, Samir

2008-09-01

The production optimization of alpha-amylase (E.C.3.2.1.1) from Aspergillus oryzae CBS 819.72 fungus, using a by-product of wheat grinding (gruel) as sole carbon source, was performed with statistical methodology based on three experimental designs. The optimisation of temperature, agitation and inoculum size was attempted using a Box-Behnken design under the response surface methodology. The screening of nineteen nutrients for their influence on alpha-amylase production was achieved using a Plackett-Burman design. KH(2)PO(4), urea, glycerol, (NH(4))(2)SO(4), CoCl(2), casein hydrolysate, soybean meal hydrolysate, MgSO(4) were selected based on their positive influence on enzyme formation. The optimized nutrients concentration was obtained using a Taguchi experimental design and the analysis of the data predicts a theoretical increase in the alpha-amylase expression of 73.2% (from 40.1 to 151.1 U/ml). These conditions were validated experimentally and revealed an enhanced alpha-amylase yield of 72.7%.

Chemical composition and α-amylase inhibitory activity of the essential oil from Sabina chinensis cv. Kaizuca leaves.

PubMed

Gu, Dongyu; Fang, Chen; Yang, Jiao; Li, Minjing; Liu, Hengming; Yang, Yi

2018-03-01

Sabina chinensis cv. Kaizuca (SCK) is a variant of S. chinensis L. The essential oil from its leaves exhibited α-amylase inhibitory activity in vitro and the IC 50 value was 187.08 ± 0.56 μg/mL. Nineteen compounds were identified from this essential oil by gas chromatography-mass spectrometry (GC-MS) analysis. The major compounds identified were bornyl acetate (42.6%), elemol (20.5%), β-myrcene (13.7%) and β-linalool (4.0%). In order to study the reason of the α-amylase inhibitory activity of this essential oil, the identified compounds were docked with α-amylase by molecular docking individually. Among these compounds, γ-eudesmol exhibited the lowest binding energy (-6.73 kcal/mol), followed by α-copaen-11-ol (-6.66 kcal/mol), cubedol (-6.39 kcal/mol) and α-acorenol (-6.12 kcal/mol). The results indicated that these compounds were the active ingredients responsible for the α-amylase inhibitory activity of essential oil from SCK.

Enhancing arabic bread quality and shelf life stability using bread improvers.

PubMed

Aleid, S M; Al-Hulaibi, A A; Ghoush, M Abu; Al-Shathri, A A

2015-08-01

Arabic breads is produced mainly from hard red winter wheat (HRWW) and have relatively little crumb, dense texture, form pocket and are often round with golden brown crust color. The objectives of this research were to investigate the effect of different bread improvers combinations addition in enhancing the quality parameters of Arabic bread. Therefore, the ability of Arabic bread for rolling, folding and overall quality were evaluated during the Arabic bread storage period for 2 days. It was found that there was significant effect of bread improvers combinations (Arabic gum "AG" * Mongglycerides "MG" *alpha-amylase) addition on the ability of Arabic bread for rolling and folding on the second day (P ≤ 0.1). The highest white Arabic bread quality was obtained significantly from addition of low AG, high of MG and high alpha-amylase combination and high AG, low of MG and high alpha-amylase combination. While, low of AG, high of MG and low alpha-amylase combination and high of AG, high of MG and low alpha-amylase combination significantly exhibited the highest overall quality for the Arabic bread made from whole flour.

Isolation of a novel amylase and lipase-producing Pseudomonas luteola strain: study of amylase production conditions

PubMed Central

2014-01-01

An amylase and lipase producing bacterium (strain C2) was enriched and isolated from soil regularly contaminated with olive washing wastewater in Sfax, Tunisia. Cell was aerobic, mesophilic, Gram-negative, motile, non-sporulating bacterium, capable of growing optimally at pH 7 and 30°C and tolerated maximally 10% (W/V) NaCl. The predominant fatty acids were found to be C18:1ω7c (32.8%), C16:1ω7c (27.3%) and C16:0 (23.1%). Phylogenetic analysis of the 16S rRNA gene revealed that this strain belonging to the genus Pseudomonas. Strain C2 was found to be closely related to Pseudomonas luteola with more than 99% of similarity. Amylase optimization extraction was carried out using Box Behnken Design (BBD). Its maximal activity was found when the pH and temperature ranged from 5.5 to 6.5 and from 33 to 37°C, respectively. Under these conditions, amylase activity was found to be about 9.48 U/ml. PMID:24405763

Enzyme-assisted extraction and identification of antioxidative and α-amylase inhibitory peptides from Pinto beans (Phaseolus vulgaris cv. Pinto).

PubMed

Ngoh, Ying-Yuan; Gan, Chee-Yuen

2016-01-01

Antioxidant and α-amylase inhibitor peptides were successfully extracted from Pinto bean protein isolate (PBPI) using Protamex. A factorial design experiment was conducted and the effects of extraction time, pH and temperature were studied. pH 7.5, extraction time of 1h, S/E ratio of 10 (w/w) and temperature of 50 °C gave the highest antioxidant activities (i.e., ABTS scavenging activity (53.3%) and FRAP value (3.71 mM)), whereas pH 6.5 with the same extraction time, S/E ratio and temperature, gave the highest α-amylase inhibitory activity (57.5%). It was then fractioned using membrane ultrafiltration with molecular weight cutoffs of 100, 50, 30, 10 and 3 kDa. Peptide fraction <3 kDa, which exhibited the highest antioxidant activities (i.e., ABTS (42.2%) and FRAP (0.81 mM)) and α-amylase inhibitory activity (62.1%), was then subjected to LCMS and MS/MS analyses. Six sequences were identified for antioxidant peptides, whereas seven peptides for α-amylase inhibitor. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of exogenous gamma-aminobutyric acid on α-amylase activity in the aleurone of barley seeds.

PubMed

Sheng, Yidi; Xiao, Huiyuan; Guo, Chunli; Wu, Hong; Wang, Xiaojing

2018-03-03

Gamma-aminobutyric acid (GABA), a nonprotein amino acid, often accumulates in plants exposed to certain environmental stimuli. Previous studies indicated that a closed relationship existed between endogenous GABA and seed germination. However, there are few studies on the effect of exogenous GABA on seed germination. The objective of this study was to explore whether exogenous GABA affected α-amylase activity which the activation is an important stage in seed germination. The level of endogenous GABA in barley seeds rose gradually during germination, suggesting that endogenous GABA was involved in germination. We measured starch degradation under application of various concentration GABA and found that GABA promoted seed starch degradation with a dose-responsive effect. The relationship between GABA and α-amylase activity was investigated by treating barley aleurone with exogenous GABA. The result showed that α-amylase activity began to rise after about 24 h and reached a peak at 48 h. Molecular evidence suggested that GABA increased α-amylase gene expression. We explore the possible roles played by GABA in signal transduction. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Two forms of alpha-amylase in mantle tissue of Mytilus galloprovincialis: purification and molecular properties of form II.

PubMed

Lombraña, M; Suárez, P; San Juan, F

2005-09-01

alpha-Amylase activity has been shown for the first time in a non-digestive tissue from Mytilus galloprovincialis. alpha-amylase from mussel mantle tissue has been purified by affinity chromatography on insoluble starch, followed by gel-filtration chromatography on Superdex-200. The chromatographic and electrophoretic behaviour of M. galloprovincialis alpha-amylase and stability characteristics suggest two forms of this enzyme: one form forming stable aggregates (form I) and a monomeric form (form II) that is more abundant, active and unstable. Both forms show an inverse quantitative variation. Purified form II was highly unstable and the molecular mass was estimated to be 66 kDa by sodium dodecyl sulphate (SDS)-gel electrophoresis. Maximum activity was noted at pH 6.5 and 35 degrees C.

Unexpected High Digestion Rate of Cooked Starch by the Ct-Maltase-Glucoamylase Small Intestine Mucosal α-Glucosidase Subunit

PubMed Central

Lin, Amy Hui-Mei; Nichols, Buford L.; Quezada-Calvillo, Roberto; Avery, Stephen E.; Sim, Lyann; Rose, David R.; Naim, Hassan Y.; Hamaker, Bruce R.

2012-01-01

For starch digestion to glucose, two luminal α-amylases and four gut mucosal α-glucosidase subunits are employed. The aim of this research was to investigate, for the first time, direct digestion capability of individual mucosal α-glucosidases on cooked (gelatinized) starch. Gelatinized normal maize starch was digested with N- and C-terminal subunits of recombinant mammalian maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) of varying amounts and digestion periods. Without the aid of α-amylase, Ct-MGAM demonstrated an unexpected rapid and high digestion degree near 80%, while other subunits showed 20 to 30% digestion. These findings suggest that Ct-MGAM assists α-amylase in digesting starch molecules and potentially may compensate for developmental or pathological amylase deficiencies. PMID:22563462

Unexpected high digestion rate of cooked starch by the Ct-maltase-glucoamylase small intestine mucosal α-glucosidase subunit.

PubMed

Lin, Amy Hui-Mei; Nichols, Buford L; Quezada-Calvillo, Roberto; Avery, Stephen E; Sim, Lyann; Rose, David R; Naim, Hassan Y; Hamaker, Bruce R

2012-01-01

For starch digestion to glucose, two luminal α-amylases and four gut mucosal α-glucosidase subunits are employed. The aim of this research was to investigate, for the first time, direct digestion capability of individual mucosal α-glucosidases on cooked (gelatinized) starch. Gelatinized normal maize starch was digested with N- and C-terminal subunits of recombinant mammalian maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) of varying amounts and digestion periods. Without the aid of α-amylase, Ct-MGAM demonstrated an unexpected rapid and high digestion degree near 80%, while other subunits showed 20 to 30% digestion. These findings suggest that Ct-MGAM assists α-amylase in digesting starch molecules and potentially may compensate for developmental or pathological amylase deficiencies.

Improved α-Amylase Production by Dephosphorylation Mutation of CreD, an Arrestin-Like Protein Required for Glucose-Induced Endocytosis of Maltose Permease and Carbon Catabolite Derepression in Aspergillus oryzae

PubMed Central

Tanaka, Mizuki; Hiramoto, Tetsuya; Tada, Hinako; Shintani, Takahiro

2017-01-01

ABSTRACT Aspergillus oryzae produces copious amount of amylolytic enzymes, and MalP, a major maltose permease, is required for the expression of amylase-encoding genes. The expression of these genes is strongly repressed by carbon catabolite repression (CCR) in the presence of glucose. MalP is transported from the plasma membrane to the vacuole by endocytosis, which requires the homolog of E6-AP carboxyl terminus ubiquitin ligase HulA, an ortholog of yeast Rsp5. In yeast, arrestin-like proteins mediate endocytosis as adaptors of Rsp5 and transporters. In the present study, we examined the involvement of CreD, an arrestin-like protein, in glucose-induced MalP endocytosis and CCR of amylase-encoding genes. Deletion of creD inhibited the glucose-induced endocytosis of MalP, and CreD showed physical interaction with HulA. Phosphorylation of CreD was detected by Western blotting, and two serine residues were determined as the putative phosphorylation sites. However, the phosphorylation state of the serine residues did not regulate MalP endocytosis and its interaction with HulA. Although α-amylase production was significantly repressed by creD deletion, both phosphorylation and dephosphorylation mimics of CreD had a negligible effect on α-amylase activity. Interestingly, dephosphorylation of CreD was required for CCR relief of amylase genes that was triggered by disruption of the deubiquitinating enzyme-encoding gene creB. The α-amylase activity of the creB mutant was 1.6-fold higher than that of the wild type, and the dephosphorylation mimic of CreD further improved the α-amylase activity by 2.6-fold. These results indicate that a combination of the dephosphorylation mutation of CreD and creB disruption increased the production of amylolytic enzymes in A. oryzae. IMPORTANCE In eukaryotes, glucose induces carbon catabolite repression (CCR) and proteolytic degradation of plasma membrane transporters via endocytosis. Glucose-induced endocytosis of transporters is mediated by their ubiquitination, and arrestin-like proteins act as adaptors of transporters and ubiquitin ligases. In this study, we showed that CreD, an arrestin-like protein, is involved in glucose-induced endocytosis of maltose permease and carbon catabolite derepression of amylase gene expression in Aspergillus oryzae. Dephosphorylation of CreD was required for CCR relief triggered by the disruption of creB, which encodes a deubiquitinating enzyme; a combination of the phosphorylation-defective mutation of CreD and creB disruption dramatically improved α-amylase production. This study shows the dual function of an arrestin-like protein and provides a novel approach for improving the production of amylolytic enzymes in A. oryzae. PMID:28455339

Improved α-Amylase Production by Dephosphorylation Mutation of CreD, an Arrestin-Like Protein Required for Glucose-Induced Endocytosis of Maltose Permease and Carbon Catabolite Derepression in Aspergillus oryzae.

PubMed

Tanaka, Mizuki; Hiramoto, Tetsuya; Tada, Hinako; Shintani, Takahiro; Gomi, Katsuya

2017-07-01

Aspergillus oryzae produces copious amount of amylolytic enzymes, and MalP, a major maltose permease, is required for the expression of amylase-encoding genes. The expression of these genes is strongly repressed by carbon catabolite repression (CCR) in the presence of glucose. MalP is transported from the plasma membrane to the vacuole by endocytosis, which requires the homolog of E6-AP carboxyl terminus ubiquitin ligase HulA, an ortholog of yeast Rsp5. In yeast, arrestin-like proteins mediate endocytosis as adaptors of Rsp5 and transporters. In the present study, we examined the involvement of CreD, an arrestin-like protein, in glucose-induced MalP endocytosis and CCR of amylase-encoding genes. Deletion of creD inhibited the glucose-induced endocytosis of MalP, and CreD showed physical interaction with HulA. Phosphorylation of CreD was detected by Western blotting, and two serine residues were determined as the putative phosphorylation sites. However, the phosphorylation state of the serine residues did not regulate MalP endocytosis and its interaction with HulA. Although α-amylase production was significantly repressed by creD deletion, both phosphorylation and dephosphorylation mimics of CreD had a negligible effect on α-amylase activity. Interestingly, dephosphorylation of CreD was required for CCR relief of amylase genes that was triggered by disruption of the deubiquitinating enzyme-encoding gene creB The α-amylase activity of the creB mutant was 1.6-fold higher than that of the wild type, and the dephosphorylation mimic of CreD further improved the α-amylase activity by 2.6-fold. These results indicate that a combination of the dephosphorylation mutation of CreD and creB disruption increased the production of amylolytic enzymes in A. oryzae IMPORTANCE In eukaryotes, glucose induces carbon catabolite repression (CCR) and proteolytic degradation of plasma membrane transporters via endocytosis. Glucose-induced endocytosis of transporters is mediated by their ubiquitination, and arrestin-like proteins act as adaptors of transporters and ubiquitin ligases. In this study, we showed that CreD, an arrestin-like protein, is involved in glucose-induced endocytosis of maltose permease and carbon catabolite derepression of amylase gene expression in Aspergillus oryzae Dephosphorylation of CreD was required for CCR relief triggered by the disruption of creB , which encodes a deubiquitinating enzyme; a combination of the phosphorylation-defective mutation of CreD and creB disruption dramatically improved α-amylase production. This study shows the dual function of an arrestin-like protein and provides a novel approach for improving the production of amylolytic enzymes in A. oryzae . Copyright © 2017 American Society for Microbiology.

Engineering Saccharomyces cerevisiae for direct conversion of raw, uncooked or granular starch to ethanol.

PubMed

Görgens, Johann F; Bressler, David C; van Rensburg, Eugéne

2015-01-01

The production of raw starch-degrading amylases by recombinant Saccharomyces cerevisiae provides opportunities for the direct hydrolysis and fermentation of raw starch to ethanol without cooking or exogenous enzyme addition. Such a consolidated bioprocess (CBP) for raw starch fermentation will substantially reduce costs associated with energy usage and commercial granular starch hydrolyzing (GSH) enzymes. The core purpose of this review is to provide comprehensive insight into the physiological impact of recombinant amylase production on the ethanol-producing yeast. Key production parameters, based on outcomes from modifications to the yeast genome and levels of amylase production, were compared to key benchmark data. In turn, these outcomes are of significance from a process point of view to highlight shortcomings in the current state of the art of raw starch fermentation yeast compared to a set of industrial standards. Therefore, this study provides an integrated critical assessment of physiology, genetics and process aspects of recombinant raw starch fermenting yeast in relation to presently used technology. Various approaches to strain development were compared on a common basis of quantitative performance measures, including the extent of hydrolysis, fermentation-hydrolysis yield and productivity. Key findings showed that levels of α-amylase required for raw starch hydrolysis far exceeded enzyme levels for soluble starch hydrolysis, pointing to a pre-requisite for excess α-amylase compared to glucoamylase for efficient raw starch hydrolysis. However, the physiological limitations of amylase production by yeast, requiring high biomass concentrations and long cultivation periods for sufficient enzyme accumulation under anaerobic conditions, remained a substantial challenge. Accordingly, the fermentation performance of the recombinant S. cerevisiae strains reviewed in this study could not match the performance of conventional starch fermentation processes, based either on starch cooking and/or exogenous amylase enzyme addition. As an alternative strategy, the addition of exogenous GSH enzymes during early stages of raw starch fermentation may prove to be a viable approach for industrial application of recombinant S. cerevisiae, with the process still benefitting from amylase production by CBP yeast during later stages of cultivation.

Lactucaxanthin - a potential anti-diabetic carotenoid from lettuce (Lactuca sativa) inhibits α-amylase and α-glucosidase activity in vitro and in diabetic rats.

PubMed

Gopal, Sowmya Shree; Lakshmi, Magisetty Jhansi; Sharavana, Gurunathan; Sathaiah, Gunaseelan; Sreerama, Yadahally N; Baskaran, Vallikannan

2017-03-22

Intestinal and pancreatic α-amylase and α-glucosidase inhibitors offer an approach to lower the levels of post-prandial hyperglycemia through the control of dietary starch breakdown in digestion. This study hypothesized that lactucaxanthin (Lxn) in lettuce (Lactuca sativa) inhibits the activity of α-amylase and α-glucosidase. In this study, the interaction of Lxn with α-amylase and α-glucosidase in silico and its inhibitory effect on these enzymes were studied using in vitro and STZ-induced diabetic rat models. Lxn was isolated from lettuce with 96% purity confirmed by HPLC and LCMS. The in silico analysis showed that Lxn has a lower binding energy (-6.05 and -6.34 kcal mol -1 ) with α-amylase and α-glucosidase compared to their synthetic inhibitors, acarbose (-0.21 kcal mol -1 ) and miglitol (-2.78 kcal mol -1 ), respectively. In vitro α-amylase and α-glucosidase inhibition assays revealed that Lxn had IC 50 values of 435.5 μg mL -1 and 1.84 mg mL -1 , but acarbose has values of 2.5 and 16.19 μg mL -1 . The in vivo results showed an increased activity for α-amylase and α-glucosidase in the intestine (4.7 and 1.30 fold, p < 0.05) and pancreas (1.3 and 1.48 fold, p < 0.05) of STZ induced diabetic rats compared to normal rats. Whereas the activity decreased (p < 0.05) in the Lxn fed diabetic rats, except for the intestinal α-glucosidase activity (1.69 ± 0.12 PNP per min per mg protein). This was confirmed by the low blood glucose level (239.4 ± 18.2 mg dL -1 ) in diabetic rats fed Lxn compared to the diabetic group (572.2 ± 30.5 mg dL -1 , p < 0.05). Lxn significantly inhibited (p < 0.05) the activity of α-amylase and α-glucosidase and could be of medical and nutritional relevance in the treatment of diabetes.

Effects of ethanol and folic acid consumption during pregnancy and lactation on basal enzymatic secretion in the duodenal juice of offspring rats.

PubMed

Cano, Ma José; Murillo, Ma Luisa; Delgado, Ma José; Carreras, Olimpia

2003-09-01

Studies on duodenal juice enzyme activities were carried out on suckling Wistar rats born to dams given ethanol during gestation and suckling. The results were compared with offspring of dams given diets containing no ethanol. Comparisons were also made with offspring of dams given ethanol and folic acid supplementation to observe whether a folate supplement could sufficiently reverse the negative effect of ethanol consumption. The dams were fed increased amounts of ethanol (5% to 20%, vol/vol) in tap water for 4 wk. The maximum quantity, 20% ethanol, was given to the dams during pregnancy and lactation. Offspring animals were randomized into three groups: control (CG), ethanol treated (EG), and ethanol plus folic acid (EFG). Body weight at birth and at 21 d after birth and pancreatic weight were lower in offspring after ethanol treatment. Folic acid supplement increased these parameters in the EFG. Under basal conditions, decreases in amylase, lipase, and chymotrypsin activities in the duodenal juice after ethanol treatment were detected. Serum and urine amylase activities also decreased in the EG and EFG. These changes were different in the ethanol-treated progenitors. In these progenitors, ethanol treatment increased serum amylase levels. In the offspring, amylase activities in the EFG decreased with respect to the CG; however, an increase in the EG was observed. In dams the folic acid supplement did not significantly alter the serum amylase activities. Lipase and chymotrypsin activities in the EFG were similar to those in the EG. An increase of serum and urine amylase in the EFG with respect to the EG was found. Our findings indicated that, under basal conditions, ethanol treatment during gestation and lactation negatively affects the digestive function in offspring. The effects of ethanol were slightly attenuated in rats supplemented with folic acid for amylase activities. Although extrapolation from animal studies can be tenuous, the present findings may explain the use of folic acid in the prevention of damage induced by ethanol to increase the amylase levels to physiologic concentrations.

Bacterial and Archaeal α-Amylases: Diversity and Amelioration of the Desirable Characteristics for Industrial Applications

PubMed Central

Mehta, Deepika; Satyanarayana, Tulasi

2016-01-01

Industrial enzyme market has been projected to reach US$ 6.2 billion by 2020. Major reasons for continuous rise in the global sales of microbial enzymes are because of increase in the demand for consumer goods and biofuels. Among major industrial enzymes that find applications in baking, alcohol, detergent, and textile industries are α-amylases. These are produced by a variety of microbes, which randomly cleave α-1,4-glycosidic linkages in starch leading to the formation of limit dextrins. α-Amylases from different microbial sources vary in their properties, thus, suit specific applications. This review focuses on the native and recombinant α-amylases from bacteria and archaea, their production and the advancements in the molecular biology, protein engineering and structural studies, which aid in ameliorating their properties to suit the targeted industrial applications. PMID:27516755

The amylase inhibitor montbretin A reveals a new glycosidase inhibition motif.

PubMed

Williams, Leslie K; Zhang, Xiaohua; Caner, Sami; Tysoe, Christina; Nguyen, Nham T; Wicki, Jacqueline; Williams, David E; Coleman, John; McNeill, John H; Yuen, Violet; Andersen, Raymond J; Withers, Stephen G; Brayer, Gary D

2015-09-01

The complex plant flavonol glycoside montbretin A is a potent (Ki = 8 nM) and specific inhibitor of human pancreatic α-amylase with potential as a therapeutic for diabetes and obesity. Controlled degradation studies on montbretin A, coupled with inhibition analyses, identified an essential high-affinity core structure comprising the myricetin and caffeic acid moieties linked via a disaccharide. X-ray structural analyses of the montbretin A-human α-amylase complex confirmed the importance of this core structure and revealed a novel mode of glycosidase inhibition wherein internal π-stacking interactions between the myricetin and caffeic acid organize their ring hydroxyls for optimal hydrogen bonding to the α-amylase catalytic residues D197 and E233. This novel inhibitory motif can be reproduced in a greatly simplified analog, offering potential for new strategies for glycosidase inhibition and therapeutic development.

Supplementation of methionine to a low soybean protein diet strikingly increases pancreatic amylase activity in rats.

PubMed

Hara, H; Kiriyama, S; Kasai, T

1997-02-01

Feed efficiency in rats fed a low soybean protein isolate (SPI) diet (100 g/kg diet) was dramatically improved with the supplementation of L-methionine (3 g/kg diet). Pancreatic amylase activity was low in rats fed a low SPI diet, and was much higher in the supplemented group than in the non-supplemented group. Pancreatic trypsinogen and chymotrypsinogen contents (as activities of trypsin and chymotrypsin) were not changed with the methionine supplementation. In the small intestine, sucrase and leucine aminopeptidase in the jejunum and ileum were not clearly changed. In conclusion, a small amount of methionine supplemented to a low SPI diet especially induced pancreatic amylase among digestive enzymes. The factor involved in nutritional status, not the physiological action of methionine itself, may contribute the induction of amylase.

Bacterial and Archaeal α-Amylases: Diversity and Amelioration of the Desirable Characteristics for Industrial Applications.

PubMed

Mehta, Deepika; Satyanarayana, Tulasi

2016-01-01

Industrial enzyme market has been projected to reach US$ 6.2 billion by 2020. Major reasons for continuous rise in the global sales of microbial enzymes are because of increase in the demand for consumer goods and biofuels. Among major industrial enzymes that find applications in baking, alcohol, detergent, and textile industries are α-amylases. These are produced by a variety of microbes, which randomly cleave α-1,4-glycosidic linkages in starch leading to the formation of limit dextrins. α-Amylases from different microbial sources vary in their properties, thus, suit specific applications. This review focuses on the native and recombinant α-amylases from bacteria and archaea, their production and the advancements in the molecular biology, protein engineering and structural studies, which aid in ameliorating their properties to suit the targeted industrial applications.

Characterization of a thermostable raw-starch hydrolyzing α-amylase from deep-sea thermophile Geobacillus sp.

PubMed

Jiang, Tao; Cai, Menghao; Huang, Mengmeng; He, Hao; Lu, Jian; Zhou, Xiangshan; Zhang, Yuanxing

2015-10-01

A deep-sea thermophile, Geobacillus sp. 4j, was identified to grow on starch and produce thermostable amylase. N-terminally truncated form of Geobacillus sp. 4j α-amylase (Gs4j-amyA) was fused at its N-terminal end with the signal peptide of outer membrane protein A (OmpA) of Escherichia coli. The enzyme was over-expressed in E. coli BL21 with a maximum extracellular production of 130U/ml in shake flask. The yield of the transformant increased 22-fold as compared with that of the wild strain. The recombinant enzyme purified to apparent homogeneity by metal-affinity chromatography, exhibited a molecular mass of 62kDa. It displayed the maximal activity at 60-65°C and pH 5.5. Its half-life (t1/2) at 80°C was 4.25h with a temperature deactivation energy of 166.3kJ/mol. Compared to three commonly used commercial α-amylases, the Gs4j-amyA exhibited similar thermostable performance to BLA but better than BAA and BSA. It also showed a universally efficient raw starch hydrolysis performance superior to commercial α-amylases at an acidic pH approaching nature of starch slurry. As a new acidic-resistant thermostable α-amylase, it has the potential to bypass the industrial gelatinization step in raw starch hydrolysis. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparing Dental Stress in New Child Patients and Returning Patients Using Salivary Cortisol, Immunoglobulin-A and Alpha- Amylase.

PubMed

Alaki, Sumer M; Safi, Ayman; Ouda, Soliman; Nadhreen, Alaa

this study was aimed at comparing dental stress in children having their first dental visit to those returning for dental treatment using salivary biomarkers of stress including salivary cortisol (s-cortisol), Immunoglobulin-A (s-IgA) and alpha-amylase (s-α-amylase). Additionally, the study was aimed at monitoring the change in stress in new patients as they progressed from the waiting to the clinical areas. salivary samples were collected from 40 children who had not been to a dentist before and similar samples were collected from 40 children who were returning for completion of dental treatment. Salivary cortisol, s-IgA and s-α-amylase concentrations were obtained by Enzyme-linked Immunosorbent Assay (ELISA). salivary cortisol levels were higher for new patients at the waiting area compared to that at the dental chair (p=0.05). Salivary alpha-amylase significantly increased in new patients while being seated in the dental chair. Returning patients had higher s-α-amylase (p=0.001) and s-IgA (p=0.016) compared to new patients. Returning patients had the lowest level of s-cortisol when providers were faculty pediatric dentists than with students and interns (p=0.035). children coming in for their first dental visit may experience dental stress at the waiting area before being seated for dental examination. Returning children may experience higher levels of stress compared to new child patients possibly due to previous dental exposure.

Progress of pancreatitis disease biomarker alpha amylase enzyme by new nano optical sensor.

PubMed

Attia, M S; Al-Radadi, Najlaa S

2016-12-15

A new nano optical sensor binuclear Pd-(2-aminothiazole) (urea), Pd(atz,ur) complex was prepared and characterized for the assessment of the activity of alpha amylase enzyme in urine and serum samples for early diagnosis of Pancreatitis disease. The assessment of alpha amylase activity is carried out by the quenching of the luminescence intensity of the nano optical sensor binuclear Pd(atz,ur) complex at 457nm by the 2-chloro-4-nitrophenol (2-CNP) which produced from the reaction of the enzyme with 2-chloro-4-nitrophenyl-α-d-maltotrioside (CNPG3) substrate. The remarkable quenching of the luminescence intensity at 457nm of nano Pd(atz,ur) doped in sol-gel matrix by various concentrations of the 2-CNP was successfully used as an optical sensor for the assessment of α-amylase activity. The calibration plot was achieved over the concentration range 8.5×10(-6) to 1.9×10(-9)molL(-1) 2-CNP with a correlation coefficient of (0.999) and a detection limit of (7.4×10(-10)molL(-1)). The method was used satisfactorily for the assessment of the α-amylase activity over activity range (3-321U/L) in different urine and serum samples of pancreatitis patients. The assessment of the alpha amylase biomarker by the proposed method increases its sensitivity (96.88%) and specificity (94.41%) for early diagnosis of pancreatitis diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Crystal structures of the psychrophilic alpha-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor.

PubMed Central

Aghajari, N.; Feller, G.; Gerday, C.; Haser, R.

1998-01-01

Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the alpha-amylase (AHA) secreted by this bacterium, in its native state to 2.0 angstroms resolution as well as in complex with Tris to 1.85 angstroms resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known alpha-amylases of various origins with a surprisingly greatest similarity to mammalian alpha-amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate alpha-1,4-glycosidic bonds. The chloride binding site is situated approximately 5 angstroms from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found approximately 22 angstroms from the active site. We found that this triad is equally present in other chloride dependent alpha-amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted alpha-amylase. PMID:9541387

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, T.; Teo, L.H.; Sin, Y.M.

A number of digestive enzymes in the green mussel, Perna viridis L., have been reported, and {alpha}-amylase is believed to have a higher activity than the others. Small plankton, on which the green mussel feeds, may supply plenty of starch and glycogen. They may be an important source of nutrients for the green mussel and the ability of the latter to make good use of them depends mainly on the activities of amylase. The effect of heavy metals on amylase activity is also important as the ability of the mussel`s digestive gland to accumulate these metals is well known. Highmore » concentrations of heavy metals, especially lead, have been observed in the water around Singapore. The in vitro inhibition of some metals on the activities of digestive enzymes from the green mussel has been observed, but kinetic properties of the inhibition and the in vivo inhibition of the heavy metals on digestive enzymes are little understood. In the present study, in vitro inhibition of four metals (Pb, Cd, Zn and Hg) on the activity of {alpha}-amylase from the digestive gland of the green mussel will be compared. Their effects on the K{sub M} and V{sub max} values of {alpha}-amylase will also be compared. Finally, lead is either added to the food or water, to see how it affects the activity of {alpha}-amylase and how this effect acts in combination with starvation. 12 refs., 3 figs., 3 tabs.« less

A Proposed Mechanism for the Thermal Denaturation of a Recombinant Bacillus Halmapalus Alpha-amylase - the Effect of Calcium Ions

NASA Technical Reports Server (NTRS)

Nielsen, Anders D.; Pusey, Marc L.; Fuglsang, Claus C.; Westh, Peter

2003-01-01

The thermal stability of a recombinant alpha-amylase from Bacillus halmapalus alpha-amylase (BHA) has been investigated using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). This alpha-amylase is homologous to other Bacillus alpha-amylases where previous crystallographic studies have identified the existence of 3 calcium binding sites in the structure. Denaturation of BHA is irreversible with a Tm of approximately 89 C, and DSC thermograms can be described using a one-step irreversible model. A 5 C increase in T(sub m) in the presence of 10 fold excess CaCl2 was observed. However, a concomitant increase in the tendency to aggregate was also observed. The presence of 30-40 fold excess calcium chelator (EDTA or EGTA) results in a large destabilization of BHA corresponding to about 40 C lower T(sub m), as determined by both CD and DSC. Ten fold excess EGTA reveals complex DSC thermograms corresponding to both reversible and irreversible transitions, which possibly originate from different populations of BHA:calcium complexes. The observations in the present study have, in combination with structural information of homologous alpha-amylases, provided the basis for the proposal of a simple denaturation mechanism of BHA. The proposed mechanism describes the irreversible thermal denaturation of different BHA:calcium complexes and the calcium binding equilibrium involved. Furthermore, the model accounts for a temperature induced reversible structural change associated with calcium binding.

Genetic diversity of Bacillus sp producers of amylase isolated from the soil.

PubMed

Xavier, A R E O; Lima, E R; Oliveira, A M E; Cardoso, L; Santos, J; Cangussu, C H C; Leite, L N; Quirino, M C L; Júnior, I G C; Oliveira, D A; Xavier, M A S

2017-09-27

The microorganisms are the best source of extracellular enzymes since they allow an economical technology with low-resource consumption compared to animals and plants. The amylases are among the most important enzymes being the genus Bacillus one of the most investigated due to its ability to produce this enzyme. The objective of this study was to isolate and analyze the genetic diversity among bacteria of the genus Bacillus sp producer of amylase originated from the soil. To this end, soil samples were collected and submitted to the condition of extreme temperature. The serial dilution procedure followed by seeding on solid medium containing starch was used for isolation of strains that produce amylase. The microorganisms isolated were subjected to standard morphological methods for presumptive identification of the genus Bacillus. The PCR assay with the universal genetic marker 16S rDNA was used for confirmation of bacterial strain. All the 10 isolates presumptively identified as bacteria amplified a fragment of 370 bp corresponding to the 16S rDNA gene. The enzymatic activity was expressed as an enzymatic index (EI), after 24 h of incubation. All isolate producers of amylase exhibited EI ≥ 2.0. The determination of the genetic profile and the clonal relationship among the isolates were performed by the method of ERIC-PCR polymorphism. The isolates of Bacillus spp were divided into 2 groups (I and II). Through this method, the discriminatory capacity of this analysis of polymorphisms was verified in differing producer strains from those not producing amylase.

Purification and Characterization of Thermostable and Detergent-Stable α-Amylase from Anoxybacillus sp. AH1

PubMed Central

Bekler, Fatma Matpan; Pirinççioğlu, Hemşe; Güven, Reyhan Gül; Güven, Kemal

2016-01-01

Summary A thermostable and detergent-stable α-amylase from a newly isolated Anoxybacillus sp. AH1 was purified and characterized. Maximum enzyme production (1874.8 U/mL) was obtained at 24 h of incubation. The amylase was purified by using Sephadex G-75 gel filtration, after which an 18-fold increase in specific activity and a yield of 9% were achieved. The molecular mass of the purified enzyme was estimated at 85 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature values of the enzyme were 7.0 and 60 °C, respectively. The enzyme was highly stable in the presence of 30% glycerol, retaining 85% of its original activity at 60 °C within 120 min. Km and vmax values were 0.102 µmol and 0.929 µmol/min, respectively, using Lineweaver-Burk plot. The enzyme activity was increased by various detergents, but it was significantly inhibited in the presence of urea. Mg2+ and Ca2+ also significantly activated α-amylase, while Zn2+, Cu2+ and metal ion chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline (phen) greatly inhibited the enzyme activity. α-Amylase activity was enhanced by β-mercaptoethanol (β-ME) and dithiothreitol (DTT) to a great extent, but inhibited by p-chloromercuribenzoic acid (PCMB). Iodoacetamide (IAA) and N-ethylmaleimide (NEM) had a slight, whereas phenylmethylsulfonyl fluoride (PMSF) had a strong inhibitory effect on the amylase activity. PMID:27904395

Pouteria torta epicarp as a useful source of α-amylase inhibitor in the control of type 2 diabetes.

PubMed

de Sales, Paloma Michelle; de Souza, Paula Monteiro; Dartora, Mariana; Resck, Inês Sabioni; Simeoni, Luiz Alberto; Fonseca-Bazzo, Yris Maria; de Oliveira Magalhães, Pérola; Silveira, Dâmaris

2017-11-01

Type 2 diabetes plays a major role in public health, affecting about 400 million adults. One of the used strategies to control type 2 diabetes is the inhibition of α-amylase activity to reduce post-prandial blood glucose levels. Therefore, in past decades, the search of new α-amylase inhibitors has led to the evaluation of natural products as a source of these compounds. Pouteria torta (Sapotaceae) is widespread in Brazil and bears edible fruits. Epicarp and pulp crude extracts of fresh fruits were studied for in vitro α-amylase inhibition activity. The pulp did not present activity while epicarp, usually considered as waste, showed a high α-amylase inhibitory capacity when compared with acarbose and Triticum aestivum. Therefore, an assay-guided fractionation study of epicarp crude extract was performed. Fraction VI shows very high inhibitory activity with IC 50 of 9 μg/mL. However, subsequent fractionation led to lower inhibition potential (IC 50 of 22.1 μg/mL). The qualitative characterization of fraction VI were performed by chromatographic and spectrometric analysis and showed the presence of epicatechin, catechin, sucrose, glucose, and fructose. Total phenolic and flavonoid contents and antioxidant capacity were also assessed and there seemed to be no correlation between phenolic or flavonoids-rich fractions and antioxidant capacity or α-amylase inhibitory activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

New insight in the structural features of haloadaptation in α-amylases from halophilic Archaea following homology modeling strategy: folded and stable conformation maintained through low hydrophobicity and highly negative charged surface

NASA Astrophysics Data System (ADS)

Zorgani, Mohamed Amine; Patron, Kevin; Desvaux, Mickaël

2014-07-01

Proteins from halophilic archaea, which live in extreme saline conditions, have evolved to remain folded, active and stable at very high ionic strengths. Understanding the mechanism of haloadaptation is the first step toward engineering of halostable biomolecules. Amylases are one of the main enzymes used in industry. Yet, no three-dimensional structure has been experimentally resolved for α-amylases from halophilic archaea. In this study, homology structure modeling of α-amylases from the halophilic archaea Haloarcula marismortui, Haloarcula hispanica, and Halalkalicoccus jeotgali were performed. The resulting models were subjected to energy minimization, evaluation, and structural analysis. Calculations of the amino acid composition, salt bridges and hydrophobic interactions were also performed and compared to a set of non-halophilic counterparts. It clearly appeared that haloarchaeal α-amylases exhibited lower propensities for helix formation and higher propensities for coil-forming regions. Furthermore, they could maintain a folded and stable conformation in high salt concentration through highly negative charged surface with over representation of acidic residues, especially Asp, and low hydrophobicity with increase of salt bridges and decrease in hydrophobic interactions on the protein surface. This study sheds some light on the stability of α-amylases from halophilic archaea and provides strong basis not only to understand haloadaptation mechanisms of proteins in microorganisms from hypersalines environments but also for biotechnological applications.

New insight in the structural features of haloadaptation in α-amylases from halophilic Archaea following homology modeling strategy: folded and stable conformation maintained through low hydrophobicity and highly negative charged surface.

PubMed

Zorgani, Mohamed Amine; Patron, Kevin; Desvaux, Mickaël

2014-07-01

Proteins from halophilic archaea, which live in extreme saline conditions, have evolved to remain folded, active and stable at very high ionic strengths. Understanding the mechanism of haloadaptation is the first step toward engineering of halostable biomolecules. Amylases are one of the main enzymes used in industry. Yet, no three-dimensional structure has been experimentally resolved for α-amylases from halophilic archaea. In this study, homology structure modeling of α-amylases from the halophilic archaea Haloarcula marismortui, Haloarcula hispanica, and Halalkalicoccus jeotgali were performed. The resulting models were subjected to energy minimization, evaluation, and structural analysis. Calculations of the amino acid composition, salt bridges and hydrophobic interactions were also performed and compared to a set of non-halophilic counterparts. It clearly appeared that haloarchaeal α-amylases exhibited lower propensities for helix formation and higher propensities for coil-forming regions. Furthermore, they could maintain a folded and stable conformation in high salt concentration through highly negative charged surface with over representation of acidic residues, especially Asp, and low hydrophobicity with increase of salt bridges and decrease in hydrophobic interactions on the protein surface. This study sheds some light on the stability of α-amylases from halophilic archaea and provides strong basis not only to understand haloadaptation mechanisms of proteins in microorganisms from hypersalines environments but also for biotechnological applications.

Evaluation of adenosine deaminase in saliva and serum, and salivary α-amylase, in canine pyometra at diagnosis and after ovariohysterectomy.

PubMed

Tecles, F; Escribano, D; Contreras-Aguilar, M D; Rubio, C P; Szczubiał, M; Cerón, J J; Dąbrowski, R; Tvarijonaviciute, A

2018-06-01

An assay for adenosine deaminase (ADA) was validated in serum and saliva in dogs. Changes in ADA and salivary α-amylase activities were analysed in 26 bitches diagnosed with pyometra and compared with activities in 19 healthy bitches. All animals were classified according to the American Society of Anaesthesiologists (ASA) scoring for physical status. In the validation study, the ADA assay had an imprecision<12% and determination coefficients>0.90 in linearity under dilution experiments, with recoveries of 99.2-114.4%. On the day of presentation, salivary ADA activity was significantly higher in dogs with pyometra than in healthy dogs (median values 7.1IU/L vs. 0.8IU/L, respectively; P<0.01). ADA had a moderate positive correlation with leucocyte and band neutrophil counts, haptoglobin, salivary α-amylase and ASA score, and a low positive correlation with C-reactive protein. There were no significant differences in salivary α-amylase activity between dogs with pyometra and healthy dogs (57.3IU/L vs. 27.4IU/L, respectively). Salivary α-amylase had a low correlation with ASA grade, and leucocyte and band neutrophil counts. In 7/26 bitches with pyometra that were sampled 3 and 10days after ovariohysterectomy, there were no significant changes in α-amylase or ADA activities. These results indicate that ADA activity is increased in the saliva of bitches with pyometra, probably related to systemic inflammation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Digestion of Starch Granules from Maize, Potato and Wheat by Larvae of the the Yellow Mealworm, Tenebrio molitor and the Mexican Bean Weevil, Zabrotes subfasciatus

PubMed Central

Meireles, Elaine A.; Carneiro, Cíntia N. B.; DaMatta, Renato A.; Samuels, Richard I.; Silva, Carlos P.

2009-01-01

Scanning electron microscopy images were taken of starch granules from different sources following exposure in vivo and in vitro to gut α-amylases isolated from Tenebrio molitor L. (Coleoptera: Tenebrionidae) and Zabrotes subfasciatus Boheman (Coleoptera: Bruchidae). One α-amylase was isolated from whole larval midguts of T. molitor using non-denaturing SDS-PAGE, while two other α-amylase fractions were isolated from whole larval midguts of Z. subfasciatus using hydrophobic interaction chromatography., Digested starch granules from larvae fed on maize, potato or wheat were isolated from midgut contents. Combinations of starch granules with isolated α-amylases from both species showed similar patterns of granule degradation. In vitro enzymatic degradation of maize starch granules by the three different α-amylase fractions began by creating small holes and crater-like areas on the surface of the granules. Over time, these holes increased in number and area resulting in extensive degradation of the granule structure. Granules from potato did not show formation of pits and craters on their surface, but presented extensive erosion in their interior. For all types of starch, as soon as the interior of the starch granule was reached, the inner layers of amylose and amylopectin were differentially hydrolyzed, resulting in a striated pattern. These data support the hypothesis that the pattern of starch degradation depends more on the granule type than on the α-amylase involved. PMID:19619014

Amylase in drain fluid for the diagnosis of pancreatic leak in post-pancreatic resection.

PubMed

Davidson, Tsetsegdemberel Bat-Ulzii; Yaghoobi, Mohammad; Davidson, Brian R; Gurusamy, Kurinchi Selvan

2017-04-07

The treatment of people with clinically significant postoperative pancreatic leaks is different from those without clinically significant pancreatic leaks. It is important to know the diagnostic accuracy of drain fluid amylase as a triage test for the detection of clinically significant pancreatic leaks, so that an informed decision can be made as to whether the patient with a suspected pancreatic leak needs further investigations and treatment. There is currently no systematic review of the diagnostic test accuracy of drain fluid amylase for the diagnosis of clinically relevant pancreatic leak. To determine the diagnostic accuracy of amylase in drain fluid at 48 hours or more for the diagnosis of pancreatic leak in people who had undergone pancreatic resection. We searched MEDLINE, Embase, the Science Citation Index Expanded, and the National Institute for Health Research Health Technology Assessment (NIHR HTA) websites up to 20 February 2017. We searched the references of the included studies to identify additional studies. We did not restrict studies based on language or publication status, or whether data were collected prospectively or retrospectively. We also performed a 'related search' and 'citing reference' search in MEDLINE and Embase. We included all studies that evaluated the diagnostic test accuracy of amylase in the drain fluid at 48 hours or more for the diagnosis of pancreatic leak in people who had undergone pancreatic resection excluding total pancreatectomy. We planned to exclude case-control studies because these studies are prone to bias, but did not find any. At least two authors independently searched and screened the references produced by the search to identify relevant studies. Two review authors independently extracted data from the included studies. The included studies reported drain fluid amylase on different postoperative days and measured at different cut-off levels, so it was not possible to perform a meta-analysis using the bivariate model as planned. We have reported the sensitivity, specificity, post-test probability of a positive and negative drain fluid amylase along with 95% confidence interval (CI) on each of the different postoperative days and measured at different cut-off levels. A total of five studies including 868 participants met the inclusion criteria for this review. The five studies included in this review reported the value of drain fluid amylase at different thresholds and different postoperative days. The sensitivities and specificities were variable; the sensitivities ranged between 0.72 and 1.00 while the specificities ranged between 0.73 and 0.99 for different thresholds on different postoperative days. At the median prevalence (pre-test probability) of 15.9%, the post-test probabilities for pancreatic leak ranged between 35.9% and 95.4% for a positive drain fluid amylase test and ranged between 0% and 5.5% for a negative drain fluid amylase test.None of the studies used the reference standard of confirmation by surgery or by a combination of surgery and clinical follow-up, but used the International Study Group on Pancreatic Fistula (ISGPF) grade B and C as the reference standard. The overall methodological quality was unclear or high in all the studies. Because of the paucity of data and methodological deficiencies in the studies, we are uncertain whether drain fluid amylase should be used as a method for testing for pancreatic leak in an unselected population after pancreatic resection; and we judge that the optimal cut-off of drain fluid amylase for making the diagnosis of pancreatic leak is also not clear. Further well-designed diagnostic test accuracy studies with pre-specified index test threshold of drain fluid amylase (at three times more on postoperative day 5 or another suitable pre-specified threshold), appropriate follow-up (for at least six to eight weeks to ensure that there are no pancreatic leaks), and clearly defined reference standards (of surgical, clinical, and radiological confirmation of pancreatic leak) are important to reliably determine the diagnostic accuracy of drain fluid amylase in the diagnosis of pancreatic leak.

Functional expression of a novel α-amylase from Antarctic psychrotolerant fungus for baking industry and its magnetic immobilization.

PubMed

He, Lei; Mao, Youzhi; Zhang, Lujia; Wang, Hualei; Alias, Siti Aisyah; Gao, Bei; Wei, Dongzhi

2017-02-28

α-Amylase plays a pivotal role in a broad range of industrial processes. To meet increasing demands of biocatalytic tasks, considerable efforts have been made to isolate enzymes produced by extremophiles. However, the relevant data of α-amylases from cold-adapted fungi are still insufficient. In addition, bread quality presents a particular interest due to its high consummation. Thus developing amylases to improve textural properties could combine health benefits with good sensory properties. Furthermore, iron oxide nanoparticles provide an economical and convenient method for separation of biomacromolecules. In order to maximize the catalytic efficiency of α-amylase and support further applications, a comprehensive characterization of magnetic immobilization of α-amylase is crucial and needed. A novel α-amylase (AmyA1) containing an open reading frame of 1482 bp was cloned from Antarctic psychrotolerant fungus G. pannorum and then expressed in the newly constructed Aspergillus oryzae system. The purified recombinant AmyA1 was approximate 52 kDa. AmyA1 was optimally active at pH 5.0 and 40 °C, and retained over 20% of maximal activity at 0-20 °C. The K m and V max values toward soluble starch were 2.51 mg/mL and 8.24 × 10 -2 mg/(mL min) respectively, with specific activity of 12.8 × 10 3 U/mg. AmyA1 presented broad substrate specificity, and the main hydrolysis products were glucose, maltose, and maltotetraose. The influence of AmyA1 on the quality of bread was further investigated. The application study shows a 26% increase in specific volume, 14.5% increase in cohesiveness and 14.1% decrease in gumminess in comparison with the control. AmyA1 was immobilized on magnetic nanoparticles and characterized. The immobilized enzyme showed improved thermostability and enhanced pH tolerance under neutral conditions. Also, magnetically immobilized AmyA1 can be easily recovered and reused for maximum utilization. A novel α-amylase (AmyA1) from Antarctic psychrotolerant fungus was cloned, heterologous expression in Aspergillus oryzae, and characterized. The detailed report of the enzymatic properties of AmyA1 gives new insights into fungal cold-adapted amylase. Application study showed potential value of AmyA1 in the food and starch fields. In addition, AmyA1 was immobilized on magnetic nanoparticles and characterized. The improved stability and longer service life of AmyA1 could potentially benefit industrial applications.

Synthesis and biological evaluation of indole derivatives as α-amylase inhibitor.

PubMed

Imran, Syahrul; Taha, Muhammad; Selvaraj, Manikandan; Ismail, Nor Hadiani; Chigurupati, Sridevi; Mohammad, Jahidul Islam

2017-08-01

A series of twenty indole hydrazone analogs (1-21) were synthesized, characterized by different spectroscopic techniques such as 1 H NMR and EI-MS, and screened for α-amylase inhibitory activity. All analogs showed a variable degree of α-amylase inhibition with IC 50 values ranging between 1.66 and 2.65μM. Nine compounds that are 1 (2.23±0.01μM), 8 (2.44±0.12μM), 10 (1.92±0.12μM), 12 (2.49±0.17μM), 13 (1.66±0.09μM), 17 (2.25±0.1μM), 18 (1.87±0.25μM), 20 (1.83±0.63μM), and 19 (1.97±0.02μM) showed potent α-amylase inhibition when compared with the standard acarbose (1.05±0.29μM). Other analogs showed good to moderate α-amylase inhibition. The structure activity relationship is mainly focusing on difference of substituents on phenyl part. Molecular docking studies were carried out to understand the binding interaction of the most active compounds. Copyright © 2017 Elsevier Inc. All rights reserved.

Cell surface engineering of Bacillus subtilis improves production yields of heterologously expressed α-amylases.

PubMed

Cao, Haojie; van Heel, Auke J; Ahmed, Hifza; Mols, Maarten; Kuipers, Oscar P

2017-04-04

Bacillus subtilis is widely used as a cell factory for numerous heterologous proteins of commercial value and medical interest. To explore the possibility of further enhancing the secretion potential of this model bacterium, a library of engineered strains with modified cell surface components was constructed, and the corresponding influences on protein secretion were investigated by analyzing the secretion of α-amylase variants with either low-, neutral- or high- isoelectric points (pI). Relative to the wild-type strain, the presence of overall anionic membrane phospholipids (phosphatidylglycerol and cardiolipin) increased dramatically in the PssA-, ClsA- and double KO mutants, which resulted in an up to 47% higher secretion of α-amylase. Additionally, we demonstrated that the appropriate net charge of secreted targets (AmyTS-23, AmyBs and AmyBm) was beneficial for secretion efficiency as well. In B. subtilis, the characteristics of cell membrane phospholipid bilayer and the pIs of heterologous α-amylases appear to be important for their secretion efficiency. These two factors can be engineered to reduce the electrostatic interaction between each other during the secretion process, which finally leads to a better secretion yield of α-amylases.

Potent α-glucosidase and α-amylase inhibitory activities of standardized 50% ethanolic extracts and sinensetin from Orthosiphon stamineus Benth as anti-diabetic mechanism

PubMed Central

2012-01-01

Background In the present study, we tested a 50% ethanolic extract of Orthosiphon stamineus plants and its isolated bioactive compound with respect to their α-glucosidase and α-amylase inhibitory activities. Methods Bioactive flavonoid sinensetin was isolated from 50% ethanolic extract of Orthosiphon stamineus. The structure of this pure compound was determined on the NMR data and the α-glucosidase and α-amylase inhibitory activities of isolated sinensetin and 50% ethanolic extract of Orthosiphon stamineus were evaluated. Results In vitro studies of a 50% ethanolic extract of O. stamineus and the isolated sinensetin compound showed inhibitory activity on α-glucosidase (IC50: 4.63 and 0.66 mg/ml, respectively) and α-amylase (IC50: 36.70 mg/ml and 1.13 mg/ml, respectively). Inhibition of these enzymes provides a strong biochemical basis for the management of type 2 diabetes via the control of glucose absorption. Conclusion Alpha-glucosidase and α-amylase inhibition could the mechanisms through which the 50% ethanolic extract of O. stamineus and sinensetin exert their antidiabetic activity, indicating that it could have potential use in the management of non-insulin-dependent diabetes. PMID:23039079

Permissive role of the acidification caused by wheat aleurone layers upon. alpha. -amylase induction by GA sub 3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodriguez-Campos, E.; Bernal-Lugo, I.; Hamabata, A.

1989-04-01

Wheat aleurone has the capacity of acidifying the incubation medium in 1 to 2 pH units. The {alpha}-amylase induction by GA{sub 3} in isolated wheat aleurone layers is strongly dependent on acidic pH of the medium (pH < 5). To examine possible mechanisms {sup 35}-Met incorporation into proteins and {alpha}-amylase, in the presence of GA{sub 3} and Ca{sup 2+} at pH, 4, 5 and 6 was studied. Although {sup 35}-Met uptake decreased markedly ({approx} 90%) at pH 4 in thepresence of GA{sub 3}, incorporation into total protein did not change significantly from other conditions. Auto-radiography of SDS-PAGE showed that mostmore » of the amino acid was in the {alpha}-amylase band, meaning that the effect of acidic pH is specific for GA{sub 3} actions on aleurone tissue. On the other hand, an increase of protonated GA{sub 3} diffusion could be ruled out. Also, there was not {alpha}-amylase inactivation at pH 6. These findings point out to the important physiological role of the acidification caused by the aleurone.« less

Development of a workflow for screening and identification of α-amylase inhibitory peptides from food source using an integrated Bioinformatics-phage display approach: Case study - Cumin seed.

PubMed

Siow, Hwee-Leng; Lim, Theam Soon; Gan, Chee-Yuen

2017-01-01

The main objective of this study was to develop an efficient workflow to discover α-amylase inhibitory peptides from cumin seed. A total of 56 unknown peptides was initially found in the cumin seed protein hydrolysate. They were subjected to 2 different in silico screenings and 6 peptides were shortlisted. The peptides were then subjected to in vitro selection using phage display technique and 3 clones (CSP3, CSP4 and CSP6) showed high affinity in binding α-amylase. These clones were subjected to the inhibitory test and only CSP4 and CSP6 exhibited high inhibitory activity. Therefore, these peptides were chemically synthesized for validation purposes. CSP4 exhibited inhibition of bacterial and human salivary α-amylases with IC50 values of 0.11 and 0.04μmol, respectively, whereas CSP6 was about 0.10 and 0.15μmol, respectively. Results showed that the strength of each protocol has been successfully combined as deemed fit to enhance the α-amylase inhibitor peptide discovery. Copyright © 2016 Elsevier Ltd. All rights reserved.

Functional characterization and crystal structure of thermostable amylase from Thermotoga petrophila, reveals high thermostability and an unusual form of dimerization.

PubMed

Hameed, Uzma; Price, Ian; Ikram-Ul-Haq; Ke, Ailong; Wilson, David B; Mirza, Osman

2017-10-01

Thermostable α-amylases have many industrial applications and are therefore continuously explored from novel sources. We present the characterization of a novel putative α-amylase gene product (Tp-AmyS) cloned from Thermotoga petrophila. The purified recombinant enzyme is highly thermostable and able to hydrolyze starch into dextrin between 90 and 100°C, with optimum activity at 98°C and pH8.5. The activity increased in the presence of Rb 1+ , K 1+ and Ca 2+ ions, whereas other ions inhibited activity. The crystal structure of Tp-AmyS at 1.7Å resolution showed common features of the GH-13 family, however was apparently found to be a dimer. Several residues from one monomer interacted with a docked acarbose, an inhibitor of Tp-AmyS, in the other monomer, suggesting catalytic cooperativity within the dimer. The most striking feature of the dimer was that it resembled the dimerization of salivary amylase from a previous crystal structure, and thus could be a functional feature of some amylases. Copyright © 2017 Elsevier B.V. All rights reserved.

The green tea polyphenol (-)-epigallocatechin gallate precipitates salivary proteins including alpha-amylase: biochemical implications for oral health.

PubMed

Hara, Kumiko; Ohara, Masaru; Hayashi, Ikue; Hino, Takamune; Nishimura, Rumi; Iwasaki, Yoriko; Ogawa, Tetsuji; Ohyama, Yoshihiko; Sugiyama, Masaru; Amano, Hideaki

2012-04-01

Green tea is a popular drink throughout the world, and it contains various components, including the green tea polyphenol (-)-epigallocatechin gallate (EGCG). Tea interacts with saliva upon entering the mouth, so the interaction between saliva and EGCG interested us, especially with respect to EGCG-protein binding. SDS-PAGE revealed that several salivary proteins were precipitated after adding EGCG to saliva. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting indicated that the major proteins precipitated by EGCG were alpha-amylase, S100, and cystatins. Surface plasmon resonance revealed that EGCG bound to alpha-amylase at dissociation constant (K(d)) = 2.74 × 10(-6) M, suggesting that EGCG interacts with salivary proteins with a relatively strong affinity. In addition, EGCG inhibited the activity of alpha-amylase by non-competitive inhibition, indicating that EGCG is effective at inhibiting the formation of fermentable carbohydrates involved in caries formation. Interestingly, alpha-amylase reduced the antimicrobial activity of EGCG against the periodontal bacterium Aggregatibacter actinomycetemcomitans. Therefore, we considered that EGCG-salivary protein interactions might have both protective and detrimental effects with respect to oral health. © 2012 Eur J Oral Sci.

Effect of carbon and nitrogen sources on simultaneous production of α-amylase and green food packaging polymer by Bacillus sp. CFR 67.

PubMed

Sreekanth, M S; Vijayendra, S V N; Joshi, G J; Shamala, T R

2013-04-01

In this paper, effect of different carbon and nitrogen sources, including hydrolysates of rice bran and wheat bran, on simultaneous production of α-amylase (for hydrolysis of starch in food systems) and polyhydroxyalkanoates (PHA, a green biopolymer, which can be used as a packing material for foods) by Bacillus sp. CFR 67 was studied by submerged fermentation. Amongst various carbon sources tested, glucose and sucrose supported production of significantly (P < 0.05) higher amount of α-amylase (66 U/ml) and PHA (444 mg/l), respectively. Of the nitrogen sources tested, ammonium acetate and beef extract led to the production of maximum amount of amylase (36 U/ml) and PHA (592 mg/l), respectively. Supplementation of the production medium with wheat bran hydrolysate (50 ml/l) produced significantly higher amounts of amylase (73 U/ml) and PHA (524 mg/l). Thus this study indicated the potential of agro-residues for the production of value added biomolecules, which can reduce the cost of production of these molecules and enables to reduce the pollution mainly caused by the use of non biodegradable plastics.

Comparison of Salivary Cortisol and α-amylase Levels and Psychological Profiles in Patients with Burning Mouth Syndrome.

PubMed

Nosratzehi, Tahereh; Salimi, Saeedeh; Parvaee, Azadeh

2017-05-01

The aim of the study was to compare salivary cortisol and α-amylase levels and psychological profiles in patients with burning mouth syndrome (BMS) and healthy subjects. Spitting method was used to collect unstimulated salivary. Salivary cortisol and α-amylase levels were determined using ELISA and the Caraway starch-iodine technique. SLC-90 questionnaire was used to determine the psychological profiles. The difference between mean of salivary cortisol levels in patients with BMS and in healthy subjects was not significant (4.50 ± 3.68 ng/mL vs. 3.69 ± 3.07 ng/mL, p = 0.377). The difference between mean of α-amylase levels in the BMS patients and healthy subjects was significant (351.68 ± 142.5 and 146.22 ± 130.4, p = 0.001). Note that, 83.4% and 18% of the patients in the case and control groups had psychological problems (p = 0.001). The results showed higher salivary levels of cortisol and α-amylase in patients with BMS compared to healthy individuals. Psychological problem was prevalent in patients. © 2017 Special Care Dentistry Association and Wiley Periodicals, Inc.

Persimmon-Tannin, an α-Amylase Inhibitor, Retards Carbohydrate Absorption in Rats.

PubMed

Tsujita, Takahiro

2016-01-01

Inhibitors of carbohydrate-hydrolyzing enzymes play an important role in controlling postprandial blood glucose levels. Thus the effect of persimmon tannin on pancreatic α-amylase and intestinal α-glucosidase has been investigated. Persimmon tannin inhibits pancreatic α-amylase and intestinal α-glucosidase in a concentration-dependent manner with the 50% inhibition concentration (IC50) for amylase, maltase and sucrase being 1.7 μg/mL, 632 μg/mL and 308 μg/mL, respectively. The effect of persimmon-tannin extract on carbohydrate absorption in rats has also been investigated. Oral administration of persimmon tannin to normal rats fed cornstarch (2 g/kg body weight) significantly suppressed the increase in blood glucose levels and the area under the curve (AUC) after starch loading in a dose-dependent manner. The effective dose of persimmon tannin required to achieve 50% suppression of the rise in blood glucose level was estimated to be 300 mg/kg body weight. Administration of persimmon tannin to rats fed maltose or sucrose delayed the increase of blood glucose level and slightly suppressed AUC, but not significantly. These results suggest that persimmon tannin retards absorption of carbohydrate and reduces post-prandial hyperglycemia mainly through inhibition of α-amylase.

Salivary biomarkers of neural hypervigilance in trauma-exposed women.

PubMed

Yoon, Seungyeon A; Weierich, Mariann R

2016-01-01

More than half of all adults will be exposed to a traumatic event at some point in their lives, yet we do not yet have reliable biomarkers to help predict who experiences trauma-related symptoms in response to exposure. We tested the utility of salivary cortisol and salivary alpha amylase as markers of (1) neural reactivity to negative affective information and (2) neural hypervigilance in the absence of threat. 20 women (mean age 23.6 +/- 5.8 years) with a history of trauma exposure. Salivary cortisol and alpha amylase reactivity were measured in response to a trauma reminder during a clinical interview. Neural reactivity to novel and familiar affective scenes was measured in a later session using functional magnetic resonance imaging. Salivary alpha amylase, but not cortisol, increased in response to the trauma reminder. Salivary alpha amylase reactivity was associated with neural reactivity in the salience network in response to novel negative scenes and neural hypervigilance as indexed by reactivity to novel neutral scenes. Salivary alpha amylase might serve as a more reliable marker of trauma-related reactivity to negative affective information, and also as a marker of hypervigilance in the absence of threatening information. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bioprocess exploration for thermostable α-amylase production of a deep-sea thermophile Geobacillus sp. in high-temperature bioreactor.

PubMed

Jiang, Tao; Huang, Mengmeng; He, Hao; Lu, Jian; Zhou, Xiangshan; Cai, Menghao; Zhang, Yuanxing

2016-08-17

Geobacillus sp. 4j, a deep-sea high-salt thermophile, was found to produce thermostable α-amylase. In this work, culture medium and conditions were first optimized to enhance the production of thermostable α-amylase by statistical methodologies. The resulting extracellular production was increased by five times and reached 6.40 U/ml. Then, a high-temperature batch culture of the thermophile in a 15 l in-house-designed bioreactor was studied. The results showed that a relatively high dissolved oxygen (600 rpm and 15 l/min) and culture temperature of 60°C facilitated both cell growth and α-amylase production. Thus, an efficient fermentation process was established with initial medium of pH 6.0, culture temperature of 60°C, and dissolved oxygen above 20%. It gave an α-amylase production of 79 U/ml and productivity of 19804 U/l·hr, which were 10.8 and 208 times higher than those in shake flask, respectively. This work is useful for deep-sea high-salt thermophile culture, where efforts are lacking presently.

Human α-amylase present in lower-genital-tract mucosal fluid processes glycogen to support vaginal colonization by Lactobacillus.

PubMed

Spear, Gregory T; French, Audrey L; Gilbert, Douglas; Zariffard, M Reza; Mirmonsef, Paria; Sullivan, Thomas H; Spear, William W; Landay, Alan; Micci, Sandra; Lee, Byung-Hoo; Hamaker, Bruce R

2014-10-01

Lactobacillus colonization of the lower female genital tract provides protection from the acquisition of sexually transmitted diseases, including human immunodeficiency virus, and from adverse pregnancy outcomes. While glycogen in vaginal epithelium is thought to support Lactobacillus colonization in vivo, many Lactobacillus isolates cannot utilize glycogen in vitro. This study investigated how glycogen could be utilized by vaginal lactobacilli in the genital tract. Several Lactobacillus isolates were confirmed to not grow in glycogen, but did grow in glycogen-breakdown products, including maltose, maltotriose, maltopentaose, maltodextrins, and glycogen treated with salivary α-amylase. A temperature-dependent glycogen-degrading activity was detected in genital fluids that correlated with levels of α-amylase. Treatment of glycogen with genital fluids resulted in production of maltose, maltotriose, and maltotetraose, the major products of α-amylase digestion. These studies show that human α-amylase is present in the female lower genital tract and elucidates how epithelial glycogen can support Lactobacillus colonization in the genital tract. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Potent α-glucosidase and α-amylase inhibitory activities of standardized 50% ethanolic extracts and sinensetin from Orthosiphon stamineus Benth as anti-diabetic mechanism.

PubMed

Mohamed, Elsnoussi Ali Hussin; Siddiqui, Mohammad Jamshed Ahmad; Ang, Lee Fung; Sadikun, Amirin; Chan, Sue Hay; Tan, Soo Choon; Asmawi, Mohd Zaini; Yam, Mun Fei

2012-10-08

In the present study, we tested a 50% ethanolic extract of Orthosiphon stamineus plants and its isolated bioactive compound with respect to their α-glucosidase and α-amylase inhibitory activities. Bioactive flavonoid sinensetin was isolated from 50% ethanolic extract of Orthosiphon stamineus. The structure of this pure compound was determined on the NMR data and the α-glucosidase and α-amylase inhibitory activities of isolated sinensetin and 50% ethanolic extract of Orthosiphon stamineus were evaluated. In vitro studies of a 50% ethanolic extract of O. stamineus and the isolated sinensetin compound showed inhibitory activity on α-glucosidase (IC50: 4.63 and 0.66 mg/ml, respectively) and α-amylase (IC50: 36.70 mg/ml and 1.13 mg/ml, respectively). Inhibition of these enzymes provides a strong biochemical basis for the management of type 2 diabetes via the control of glucose absorption. Alpha-glucosidase and α-amylase inhibition could the mechanisms through which the 50% ethanolic extract of O. stamineus and sinensetin exert their antidiabetic activity, indicating that it could have potential use in the management of non-insulin-dependent diabetes.

Amylase production by Preussia minima, a fungus of endophytic origin: optimization of fermentation conditions and analysis of fungal secretome by LC-MS

PubMed Central

2014-01-01

Background Environmental screening programs are used to find new enzymes that may be utilized in large-scale industrial processes. Among microbial sources of new enzymes, the rationale for screening fungal endophytes as a potential source of such enzymes relates to the hypothesised mutualistic relationship between the endophyte and its host plant. There is a need for new microbial amylases that are active at low temperature and alkaline conditions as these would find industrial applications as detergents. Results An α-amylase produced by Preussia minima, isolated from the Australian native plant, Eremophilia longifolia, was purified to homogeneity through fractional acetone precipitation and Sephadex G-200 gel filtration, followed by DEAE-Sepharose ion exchange chromatography. The purified α-amylase showed a molecular mass of 70 kDa which was confirmed by zymography. Temperature and pH optima were 25°C and pH 9, respectively. The enzyme was activated and stabilized mainly by the metal ions manganese and calcium. Enzyme activity was also studied using different carbon and nitrogen sources. It was observed that enzyme activity was highest (138 U/mg) with starch as the carbon source and L-asparagine as the nitrogen source. Bioreactor studies showed that enzyme activity was comparable to that obtained in shaker cultures, which encourages scale-up fermentation for enzyme production. Following in-gel digestion of the purified protein by trypsin, a 9-mer peptide was sequenced and analysed by LC-ESI-MS/MS. The partial amino acid sequence of the purified enzyme presented similarity to α-amylase from Magnaporthe oryzae. Conclusions The findings of the present study indicate that the purified α-amylase exhibits a number of promising properties that make it a strong candidate for application in the detergent industry. To our knowledge, this is the first amylase isolated from a Preussia minima strain of endophytic origin. PMID:24602289

Amylase production by Preussia minima, a fungus of endophytic origin: optimization of fermentation conditions and analysis of fungal secretome by LC-MS.

PubMed

Zaferanloo, Bita; Bhattacharjee, Shatabdi; Ghorbani, Mahmood M; Mahon, Peter J; Palombo, Enzo A

2014-03-07

Environmental screening programs are used to find new enzymes that may be utilized in large-scale industrial processes. Among microbial sources of new enzymes, the rationale for screening fungal endophytes as a potential source of such enzymes relates to the hypothesised mutualistic relationship between the endophyte and its host plant. There is a need for new microbial amylases that are active at low temperature and alkaline conditions as these would find industrial applications as detergents. An α-amylase produced by Preussia minima, isolated from the Australian native plant, Eremophilia longifolia, was purified to homogeneity through fractional acetone precipitation and Sephadex G-200 gel filtration, followed by DEAE-Sepharose ion exchange chromatography. The purified α-amylase showed a molecular mass of 70 kDa which was confirmed by zymography. Temperature and pH optima were 25°C and pH 9, respectively. The enzyme was activated and stabilized mainly by the metal ions manganese and calcium. Enzyme activity was also studied using different carbon and nitrogen sources. It was observed that enzyme activity was highest (138 U/mg) with starch as the carbon source and L-asparagine as the nitrogen source. Bioreactor studies showed that enzyme activity was comparable to that obtained in shaker cultures, which encourages scale-up fermentation for enzyme production. Following in-gel digestion of the purified protein by trypsin, a 9-mer peptide was sequenced and analysed by LC-ESI-MS/MS. The partial amino acid sequence of the purified enzyme presented similarity to α-amylase from Magnaporthe oryzae. The findings of the present study indicate that the purified α-amylase exhibits a number of promising properties that make it a strong candidate for application in the detergent industry. To our knowledge, this is the first amylase isolated from a Preussia minima strain of endophytic origin.

Oral Digestion and Perception of Starch: Effects of Cooking, Tasting Time, and Salivary α-Amylase Activity.

PubMed

Lapis, Trina J; Penner, Michael H; Balto, Amy S; Lim, Juyun

2017-10-01

Since starch is a significant part of human diet, its oral detection would be highly beneficial. This study was designed to determine whether starch or its degradation products can be tasted and what factors influence its perception. Subjects were asked 1) to taste 8% raw and cooked starch samples for 5, 15, and 35 s and rate perceived intensities of sweetness and "other" taste (i.e., other than sweet), 2) to donate saliva to obtain salivary flow rate (mg/s) and salivary α-amylase activity (per mg saliva), and 3) to fill out a carbohydrate consumption survey. Subsequently, in vitro hydrolysis of starch was performed; saliva was collected from 5 subjects with low and high amylase activities and reacted with 8% raw and cooked starch at 2, 15, and 30 s. Hydrolysis products were then quantified using a High performance liquid chromatography. The results showed cooking increased the digestibility of starch such that the amount of hydrolysis products increased with reaction time. However, cooking did not influence taste ratings, nor were they influenced by tasting time. Subjects' salivary amylase activities were associated with the efficacy of their saliva to degrade starch, in particular cooked starch, and thus the amount of maltooligosaccharide products generated. Effective α-amylase activity [i.e. α-amylase activity (per mg saliva) × salivary flow rate (mg/s)] and carbohydrate consumption score (i.e. consumption frequency × number of servings) were also independently associated with sensory taste ratings. Human perception of starch is undoubtedly complex as shown in this study; the data herein point to the potential roles of salivary α-amylase activity and carbohydrate consumption in the perception of cooked starch. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Screening the thermophilic and hyperthermophilic bacterial population of three Iranian hot-springs to detect the thermostable α-amylase producing strain

PubMed Central

Fooladi, J; Sajjadian, A

2010-01-01

Background Screening is a routine procedure for isolation of microorganisms which are able to produce special metabolites. Purified thermostable α-amylase from bacterial sources is widely used in different industries. In this study we analyzed samples collected from three different hot springs in Iran to detect any strains capable of producing thermostable α-amylase. Materials and Methods Hot water samples from Larijan (67°C, pH 6.5), Mahallat (46°C, pH 7), and Meshkinshahr (82°C, pH 6), were cultivated in screening starch agar plates and incubated at 65°C for 24 hours. Thereafter, the plates were stained with Gram's iodine solution. Results and Discussion The bacterial colonies from the Meshkinshahr hot-spring produced the largest haloforming zone. Based on the phenotypic tests, the strain was identified as Bacillus sp. The culture condition was optimized for biosynthesis of α-amylase. The enzyme was produced at maximum level when it was incubated at 70°C in the presence of soluble starch (1%) at pH 6. The addition of calcium (10 mM) and peptone (1%) to the mineral medium, shortened the lag period and improved the growth and α-amylase synthesis. The addition of glucose (1%) to the culture greatly diminished the syntheses of α -amylase. Importantly, the enzyme extract retained 100% activity when incubated for 45 minutes at 100°C. Conclusion The Meshkinshahr hot-spring is rich in the Bacillus spp thermostable α-amylase producing strain of the thermophilic bacterial population. Iranian hot-springs like Meshkinshahr, have large microbial storages and can be used as sources of different biological products like enzymes. The enzyme which was produced with Bacillus sp. could hydrolyse polymers like starch and was used at laboratory scale successfully. PMID:22347550

Polyphenolic Contents and Antioxidant Activities of Underutilized Grape (Vitis vinifera L.) Pomace Extracts.

PubMed

Kabir, Faisal; Sultana, Mosammad Shahin; Kurnianta, Heri

2015-09-01

Grape pomace is an abundant source of underutilized winery by-products. Polyphenols were extracted from grape pomace using cellulase and gluco-amylase enzymes. 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Folin-Ciocalteu's assays were used to measure antioxidant activity and total polyphenolic contents. Both cellulase, and gluco-amylase digested grape pomace showed efficient radical scavenging activity. In addition, the total polyphenolic contents of cellulase digested grape pomace showed lower concentrations were effective compared to higher concentrations, whereas gluco-amylase enzyme did not show remarkable variations. The DPPH radical scavenging activity and total polyphenolic contents were significantly higher in the cellulase digested grape pomace compared to the gluco-amylase digested and the not digested grape pomace. It is notable that enzymatic digestions were efficient for extracting polyphenols from grape pomace. The underutilized grape pomace polyphenols can be further used for food safety as a natural antioxidant.

Selective sweep on human amylase genes postdates the split with Neanderthals

PubMed Central

Inchley, Charlotte E.; Larbey, Cynthia D. A.; Shwan, Nzar A. A.; Pagani, Luca; Saag, Lauri; Antão, Tiago; Jacobs, Guy; Hudjashov, Georgi; Metspalu, Ene; Mitt, Mario; Eichstaedt, Christina A.; Malyarchuk, Boris; Derenko, Miroslava; Wee, Joseph; Abdullah, Syafiq; Ricaut, François-Xavier; Mormina, Maru; Mägi, Reedik; Villems, Richard; Metspalu, Mait; Jones, Martin K.; Armour, John A. L.; Kivisild, Toomas

2016-01-01

Humans have more copies of amylase genes than other primates. It is still poorly understood, however, when the copy number expansion occurred and whether its spread was enhanced by selection. Here we assess amylase copy numbers in a global sample of 480 high coverage genomes and find that regions flanking the amylase locus show notable depression of genetic diversity both in African and non-African populations. Analysis of genetic variation in these regions supports the model of an early selective sweep in the human lineage after the split of humans from Neanderthals which led to the fixation of multiple copies of AMY1 in place of a single copy. We find evidence of multiple secondary losses of copy number with the highest frequency (52%) of a deletion of AMY2A and associated low copy number of AMY1 in Northeast Siberian populations whose diet has been low in starch content. PMID:27853181

Selective sweep on human amylase genes postdates the split with Neanderthals.

PubMed

Inchley, Charlotte E; Larbey, Cynthia D A; Shwan, Nzar A A; Pagani, Luca; Saag, Lauri; Antão, Tiago; Jacobs, Guy; Hudjashov, Georgi; Metspalu, Ene; Mitt, Mario; Eichstaedt, Christina A; Malyarchuk, Boris; Derenko, Miroslava; Wee, Joseph; Abdullah, Syafiq; Ricaut, François-Xavier; Mormina, Maru; Mägi, Reedik; Villems, Richard; Metspalu, Mait; Jones, Martin K; Armour, John A L; Kivisild, Toomas

2016-11-17

Humans have more copies of amylase genes than other primates. It is still poorly understood, however, when the copy number expansion occurred and whether its spread was enhanced by selection. Here we assess amylase copy numbers in a global sample of 480 high coverage genomes and find that regions flanking the amylase locus show notable depression of genetic diversity both in African and non-African populations. Analysis of genetic variation in these regions supports the model of an early selective sweep in the human lineage after the split of humans from Neanderthals which led to the fixation of multiple copies of AMY1 in place of a single copy. We find evidence of multiple secondary losses of copy number with the highest frequency (52%) of a deletion of AMY2A and associated low copy number of AMY1 in Northeast Siberian populations whose diet has been low in starch content.

Inhibition of pancreatic lipase and amylase by extracts of different spices and plants.

PubMed

Sellami, Mohamed; Louati, Hanen; Kamoun, Jannet; Kchaou, Ali; Damak, Mohamed; Gargouri, Youssef

2017-05-01

The aim of this study is to search new anti-obesity and anti-diabetic agents from plant and spices crude extracts as alternative to synthetic drugs. The inhibitory effect of 72 extracts was evaluated, in vitro, on lipase and amylase activities. Aqueous extracts of cinnamon and black tea exhibited an appreciable inhibitory effect on pancreatic amylase with IC 50 values of 18 and 87 μg, respectively. Aqueous extracts of cinnamon and mint showed strong inhibitory effects against pancreatic lipase with IC 50 of 45 and 62 μg, respectively. The presence of bile salts and colipase or an excess of interface failed to restore the lipase activity. Therefore, the inhibition of pancreatic lipase, by extracts of spices and plants, belongs to an irreversible inhibition. Crude extract of cinnamon showed the strongest anti-lipase and anti-amylase activities which offer a prospective therapeutic approach for the management of diabetes and obesity.

An energy-saving glutathione production method from low-temperature cooked rice using amylase-expressing Saccharomyces cerevisiae.

PubMed

Hara, Kiyotaka Y; Kim, Songhee; Kiriyama, Kentaro; Yoshida, Hideyo; Arai, Shogo; Ishii, Jun; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

2012-05-01

Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is industrially produced by fermentation using Saccharomyces cerevisiae. Before the glutathione fermentation process with S. cerevisiae, a glucose extraction process from starchy materials is required. This glucose extraction is usually carried out by converting starchy materials to starch using high-temperature cooking and subsequent hydrolysis by amylases to convert starch to glucose. In this study, to develop an energy-saving glutathione production process by reducing energy consumption during the cooking step, we efficiently produced glutathione from low-temperature cooked rice using amylase-expressing S. cerevisiae. The combination of the amylase-expressing yeast with low-temperature cooking is potentially applicable to a variety of energy-saving bio-production methods of chemicals from starchy bio-resources. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Smart phone: a popular device supports amylase activity assay in fisheries research.

PubMed

Thongprajukaew, Karun; Choodum, Aree; Sa-E, Barunee; Hayee, Ummah

2014-11-15

Colourimetric determinations of amylase activity were developed based on a standard dinitrosalicylic acid (DNS) staining method, using maltose as the analyte. Intensities and absorbances of red, green and blue (RGB) were obtained with iPhone imaging and Adobe Photoshop image analysis. Correlation of green and analyte concentrations was highly significant, and the accuracy of the developed method was excellent in analytical performance. The common iPhone has sufficient imaging ability for accurate quantification of maltose concentrations. Detection limits, sensitivity and linearity were comparable to a spectrophotometric method, but provided better inter-day precision. In quantifying amylase specific activity from a commercial source (P>0.02) and fish samples (P>0.05), differences compared with spectrophotometric measurements were not significant. We have demonstrated that iPhone imaging with image analysis in Adobe Photoshop has potential for field and laboratory studies of amylase. Copyright © 2014 Elsevier Ltd. All rights reserved.

Potent human α-amylase inhibition by the β-defensin-like protein helianthamide

DOE PAGES

Tysoe, Christina; Williams, Leslie K.; Keyzers, Robert; ...

2016-02-26

Here, selective inhibitors of human pancreatic α-amylase (HPA) are an effective means of controlling blood sugar levels in the management of diabetes. A high-throughput screen of marine natural product extracts led to the identification of a potent (K i = 10 pM) peptidic HPA inhibitor, helianthamide, from the Caribbean sea anemone Stichodactyla helianthus. Active helianthamide was produced in Escherichia coli via secretion as a barnase fusion protein. X-ray crystallographic analysis of the complex of helianthamide with porcine pancreatic α-amylase revealed that helianthamide adopts a β-defensin fold and binds into and across the amylase active site, utilizing a contiguous YIYH inhibitorymore » motif. Helianthamide represents the first of a novel class of glycosidase inhibitors and provides an unusual example of functional malleability of the β-defensin fold, which is rarely seen outside of its traditional role in antimicrobial peptides.« less

Polyphenolic Contents and Antioxidant Activities of Underutilized Grape (Vitis vinifera L.) Pomace Extracts

PubMed Central

Kabir, Faisal; Sultana, Mosammad Shahin; Kurnianta, Heri

2015-01-01

Grape pomace is an abundant source of underutilized winery by-products. Polyphenols were extracted from grape pomace using cellulase and gluco-amylase enzymes. 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Folin-Ciocalteu’s assays were used to measure antioxidant activity and total polyphenolic contents. Both cellulase, and gluco-amylase digested grape pomace showed efficient radical scavenging activity. In addition, the total polyphenolic contents of cellulase digested grape pomace showed lower concentrations were effective compared to higher concentrations, whereas gluco-amylase enzyme did not show remarkable variations. The DPPH radical scavenging activity and total polyphenolic contents were significantly higher in the cellulase digested grape pomace compared to the gluco-amylase digested and the not digested grape pomace. It is notable that enzymatic digestions were efficient for extracting polyphenols from grape pomace. The underutilized grape pomace polyphenols can be further used for food safety as a natural antioxidant. PMID:26451359

An In Vitro and In Vivo Study of the α-Amylase Activity of Phaseolamin

PubMed Central

de Gouveia, Neire Moura; Alves, Fernanda Vieira; Furtado, Fabiana Barcelos; Scherer, Danielli Luana; Mundim, Antonio Vicente

2014-01-01

Abstract We evaluated the polypeptide profiles, inhibition of human salivary α-amylase activity, and hemagglutination properties of a commercial phaseolamin sample. We also performed an in vivo assay to investigate the effects of a commercial phaseolamin treatment (100, 500, or 1500 mg/kg) over 20 days on the glycemia, body weight, and serum biochemical parameters (total cholesterol, triglycerides, alanine aminotransferase, and aspartate aminotransferase) of nondiabetic and streptozotocin-induced diabetic rats. The in vitro evaluation showed defined protein profiles, low hemagglutination activity, and high α-amylase inhibition. None of the experimental groups treated with phaseolamin or acarbose showed decreases in body weight. Our data demonstrate that phaseolamin inhibits amylase activity in vitro, reduces blood glucose levels, decreases or attenuates some of the renal and hepatic effects of diabetes in streptozotocin-induced rats, and could therefore have therapeutic potential in the treatment or prevention of the complications of diabetes. PMID:24650210

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watson, E.L.; Singh, J.C.; Jacobson, K.L.

Cholinergic-mediated amylase release in mouse parotid acini was augmented by forskolin; the potency but not the maximal response to carbachol was altered. Amylase released by carbachol plus forskolin was dependent on extracellular calcium and was mimicked by the calcium ionophore, A23187 plus forskolin. Forskolin was also shown to enhance carbachol-stimulated /sup 45/Ca/sup 2 +/ uptake into isolated acini. Hydroxylamine, nitroprusside, and 8-bromo-c-GMP each in combination with forskolin mimicked the effects of carbachol plus forskolin on amylase release. In the presence of carbachol (10/sup -8/M) forskolin did not augment c-AMP levels. However, in the presence of carbachol (5 x 10/sup -7/more » M) or hydroxylamine (50 ..mu..M) forskolin did significantly augment c-AMP accumulation. These results suggest that calcium and c-GMP may mediate the augmentation of cholinergic-mediated amylase release by effects on c-AMP metabolism. 21 references, 1 figure, 3 tables.« less

Bean α-amylase inhibitor 1 in transgenic peas (Pisum sativum) provides complete protection from pea weevil (Bruchus pisorum) under field conditions

PubMed Central

Morton, Roger L.; Schroeder, Hart E.; Bateman, Kaye S.; Chrispeels, Maarten J.; Armstrong, Eric; Higgins, Thomas J. V.

2000-01-01

Two α-amylase inhibitors, called αAI-1 and αAI-2, that share 78% amino acid sequence identity and have a differential specificity toward mammalian and insect α-amylases are present in different accessions of the common bean (Phaseolus vulgaris). Using greenhouse-grown transgenic peas (Pisum sativum), we have shown previously that expression of αAI-1 in pea seeds can provide complete protection against the pea weevil (Bruchus pisorum). Here, we report that αAI-1 also protects peas from the weevil under field conditions. The high degree of protection is explained by our finding that αAI-1 inhibits pea bruchid α-amylase by 80% over a broad pH range (pH 4.5–6.5). αAI-2, on the other hand, is a much less effective inhibitor of pea bruchid α-amylase, inhibiting the enzyme by only 40%, and only in the pH 4.0–4.5 range. Nevertheless, this inhibitor was still partially effective in protecting field-grown transgenic peas against pea weevils. The primary effect of αAI-2 appeared to be a delay in the maturation of the larvae. This contrasts with the effect of αAI-1, which results in larval mortality at the first or second instar. These results are discussed in relationship to the use of amylase inhibitors with different specificities to bring about protection of crops from their insect pests or to decrease insect pest populations below the economic injury level. PMID:10759552

Stilbene Glucoside, a Putative Sleep Promoting Constituent from Polygonum multiflorum Affects Sleep Homeostasis by Affecting the Activities of Lactate Dehydrogenase and Salivary Alpha Amylase.

PubMed

Wei, Qian; Ta, Guang; He, Wenjing; Wang, Wei; Wu, Qiucheng

2017-01-01

Chinese herbal medicine (CHM) has been used for treating insomnia for centuries. The most used CHM for insomnia was Polygonum multiflorum. However, the molecular mechanism for CHM preventing insomnia is unknown. Stilbene glucoside (THSG), an important active component of P. multiflorum, may play an important role for treating insomnia. To test the hypothesis, Kunming mice were treated with different dosages of THSG. To examine the sleep duration, a computer-controlled sleep-wake detection system was implemented. Electroencephalogram (EEG) and electromyogram (EMG) electrodes were implanted to determine sleep-wake state. RT-PCR and Western blot was used to measure the levels of lactate dehydrogenase (LDH) and saliva alpha amylase. Spearman's rank correlation coefficient was used to identify the strength of correlation between the variables. The results showed that THSG significantly prolonged the sleep time of the mice (p<0.01). THSG changed sleep profile by reducing wake and rapid eye movement (REM) period, and increasing non-REM period. RT-PCR and Western blot analysis showed that THSG could down-regulate the levels of LDH and saliva alpha amylase (p<0.05). The level of lactate and glucose was positively related with the activity of LDH and saliva alpha amylase (p<0.05), respectively. On the other hand, the activities of LDH and amylase were negatively associated with sleep duration (p<0.05). The levels of lactate and glucose affect sleep homeostasis. Thus, THSG may prevent insomnia by regulating sleep duration via LDH and salivary alpha amylase.

Expression and comparative characterization of complete and C-terminally truncated forms of saccharifying α-amylase from Lactobacillus plantarum S21.

PubMed

Kanpiengjai, Apinun; Nguyen, Thu-Ha; Haltrich, Dietmar; Khanongnuch, Chartchai

2017-10-01

Lactobacillus plantarum S21 α-amylase possesses 475 amino acids at the C-terminal region identified as the starch-binding domain (SBD) and has been previously reported to play a role in raw starch degradation. To understand the specific roles of this SBD, cloning and expression of the complete (AmyL9) and C-terminally truncated (AmyL9Δ SBD ) forms of α-amylase were conducted for enzyme purification and comparative characterization. AmyL9 and AmyL9Δ SBD were overproduced in Escherichia coli at approximately 10- and 20-times increased values of volumetric productivity when compared to α-amylase produced by the wild type, respectively. AmyL9Δ SBD was unable to hydrolyze raw starch and exhibited substrate specificity in a similar manner to that of AmyL9, but it was weakly active toward amylopectin and glycogen. The hydrolysis products obtained from the amylaceous substrates of both enzymes were the same. In addition, AmyL9Δ SBD showed comparatively higher K m values than AmyL9 when it reacted with starch and amylopectin, and lower values for other kinetic constants namely v max , k cat , and k cat /K m . The results indicated that the C-terminal SBDs of L. plantarum S21 α-amylase contribute to not only substrate preference but also substrate affinity and the catalytic efficiency of the α-amylase without any changes in the degradation mechanisms of the enzyme. Copyright © 2017 Elsevier B.V. All rights reserved.

Are salivary amylase and pH - Prognostic indicators of cancers?

PubMed

Ramya, Atmakuri Shanmukha; Uppala, Divya; Majumdar, Sumit; Surekha, Ch; Deepak, K G K

2015-01-01

Saliva, "Mirror of body's health" has long been of particular interest as a substitute for blood for disease diagnosis and monitoring. The radiation effects on salivary glands are of particular interest in which salivary amylase is a good indicator of salivary glands function. Thus, estimation of these parameters represents a reasonable approach in evaluation of patient's risk for disease occurrence, intensity and prognosis. To evaluate and compare the pH and amylase levels in saliva of cancer patients prior to treatment, patients during treatment. Saliva samples of 90 individuals were taken which were divided into 3 groups - 30 individuals without cancer, 30 cancer patients prior treatment and 30 cancer patients during treatment. Materials used were pH strips and pH meter, Salivary Amylase assay. Statistical analysis - ANOVA with post-hoc Tukey's test. 1) Significant decrease in salivary amylase levels - in cancer patients, during treatment when compared to others. 2) Significant decrease in salivary pH levels in newly diagnosed cancer patients prior to treatment. To conclude, pH strips and pH meter showed to be a useful tool in the measurement of pH of saliva in individuals with and without cancer. This study showed that cancer patients without treatment have a lower pH of saliva. Treatment increased the pH of the saliva to a more alkaline level whereas amylase levels decreased in those subjects. Therefore those parameters can be an area of further research with an increased sample size, which in-turn may help in opening the doors for new dimension in non invasive prognostic markers.

The effect of physical exercise on salivary secretion of MUC5B, amylase and lysozyme.

PubMed

Ligtenberg, Antoon J M; Brand, Henk S; van den Keijbus, Petra A M; Veerman, Enno C I

2015-11-01

Saliva secretion is regulated by the autonomic nervous system. Parasympathic stimuli increase the secretion of water and mucin MUC5B, whereas sympathetic stimuli such as physical exercise increase the secretion of amylase and other proteins. In the present study we investigated the effect of physical exercise, as a sympathetic stimulus, on salivary flow rate and output of MUC5B, amylase, lysozyme and total protein. Unstimulated whole saliva was collected before exercise (1), after 10 min exercise with moderate intensity by running with a heart rate around 130 beats per minute (2), followed by 10 min exercise with high intensity by running to exhaustion (3) and after 30 min recovery (4). Salivary flow rate, protein and MUC5B concentration, and amylase and lysozyme activity were determined. Saliva protein composition was analysed using SDS-PAGE and immunoblotting. Salivary flow rate, protein and lysozyme secretion increased after exercise with moderate intensity and increased further after exercise with high intensity (p<0.01). Amylase and MUC5B increased after exercise with moderate intensity (p<0.0001), but did not differ significantly between moderate and high exercise intensity. SDS-PAGE analysis and immunoblotting showed that, especially after exercise with high intensity, the concentrations of several other salivary proteins, including MUC7, albumin, and extra-parotid glycoprotein, also increased. Exercise may not only lead to the anticipated increase in amylase and protein secretion, but also to an increase in salivary flow rate and MUC5B secretion. Copyright © 2015 Elsevier Ltd. All rights reserved.

Soybean phenolic-rich extracts inhibit key-enzymes linked to type 2 diabetes (α-amylase and α-glucosidase) and hypertension (angiotensin I converting enzyme) in vitro.

PubMed

Ademiluyi, Adedayo O; Oboh, Ganiyu

2013-03-01

This study sought to assess the inhibitory activities of phenolic-rich extracts from soybean on α-amylase, α-glucosidase and angiotensin I converting enzyme (ACE) activities in vitro. The free phenolic extract of the soybean was obtained by extraction with 80% acetone, while that of the bound phenolic extract was done by extracting the alkaline and acid hydrolyzed residue with ethyl acetate. The inhibitory action of these extracts on the enzymes activity as well as their antioxidant properties was assessed. Both phenolic-rich extracts inhibited α-amylase, α-glucosidase and ACE enzyme activities in a dose dependent pattern. However, the bound phenolic extract exhibited significantly (P < 0.05) higher α-amylase and ACE inhibition while the free phenolic extract had significantly (P < 0.05) higher α-glucosidase inhibitory activity. Nevertheless, the free phenolic extract had higher α-glucosidase inhibitory activity when compared to that of α-amylase; this property confer an advantage on soybean phenolic-rich extracts over commercial antidiabetic drugs with little or no side effect. And inhibition of ACE suggests the antihypertension potential of soybean phenolic-rich extracts. Furthermore, the enzyme inhibitory activities of the phenolic-rich extracts were not associated with their phenolic content. Therefore, phenolic-rich extracts of soybean could inhibit key-enzyme linked to type 2 diabetes (α-amylase and α-glucosidase) and hypertension (ACE) and thus could explain in part the mechanism by which soybean renders these health promoting effect. Copyright © 2011 Elsevier GmbH. All rights reserved.

Salivary α-amylase and cortisol after exercise in menopause: influence of long-term HRT.

PubMed

Patacchioli, F R; Ghiciuc, C M; Bernardi, M; Dima-Cozma, L C; Fattorini, L; Squeo, M R; Galoppi, P; Brunelli, R; Ferrante, F; Pasquali, V; Perrone, G

2015-01-01

This observational prospective study analyzed the effect of an incremental cardiopulmonary exercise test (CPET) on the secretion of salivary biomarkers of the adrenergic nervous system and hypothalamus-pituitary-adrenal (HPA) axis activity by measuring salivary α-amylase and cortisol diurnal trajectories in the setting of long-term hormone replacement therapy (HRT). Fifteen healthy sedentary postmenopausal women who were current HRT users and 15 women who had never used HRT were consecutively recruited. α-Amylase and cortisol were measured in salivary samples collected on the CPET day and on a rest day. Cardiovascular and respiratory fitness parameters were recorded during the CPET challenge. The participants had very homogeneous somatic characteristics, and they were all in generally good health. The postmenopausal never-HRT users presented an abnormal diurnal pattern of α-amylase at baseline and a flattened response to CPET. In contrast, women on HRT had a physiological α-amylase diurnal pattern and increased salivary α-amylase production during the CPET-induced challenge. The CPET challenge physiologically activated the HPA axis activity, as shown by the increase in the concentration of salivary cortisol during the effort test. HPA axis activity was not affected by long-term HRT. Postmenopausal women using HRT exhibited a cardiorespiratory functional capacity that was significantly (p < 0.05) higher than that of non-users. Our findings show that healthy postmenopausal women present an asymmetry between adrenergic nervous system and HPA axis activities under both basal and stress conditions. HRT was able to modify the abnormal adrenergic nervous system activity, most likely by reducing the sympathetic hyperactivity that characterizes menopause.

Two Strategies for Microbial Production of an Industrial Enzyme-Alpha-Amylase

NASA Technical Reports Server (NTRS)

Bernhardsdotter, Eva C. M. J.; Garriott, Owen; Pusey, Marc L.; Ng, Joseph D.

2003-01-01

Extremophiles are microorganisms that thrive in, from an anthropocentric view, extreme environments including hot springs, soda lakes and arctic water. This ability of survival at extreme conditions has rendered extremophiles to be of interest in astrobiology, evolutionary biology as well as in industrial applications. Of particular interest to the biotechnology industry are the biological catalysts of the extremophiles, the extremozymes, whose unique stabilities at extreme conditions make them potential sources of novel enzymes in industrial applications. There are two major approaches to microbial enzyme production. This entails enzyme isolation directly from the natural host or creating a recombinant expression system whereby the targeted enzyme can be overexpressed in a mesophilic host. We are employing both methods in the effort to produce alpha-amylases from a hyperthermophilic archaeon (Thermococcus) isolated from a hydrothermal vent in the Atlantic Ocean, as well as from alkaliphilic bacteria (Bacillus) isolated from a soda lake in Tanzania. Alpha-amylases catalyze the hydrolysis of internal alpha-1,4-glycosidic linkages in starch to produce smaller sugars. Thermostable alpha-amylases are used in the liquefaction of starch for production of fructose and glucose syrups, whereas alpha-amylases stable at high pH have potential as detergent additives. The alpha-amylase encoding gene from Thermococcus was PCR amplified using carefully designed primers and analyzed using bioinformatics tools such as BLAST and Multiple Sequence Alignment for cloning and expression in E.coli. Four strains of Bacillus were grown in alkaline starch-enriched medium of which the culture supernatant was used as enzyme source. Amylolytic activity was detected using the starch-iodine method.

Effects of biomaterial-derived fibroblast conditioned medium on the α-amylase expression of parotid gland acinar cells.

PubMed

Chou, Ya-Shuan; Young, Tai-Horng; Lou, Pei-Jen

2015-11-01

Salivary gland cells are surrounded by a complex stromal environment, in which fibroblasts are the main cells in proximity to the gland cells. In this study, the interaction between parotid gland acinar cells (PGACs), fibroblasts, and biomaterials was investigated. We prepared different biomaterials, including chitosan, polyvinyl alcohol (PVA), poly (ethylene-co-vinyl alcohol) (EVAL), polyvinylidene fluoride (PVDF), and tissue culture polystyrene (TCPS) to culture fibroblasts and then collect their conditioned media to culture PGACs. We observed no difference in AQP3, AQP5, and E-cadherin expression among different fibroblast conditioned medium treatments. Interestingly, α-amylase expression was obviously enhanced in PGACs cultured in the presence of conditioned medium from fibroblasts cultured on PVDF. Higher neurotrophin-4 (NT-4) expression was observed in PVDF-derived fibroblast conditioned medium using a growth factor protein array assay. In addition, directly adding NT-4 into the culture medium significantly promoted α-amylase expression by PGACs. Finally, nestin and βIII-tubulin expression by fibroblasts cultured on PVDF was also enhanced. Together, these results suggest that PVDF could promote α-amylase expression by PGACs via the NT-4 produced by fibroblasts. To date, there is no effective therapy for patients with dry mouth with persistent salivary hypofunction. The study made use of different biomaterials to culture fibroblasts and then collect their conditioned media to culture PGACs. It was found that the effect of fibroblast conditioned medium from PVDF on the α-amylase expression of PGACs was obviously enhanced and higher neurotrophin-4 (NT-4) expression was found in PVDF-derived fibroblast conditioned medium. In addition, directly adding NT-4 into the culture medium significantly promoted the expression of α-amylase by PGACs and the expression of nestin and βIII-tubulin of fibroblasts after being cultured on PVDF was enhanced. Therefore, the present study represents the first description of the role of NT-4 in the expression of α-amylase of PGACs and the role of PVDF in the reprogramming fibroblasts into neural progenitor-like cells, indicating that PVDF could promote the expression of α-amylase by PGACs via the NT-4 produced by fibroblasts. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Diagnostic value of drain amylase for detecting intrathoracic leakage after esophagectomy

PubMed Central

Berkelmans, Gijs HK; Kouwenhoven, Ewout A; Smeets, Boudewijn JJ; Weijs, Teus J; Silva Corten, Luis C; van Det, Marc J; Nieuwenhuijzen, Grard AP; Luyer, Misha DP

2015-01-01

AIM: To investigate the value of elevated drain amylase concentrations for detecting anastomotic leakage (AL) after minimally invasive Ivor-Lewis esophagectomy (MI-ILE). METHODS: This was a retrospective analysis of prospectively collected data in two hospitals in the Netherlands. Consecutive patients undergoing MI-ILE were included. A Jackson-Pratt drain next to the dorsal side of the anastomosis and bilateral chest drains were placed at the end of the thoracoscopic procedure. Amylase levels in drain fluid were determined in all patients during at least the first four postoperative days. Contrast computed tomography scans and/or endoscopic imaging were performed in cases of a clinically suspected AL. Anastomotic leakage was defined as any sign of leakage of the esophago-gastric anastomosis on endoscopy, re-operation, radiographic investigations, post mortal examination or when gastro-intestinal contents were found in drain fluid. Receiver operator characteristic curves were used to determine the cut-off values. Sensitivity, specificity, positive predictive value, negative predictive value, risk ratio and overall test accuracy were calculated for elevated drain amylase concentrations. RESULTS: A total of 89 patients were included between March 2013 and August 2014. No differences in group characteristics were observed between patients with and without AL, except for age. Patients with AL were older than were patients without AL (P = 0.01). One patient (1.1%) without AL died within 30 d after surgery due to pneumonia and acute respiratory distress syndrome. Anastomotic leakage that required any intervention occurred in 15 patients (16.9%). Patients with proven anastomotic leakage had higher drain amylase levels than patients without anastomotic leakage [median 384 IU/L (IQR 34-6263) vs median 37 IU/L (IQR 26-66), P = 0.003]. Optimal cut-off values on postoperative days 1, 2, and 3 were 350 IU/L, 200 IU/L and 160 IU/L, respectively. An elevated amylase level was found in 9 of the 15 patients with AL. Five of these 9 patients had early elevations of their amylase levels, with a median of 2 d (IQR 2-5) before signs and symptoms occurred. CONCLUSION: Measurement of drain amylase levels is an inexpensive and easy tool that may be used to screen for anastomotic leakage soon after MI-ILE. However, clinical validation of this marker is necessary. PMID:26290638

Alpha-amylase Inhibition and Antioxidant Activity of Marine Green Algae and its Possible Role in Diabetes Management.

PubMed

Unnikrishnan, P S; Suthindhiran, K; Jayasri, M A

2015-10-01

In the continuing search for safe and efficient antidiabetic drug, marine algae become important source which provide several compounds of immense therapeutic potential. Alpha-amylase, alpha-glucosidase inhibitors, and antioxidant compounds are known to manage diabetes and have received much attention recently. In the present study, four green algae (Chaetomorpha aerea, Enteromorpha intestinalis, Chlorodesmis, and Cladophora rupestris) were chosen to evaluate alpha-amylase, alpha-glucosidase inhibitory, and antioxidant activity in vitro. The phytochemical constituents of all the extracts were qualitatively determined. Antidiabetic activity was evaluated by inhibitory potential of extracts against alpha-amylase and alpha-glucosidase by spectrophotometric assays. Antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl, hydrogen peroxide (H2O2), and nitric oxide scavenging assay. Gas chromatography-mass spectrometry (GC-MS) analysis was carried out to determine the major compound responsible for its antidiabetic action. Among the various extracts screened, chloroform extract of C. aerea (IC50 - 408.9 μg/ml) and methanol extract of Chlorodesmis (IC50 - 147.6 μg/ml) showed effective inhibition against alpha-amylase. The extracts were also evaluated for alpha-glucosidase inhibition, and no observed activity was found. Methanol extract of C. rupestris showed notable free radical scavenging activity (IC50 - 666.3 μg/ml), followed by H2O2 (34%) and nitric oxide (49%). Further, chemical profiling by GC-MS revealed the presence of major bioactive compounds. Phenol, 2,4-bis (1,1-dimethylethyl) and z, z-6,28-heptatriactontadien-2-one were predominantly found in the methanol extract of C. rupestris and chloroform extract of C. aerea. Our results demonstrate that the selected algae exhibit notable alpha-amylase inhibition and antioxidant activity. Therefore, characterization of active compounds and its in vivo assays will be noteworthy. Four green algae were chosen to evaluate alpha-amylase, alpha-glucosidase inhibitory, and antioxidant activity in vitro C. aerea and Chlorodesmis showed significant inhibition against alpha-amylase, and C. rupestris showed notable free radical scavenging activityNo observed activity was found against alpha-glucosidaseGC-MS analysis of the active extracts reveals the presence of major compounds which gives an insight on the antidiabetic and antioxidant activity of these algae. Abbreviations used: DPPH: 2,2-diphenyl-1-picrylhydrazyl, BHT: Butylated hydroxytoluene, GC-MS: Gas chromatography-mass spectrometry.

Alpha-amylase Inhibition and Antioxidant Activity of Marine Green Algae and its Possible Role in Diabetes Management

PubMed Central

Unnikrishnan, P. S.; Suthindhiran, K.; Jayasri, M. A.

2015-01-01

Aim: In the continuing search for safe and efficient antidiabetic drug, marine algae become important source which provide several compounds of immense therapeutic potential. Alpha-amylase, alpha-glucosidase inhibitors, and antioxidant compounds are known to manage diabetes and have received much attention recently. In the present study, four green algae (Chaetomorpha aerea, Enteromorpha intestinalis, Chlorodesmis, and Cladophora rupestris) were chosen to evaluate alpha-amylase, alpha-glucosidase inhibitory, and antioxidant activity in vitro. Materials and Methods: The phytochemical constituents of all the extracts were qualitatively determined. Antidiabetic activity was evaluated by inhibitory potential of extracts against alpha-amylase and alpha-glucosidase by spectrophotometric assays. Antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl, hydrogen peroxide (H2O2), and nitric oxide scavenging assay. Gas chromatography-mass spectrometry (GC-MS) analysis was carried out to determine the major compound responsible for its antidiabetic action. Results: Among the various extracts screened, chloroform extract of C. aerea (IC50 − 408.9 μg/ml) and methanol extract of Chlorodesmis (IC50 − 147.6 μg/ml) showed effective inhibition against alpha-amylase. The extracts were also evaluated for alpha-glucosidase inhibition, and no observed activity was found. Methanol extract of C. rupestris showed notable free radical scavenging activity (IC50 – 666.3 μg/ml), followed by H2O2 (34%) and nitric oxide (49%). Further, chemical profiling by GC-MS revealed the presence of major bioactive compounds. Phenol, 2,4-bis (1,1-dimethylethyl) and z, z-6,28-heptatriactontadien-2-one were predominantly found in the methanol extract of C. rupestris and chloroform extract of C. aerea. Conclusion: Our results demonstrate that the selected algae exhibit notable alpha-amylase inhibition and antioxidant activity. Therefore, characterization of active compounds and its in vivo assays will be noteworthy. SUMMARY Four green algae were chosen to evaluate alpha-amylase, alpha-glucosidase inhibitory, and antioxidant activity in vitro C. aerea and Chlorodesmis showed significant inhibition against alpha-amylase, and C. rupestris showed notable free radical scavenging activityNo observed activity was found against alpha-glucosidaseGC-MS analysis of the active extracts reveals the presence of major compounds which gives an insight on the antidiabetic and antioxidant activity of these algae. Abbreviations used: DPPH: 2,2-diphenyl-1-picrylhydrazyl, BHT: Butylated hydroxytoluene, GC-MS: Gas chromatography-mass spectrometry. PMID:27013787

Combined Expression of Aspergillus nidulans Endoxylanase X24 and Aspergillus oryzae (alpha)-Amylase in Industrial Baker's Yeasts and Their Use in Bread Making.

PubMed

Monfort, A; Blasco, A; Prieto, J A; Sanz, P

1996-10-01

The Aspergillus nidulans endoxylanase X24 and the Aspergillus oryzae (alpha)-amylase cDNAs were placed under the control of the Saccharomyces cerevisiae actin promoter (pACT1) and introduced into baker's yeast. Bread made with transformants expressing both enzymes (YEpACT-AMY-ACT-X24) showed a 30% increase in volume and reduced firmness in comparison with that produced with a commercial strain. Endoxylanase X24 and (alpha)-amylase seem to act synergistically to improve the quality of bread in terms of volume and density.

Combined Expression of Aspergillus nidulans Endoxylanase X24 and Aspergillus oryzae (alpha)-Amylase in Industrial Baker's Yeasts and Their Use in Bread Making

PubMed Central

Monfort, A.; Blasco, A.; Prieto, J. A.; Sanz, P.

1996-01-01

The Aspergillus nidulans endoxylanase X24 and the Aspergillus oryzae (alpha)-amylase cDNAs were placed under the control of the Saccharomyces cerevisiae actin promoter (pACT1) and introduced into baker's yeast. Bread made with transformants expressing both enzymes (YEpACT-AMY-ACT-X24) showed a 30% increase in volume and reduced firmness in comparison with that produced with a commercial strain. Endoxylanase X24 and (alpha)-amylase seem to act synergistically to improve the quality of bread in terms of volume and density. PMID:16535419

Quantitative digital image analysis of chromogenic assays for high throughput screening of alpha-amylase mutant libraries.

PubMed

Shankar, Manoharan; Priyadharshini, Ramachandran; Gunasekaran, Paramasamy

2009-08-01

An image analysis-based method for high throughput screening of an alpha-amylase mutant library using chromogenic assays was developed. Assays were performed in microplates and high resolution images of the assay plates were read using the Virtual Microplate Reader (VMR) script to quantify the concentration of the chromogen. This method is fast and sensitive in quantifying 0.025-0.3 mg starch/ml as well as 0.05-0.75 mg glucose/ml. It was also an effective screening method for improved alpha-amylase activity with a coefficient of variance of 18%.

Interaction mechanism between green tea extract and human α-amylase for reducing starch digestion.

PubMed

Miao, Ming; Jiang, Bo; Jiang, Huan; Zhang, Tao; Li, Xingfeng

2015-11-01

This study evaluated the inhibitory effects of the green tea extract on human pancreatic α-amylase activity and its molecular mechanism. The green tea extract was composed of epicatechin (59.2%), epigallocatechin gallate (14.6%) and epicatechin gallate (26.2%) as determined by HPLC analysis. Enzyme activity measurement showed that % inhibition and IC50 of the green tea extract (10%, based on starch) were 63.5% and 2.07 mg/ml, respectively. The Michaelis-Menten constant remained unchanged but the maximal velocity decreased from 0.43 (control) to 0.07 mg/(ml × min) (4 mg/ml of the green tea extract), indicating that the green tea extract was an effective inhibitor against α-amylase with a non-competitive mode. The fluorescence data revealed that the green tea extract bound with α-amylase to form a new complex with static quenching mechanism. Docking study showed the epicatechin gallate in the green tea extract presented stronger affinity than epigallocatechin gallate, with more number of amino acid residues involved in amylase binding with hydrogen bonds and Van der Waals forces. Thus, the green tea extract could be used to manipulate starch digestion for potential health benefits. Copyright © 2015 Elsevier Ltd. All rights reserved.

Synthesis and in vitro study of benzofuran hydrazone derivatives as novel alpha-amylase inhibitor.

PubMed

Taha, Muhammad; Shah, Syed Adnan Ali; Imran, Syahrul; Afifi, Muhammad; Chigurupati, Sridevi; Selvaraj, Manikandan; Rahim, Fazal; Ullah, Hayat; Zaman, Khalid; Vijayabalan, Shantini

2017-12-01

The α-amylase acts as attractive target to treat type-2 diabetes mellitus. Therefore in discovering a small molecule as α-amylase inhibitor, we have synthesized benzofuran carbohydrazide analogs (1-25), characterized through different spectroscopic techniques such as 1 HNMR and EI-MS. All screened analog shows good α-amylase inhibitory potentials with IC 50 value ranging between 1.078±0.19 and 2.926±0.05µM when compared with acarbose having IC 50 =0.62±0.22µM. Only nine analogs among the series such as analogs 3, 5, 7, 8, 10, 12, 21, 23 and 24 exhibit good inhibitory potential with IC 50 values 1.644±0.128, 1.078±0.19, 1.245±0.25, 1.843±0.19, 1.350±0.24, 1.629±0.015, 1.353±0.232, 1.359±0.119 and 1.488±0.07µM when compare with standard drug acarbose. All other analogs showed good to moderate α-amylase inhibitory potentials. The SAR study was conducted on the basis of substituent difference at the phenyl ring. The binding interaction between analogs and active site of enzyme was confirmed by docking studies. Copyright © 2017 Elsevier Inc. All rights reserved.

Amy63, a novel type of marine bacterial multifunctional enzyme possessing amylase, agarase and carrageenase activities

PubMed Central

Liu, Ge; Wu, Shimei; Jin, Weihua; Sun, Chaomin

2016-01-01

A multifunctional enzyme is one that performs multiple physiological functions, thus benefiting the organism. Characterization of multifunctional enzymes is important for researchers to understand how organisms adapt to different environmental challenges. In the present study, we report the discovery of a novel multifunctional enzyme Amy63 produced by marine bacterium Vibrio alginolyticus 63. Remarkably, Amy63 possesses amylase, agarase and carrageenase activities. Amy63 is a substrate promiscuous α-amylase, with the substrate priority order of starch, carrageenan and agar. Amy63 maintains considerable amylase, carrageenase and agarase activities and stabilities at wide temperature and pH ranges, and optimum activities are detected at temperature of 60 °C and pH of 6.0, respectively. Moreover, the heteroexpression of Amy63 dramatically enhances the ability of E. coli to degrade starch, carrageenan and agar. Motif searching shows three continuous glycosyl hydrolase 70 (GH70) family homologs existed in Amy63 encoding sequence. Combining serial deletions and phylogenetic analysis of Amy63, the GH70 homologs are proposed as the determinants of enzyme promiscuity. Notably, such enzymes exist in all kingdoms of life, thus providing an expanded perspective on studies of multifunctional enzymes. To our knowledge, this is the first report of an amylase having additional agarase and carrageenase activities. PMID:26725302

Pentacyclic triterpenes as α-glucosidase and α-amylase inhibitors: Structure-activity relationships and the synergism with acarbose.

PubMed

Zhang, Bo-Wei; Xing, Yan; Wen, Chen; Yu, Xiao-Xia; Sun, Wen-Long; Xiu, Zhi-Long; Dong, Yue-Sheng

2017-11-15

In this paper, the inhibition of α-amylase and α-glucosidase by nine pentacyclic triterpenes was determined. For α-amylase inhibitory activity, the IC 50 values of ursolic acid, corosolic acid, and oleanolic acid were 22.6±2.4μM, 31.2±3.4μM, and 94.1±6.7μM, respectively. For α-glucosidase inhibition, the IC 50 values of ursolic acid, corosolic acid, betulinic acid, and oleanolic acid were 12.1±1.0μM, 17.2±0.9μM, 14.9±1.9μM, and 35.6±2.6μM, respectively. The combination of corosolic acid and oleanolic acid with acarbose showed synergistic inhibition against α-amylase. The combination of the tested triterpenes with acarbose mainly exhibited additive inhibition against α-glucosidase. Kinetic studies revealed that corosolic acid and oleanolic acid showed non-competitive inhibition and acarbose showed mixed-type inhibition against α-amylase. The results provide valuable implications for the triterpenes (ursolic acid, corosolic acid, and oleanolic acid) alone or in combination with acarbose as a therapeutic agent for the treatment of diabetes mellitus. Copyright © 2017 Elsevier Ltd. All rights reserved.

Alpha-Amylase Inhibition and Antioxidative Capacity of Some Antidiabetic Plants Used by the Traditional Healers in Southeastern Nigeria

PubMed Central

Oyedemi, Blessing O.; Ijeh, Ifeoma I.; Ohanyerem, Princemartins E.; Aiyegoro, Olayinka A.

2017-01-01

Oxidative stress plays a significant role in the pathogenesis of metabolic syndrome including diabetes mellitus (DM). The inhibition of alpha-amylase is an important therapeutic target in the regulation of postprandial increase of blood glucose in diabetic patients. The present study investigated the alpha-amylase inhibitory and antioxidant potential of selected herbal drugs used in the treatment of DM by the traditional healers in Isiala Mbano and Ikwuano regions of southeastern Nigeria. Antioxidant activity was evaluated in terms of free radical scavenging, reducing power, and total phenolic (TPC) and flavonoid content (TFC) in consonance with the TLC profiling. The results showed that methanol crude extracts from Anacardium occidentale (AO) and Ceiba pentandra (CP) recorded higher TPC and TFC, potent free radical scavenging, and efficient reducing power (RP) as compared with other plant samples. All the plant extracts exhibited a relative alpha-amylase inhibition apart from Strophanthus hispidus (SH) extract with a negative effect. We discovered a mild to weak correlation between alpha-amylase inhibition or antioxidative capacity and the total phenol or flavonoid content. At least in part, the results obtained in this work support the traditional use of certain plant species in the treatment of patients with DM. PMID:28367491

Improvement of Starch Digestion Using α-Amylase Entrapped in Pectin-Polyvinyl Alcohol Blend

PubMed Central

Cruz, Maurício; Fernandes, Kátia; Cysneiros, Cristine; Nassar, Reginaldo; Caramori, Samantha

2015-01-01

Polyvinyl alcohol (PVA) and pectin blends were used to entrap α-amylase (Termamyl) using glutaraldehyde as a cross-linker. The effect of glutaraldehyde concentration (0.25, 0.5, 0.75, 1.0, and 1.25%) on the activity of the immobilized enzyme and rate of enzyme released was tested during a 24 h period. Characteristics of the material, such as scanning electron microscopy (SEM), tensile strength (TS), elongation, and rate of dissolution in water (pH 5.7), ruminal buffering solution (pH 7.0), and reactor containing 0.1 mol L−1 sodium phosphate buffer (pH 6.5), were also analyzed. SEM results showed that the surfaces of the pectin/PVA/amylase films were highly irregular and rough. TS values increased as a function of glutaraldehyde concentration, whereas percentage of elongation (%E) decreased. Pectin/PVA/amylase films presented similar values of solubility in the tested solvents. The material obtained with 0.25% glutaraldehyde performed best with repeated use (active for 24 h), in a phosphate buffer reactor. By contrast, the material obtained with 1.25% glutaraldehyde presented higher performance during in vitro testing using an artificial rumen. The results suggest that pectin/PVA/amylase is a highly promising material for biotechnological applications. PMID:25949991

Production of alpha-amylase from Aspergillus oryzae for several industrial applications in a single step.

PubMed

Porfirif, María C; Milatich, Esteban J; Farruggia, Beatriz M; Romanini, Diana

2016-06-01

A one-step method as a strategy of alpha-amylase concentration and purification was developed in this work. This methodology requires the use of a very low concentration of biodegradable polyelectrolyte (Eudragit(®) E-PO) and represents a low cost, fast, easy to scale up and non-polluting technology. Besides, this methodology allows recycling the polymer after precipitation. The formation of reversible soluble/insoluble complexes between alpha-amylase and the polymer Eudragit(®) E-PO was studied, and their precipitation in selected conditions was applied with bioseparation purposes. Turbidimetric assays allowed to determine the pH range where the complexes are insoluble (4.50-7.00); pH 5.50 yielded the highest turbidity of the system. The presence of NaCl (0.05M) in the medium totally dissociates the protein-polymer complexes. When the adequate concentration of polymer was added under these conditions to a liquid culture of Aspergillus oryzae, purification factors of alpha-amylase up to 7.43 and recoveries of 88% were obtained in a simple step without previous clarification. These results demonstrate that this methodology is suitable for the concentration and production of alpha-amylase from this source and could be applied at the beginning of downstream processing. Copyright © 2016 Elsevier B.V. All rights reserved.

Michaelis kinetic analysis of extracellular cellulase and amylase excreted by Lactobacillus plantarum during cassava fermentation

NASA Astrophysics Data System (ADS)

Frediansyah, Andri; Kurniadi, Muhamad

2017-01-01

Our previous study reveal that single culture of Lactobacillus plantarum has ability to ferment cassava tuber in relation to produce modified cassava flour (mocaf). It was used to accelerate a fermentation process. L. plantarum grow well and produce some extracellular enzymes i.e. cellulase to change the structure and breakdown the cell wall of cassava tuber. Then, the starchy materials will be hydrolyzed by i.e. amylase into simple sugar and convert to organic acid. All of these process will give new characteristic of cassava i.e. lower fiber content, good flavor, taste, aroma and texture and the amount of cyanide acid is lower. Therefore this present study was to analyze Michaelis kinetics of extracellular carboxymethyl cellulase and amylase production by L. plantarum during cassava fermentation. The maximum carboxymethyl cellulase and amylase activity of 8.60 U/ml and 14.07 U/ml, respectively, were obtained from filtrate which has been incubated at 37°C for 18 h under stationary conditions. The Vmax and Km of CMCase were 0.8506 × 10-3 U/ml and 0.9594 × 10-3 g/mL, respectively. For amylase were 9.291 × 10-3 U/ml and 0.9163 × 10-3 g/ml, respectively.

Validation of an assay for quantification of alpha-amylase in saliva of sheep

PubMed Central

Fuentes-Rubio, Maria; Fuentes, Francisco; Otal, Julio; Quiles, Alberto; Hevia, María Luisa

2016-01-01

The objective of this study was to develop a time-resolved immunofluorometric assay (TR-IFMA) for quantification of salivary alpha-amylase in sheep. For that purpose, after the design of the assay, an analytical and a clinical validation were carried out. The analytical validation of the assay showed intra- and inter-assay coefficients of variation (CVs) of 6.1% and 10.57%, respectively and an analytical limit of detection of 0.09 ng/mL. The assay also demonstrated a high level of accuracy, as determined by linearity under dilution. For clinical validation, a model of acute stress testing was conducted to determine whether expected significant changes in alpha-amylase were picked up in the newly developed assay. In that model, 11 sheep were immobilized and confronted with a sheepdog to induce stress. Saliva samples were obtained before stress induction and 15, 30, and 60 min afterwards. Salivary cortisol was measured as a reference of stress level. The results of TR-IFMA showed a significant increase (P < 0.01) in the concentration of alpha-amylase in saliva after stress induction. The assay developed in this study could be used to measure salivary alpha-amylase in the saliva of sheep and this enzyme could be a possible noninvasive biomarker of stress in sheep. PMID:27408332

Structure of Waxy Maize Starch Hydrolyzed by Maltogenic α-Amylase in Relation to Its Retrogradation.

PubMed

Grewal, Navneet; Faubion, Jon; Feng, Guohua; Kaufman, Rhett C; Wilson, Jeff D; Shi, Yong-Cheng

2015-04-29

Maltogenic α-amylase is widely used as an antistaling agent in bakery foods. The objective of this study was to determine the degree of hydrolysis (DH) and starch structure after maltogenic amylase treatments in relation to its retrogradation. Waxy maize starch was cooked and hydrolyzed to different degrees by a maltogenic amylase. High-performance anion-exchange chromatography and size exclusion chromatography were used to determine saccharides formed and the molecular weight (Mw) distributions of the residual starch structure, respectively. Chain length (CL) distributions of debranched starch samples were further related to amylopectin (AP) retrogradation. Differential scanning calorimetry (DSC) results showed the complete inhibition of retrogradation when starches were hydrolyzed to >20% DH. Mw and CL distributions of residual AP structure indicated that with an increase in %DH, a higher proportion of unit chains with degree of polymerization (DP) ≤9 and a lower proportion of unit chains with DP ≥17 were formed. A higher proportion of short outer AP chains that cannot participate in the formation of double helices supports the decrease in and eventual inhibition of retrogradation observed with the increase in %DH. These results suggest that the maltogenic amylase could play a powerful role in inhibiting the staling of baked products even at limited starch hydrolysis.

Improved energy intakes using amylase-digested weaning foods in Tanzanian children with acute diarrhea.

PubMed

Darling, J C; Kitundu, J A; Kingamkono, R R; Msengi, A E; Mduma, B; Sullivan, K R; Tomkins, A M

1995-07-01

Amylase from germinating cereal grains enables the preparation of porridge with a higher energy density than conventional weaning foods. This food can be combined with fermentation, which inhibits pathogen growth. These food technologies are inexpensive, can be implemented at the household level, and are therefore particularly appropriate for use in developing countries. In a controlled clinical trial, 75 children aged 6-25 months admitted to hospital with acute diarrhea were rehydrated and then randomly allocated to three corn porridge dietary groups: conventional, amylase-digested (AMD), and fermented and amylase-digested (FAD). The study diets were given ad libitum five times daily, and all intakes except breast milk were weighed. Mean daily energy intakes over 4 days in the conventional AMD, and FAD groups, respectively, were 32.4 (95% CI 28.7-36.6), 46.0 (CI 39.6-53.4), and 37.3 (CI 31.8-43.9) kcal/kg/day. The energy intake in the AMD group was 42% higher than the conventional group (p = 0.003). There were no significant differences between the groups for duration of diarrhea, frequency of stooling, or vomiting. Starch digestion using amylase from germination is an effective way of improving energy intake in children with acute diarrhea.

Evaluation of Laboratory Procedures to Quantify the Neutral Detergent Fiber Content in Forage, Concentrate, and Ruminant Feces.

PubMed

Barbosa, Marcília Medrado; Detmann, Edenio; Rocha, Gabriel Cipriano; de Oliveira Franco, Marcia; de Campos Valadares Filho, Sebastião

2015-01-01

A comparison was made of measurements of neutral detergent fiber concentrations obtained with AOAC Method 2002.04 and modified methods using pressurized environments or direct use of industrial heat-stable α-amylase in samples of forage (n=37), concentrate (n=30), and ruminant feces (n=39). The following method modifications were tested: AOAC Method 2002.04 with replacement of the reflux apparatus with an autoclave or Ankom(220®) extractor and F57 filter bags, and AOAC Method 2002.04 with replacement of the standardization procedures for α-amylase by a single addition of industrial α-amylase [250 μL of Termamyl 2X 240 Kilo Novo Units (KNU)-T/g] prior to heating the neutral detergent solution. For the feces and forage samples, the results obtained with the modified methods with an autoclave or modification of α-amylase use were similar to those obtained using AOAC Method 2002.04, but the use of the Ankom220 extractor resulted in overestimated values. For the concentrate samples, the modified methods using an autoclave or Ankom220 extractor resulted in positive systematic errors. However, the method using industrial α-amylase resulted in systematic error and slope bias despite that the obtained values were close to those obtained with AOAC Method 2002.04.

Vicilin-like peptides from Capsicum baccatum L. seeds are α-amylase inhibitors and exhibit antifungal activity against important yeasts in medical mycology.

PubMed

Vieira Bard, Gabriela C; Nascimento, Viviane V; Oliveira, Antônia Elenir A; Rodrigues, Rosana; Da Cunha, Maura; Dias, Germana B; Vasconcelos, Ilka M; Carvalho, Andre O; Gomes, Valdirene M

2014-07-01

The objective of this study was to isolate antimicrobial peptides from Capsicum baccatum seeds and evaluate their antimicrobial activity and inhibitory effects against α-amylase. Initially, proteins from the flour of C. baccatum seeds were extracted in sodium phosphate buffer, pH 5.4, and precipitated with ammonium sulfate at 90% saturation. The D1 and D2 fractions were subjected to antifungal tests against the yeasts Saccharomyces cerevisiae, Candida albicans, Candida tropicalis, and Kluyveromyces marxiannus, and tested against α-amylases from Callosobruchus maculates and human saliva. The D2 fraction presented higher antimicrobial activity and was subjected to further purification and seven new different fractions (H1-H7) were obtained. Peptides in the H4 fraction were sequenced and the N-terminal sequences revealed homology with previously reported storage vicilins from seeds. The H4 fraction exhibited strong antifungal activity and also promoted morphological changes in yeast, including pseudohyphae formation. All fractions, including H4, inhibited mammalian α-amylase activity but only the H4 fraction was able to inhibit C. maculatus α-amylase activity. These results suggest that the fractions isolated from the seeds of C. baccatum can act directly in plant defenses against pathogens and insects. © 2014 Wiley Periodicals, Inc.

Measurement of starch digestion of naturally 13C-enriched weaning foods, before and after partial digestion with amylase-rich flour, using a 13C breath test.

PubMed

Weaver, L T; Dibba, B; Sonko, B; Bohane, T D; Hoare, S

1995-10-01

Malnutrition in infancy is a global problem which leads to retardation of childhood growth and development. There is a pressing need to improve weaning strategies for infants of the developing world. Traditional Gambian weaning foods are watery and of low energy density, but addition of energy in the form of fat and carbohydrate leads to thick, viscous gruels which are difficult to ingest. Partial digestion with amylase (EC 3.2.1.1)-rich flour reduces their viscosity while retaining their energy density. The aim of the present study was to measure the digestibility of a maize-based weaning food, before and after amylase digestion, in malnourished children using a 13C breath test. Ten children (aged 7-16 months; mean weight-for-age Z score -0.8) received isovolumetric and isoenergetic quantities of a maize-based weaning food naturally abundant with 13C. Breath samples were collected at intervals of 30 min for 5 h thereafter and 13CO2 enrichment was measured by isotope-ratio mass spectrometry. Percentage dose of 13C recovered increased from a mean 13.7 (SD 3.7)% before, to 18.3 (SD 5.6)% after ingestion of amylase-treated weaning foods (P < 0.1). There was a significant inverse relation between age and weight, and percentage dose of 13C recovered in children receiving amylase-treated feeds. There were no differences in concentrations of amylase in saliva of infants or breast milk of their mothers. Partial digestion of supplementary foods may improve the nutrition of undernourished weaning children, not only by reducing their viscosity, thereby increasing ingestion, but also by improving their digestion and thereby their absorption.

Amylases StAmy23, StBAM1 and StBAM9 regulate cold-induced sweetening of potato tubers in distinct ways

PubMed Central

Hou, Juan; Zhang, Huiling; Liu, Jun; Reid, Stephen; Liu, Tengfei; Xu, Shijing; Tian, Zhendong; Sonnewald, Uwe

2017-01-01

Abstract Cold-induced sweetening (CIS) in potato is detrimental to the quality of processed products. Conversion of starch to reducing sugars (RS) by amylases is considered one of the main pathways in CIS but is not well studied. The amylase genes StAmy23, StBAM1, and StBAM9 were studied for their functions in potato CIS. StAmy23 is localized in the cytoplasm, whereas StBAM1 and StBAM9 are targeted to the plastid stroma and starch granules, respectively. Genetic transformation of these amylases in potatoes by RNA interference showed that β-amylase activity could be decreased in cold-stored tubers by silencing of StBAM1 and collective silencing of StBAM1 and StBAM9. However, StBAM9 silencing did not decrease β-amylase activity. Silencing StBAM1 and StBAM9 caused starch accumulation and lower RS, which was more evident in simultaneously silenced lines, suggesting functional redundancy. Soluble starch content increased in RNAi-StBAM1 lines but decreased in RNAi-StBAM9 lines, suggesting that StBAM1 may regulate CIS by hydrolysing soluble starch and StBAM9 by directly acting on starch granules. Moreover, StBAM9 interacted with StBAM1 on the starch granules. StAmy23 silencing resulted in higher phytoglycogen and lower RS accumulation in cold-stored tubers, implying that StAmy23 regulates CIS by degrading cytosolic phytoglycogen. Our findings suggest that StAmy23, StBAM1, and StBAM9 function in potato CIS with varying levels of impact. PMID:28369567

Spray-dried high-amylose sodium carboxymethyl starch: impact of α-amylase on drug-release profile.

PubMed

Nabais, Teresa; Zaraa, Sarra; Leclair, Grégoire

2016-11-01

Spray-dried high-amylose sodium carboxymethyl starch (SD HASCA) is a promising pharmaceutical excipient for sustained-release (SR) matrix tablets produced by direct compression. The presence of α-amylase in the gastrointestinal tract and the variations of the gastric residence time of non-disintegrating dosage forms may affect the presystemic metabolism of this excipient and, consequently, the drug-release profile from formulations produced with SD HASCA. In this study, the influence of α-amylase and the residence time in acidic conditions on the drug-release profile was evaluated for a once-daily acetaminophen formulation (Acetaminophen SR) and a once-daily tramadol hydrochloride formulation (Tramadol SR). Both formulations were based on SD HASCA. α-Amylase concentrations ranging from 0 IU/L to 20000 IU/L did not significantly affect the drug-release profiles of acetaminophen and tramadol hydrochloride from SD HASCA tablets (f2 > 50) for all but only one of the studied conditions (f2 = 47). Moreover, the drug-release properties from both SD HASCA formulations were not significantly different when the residence time in acidic medium was 1 h or 3 h. An increase in α-amylase concentration led to an increase in the importance of polymer erosion as the main mechanism of drug-release instead of drug diffusion, for both formulations and both residence times, even if release profiles remained comparable. As such, it is expected that α-amylase concentration and residence time in the stomach will not clinically affect the performance of both SD HASCA SR formulations, even if the mechanism of release itself may be affected.

Regulating unfolded protein response activator HAC1p for production of thermostable raw-starch hydrolyzing α-amylase in Pichia pastoris.

PubMed

Huang, Mengmeng; Gao, Yanyun; Zhou, Xiangshan; Zhang, Yuanxing; Cai, Menghao

2017-03-01

Unfolded protein response (UPR) usually happens when expressing heterologous proteins in high level, which may help cells to facilitate protein processing. Here, we evaluated the effects of the UPR activator HAC1p on a raw-starch hydrolyzing α-amylase (Gs4j-amyA), so as to improve heterologous production of the enzyme in Pichia pastoris. The gene (amyA) encoding Gs4j-amyA was first codon-optimized and expressed in P. pastoris under the control of the AOX1 promoter. A high gene dosage (12 copies) of amyA facilitated amylase expression which produced an enzyme activity of 305 U/ml. A spliced HAC1 encoding an UPR activator HAC1p was then co-expressed and the dosage effects of HAC1 on amylase expression was investigated. Six copies of HAC1 driven by AOX1 promoter produced a high amylase activity of 2200 U/ml, further increasing by 621%. However, excessive gene dosages driven by the same promoter led to a titration effect of its transcription factors and decreased the amount of amyA transcripts. Thus, constitutive expression of HAC1 by GAP promotor was further involved and Gs4j-amyA activity reached 3700 U/ml finally, which was further increased by 68.2%. Moreover, Gs4j-amyA was glycosylated in P. pastoris which generated higher enzyme activity than that in E. coli. Generally, regulating HAC1p expression by different strategies enhanced amylase production by 11.1 folds, indicating a reference for expression of other proteins in P. pastoris.

Regulation of muscarinic acetylcholine receptors in cultured guinea pig pancreatic acini

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hootman, S.R.; Brown, M.E.; Williams, J.A.

1986-07-01

Regulation of muscarinic receptors in cultured guinea pig pancreatic acini was investigated by assessing the effects of cholinergic agonists on binding of (N-methyl-TH)scopolamine ((TH)NMS) and on amylase release. Freshly dispersed acini bound (TH)NMS with a K/sub d/ of 74 pM and a maximal binding level (B/sub max/) of 908 fmol/mg DNA. Carbachol (CCh) stimulated amylase secretion and inhibited (TH)NMS binding. Incubation of acini for 30 min with 0.1 mM CCh decreased the subsequent efficacy of CCh in stimulating amylase release by threefold but had no effect on its potency. In contrast, amylase release in response to cholecystokinin octapeptide (CCK-8) wasmore » not altered by CCh preincubation. (TH)NMS binding to acini was decreased only 15-20% after 30-min incubation with CCh. However, culture of acini with 0.1 mM CCh decreased (TH)NMS binding by 50% at 3-4 h and by 85-90% at 24 h. This decrease was attributable primarily to a reduction in B/sub max/ (TH)NMS binding also was decreased to a similar extent by the cholinergic agonists bethanechol and methacholine but not by other secretagogues. The decrease in antagonist binding induced by CCh was dose dependent, with the IC50, 5.8 M, approximating the EC50 for amylase release, 4.3 M. Cultured of acini for 24 h with CCh abolished subsequent amylase release in response to CCh but not to CCK-8. The results indicate that muscarinic receptor turnover in the pancreatic acinus is regulated by receptor activation and that both a decease in receptor numbers and sensitivity to agonists follows prolonged cholinergic agonist exposure.« less

In vitro and in vivo α-amylase and α-glucosidase inhibiting activities of the protein extracts from two varieties of bitter gourd (Momordica charantia L.).

PubMed

Poovitha, Sundar; Parani, Madasamy

2016-07-18

α-amylase and α-glucosidase digest the carbohydrates and increase the postprandial glucose level in diabetic patients. Inhibiting the activity of these two enzymes can control postprandial hyperglycemia, and reduce the risk of developing diabetes. Bitter gourd or balsam pear is one of the important medicinal plants used for controlling postprandial hyperglycemia in diabetes patients. However, there is limited information available on the presence of α-amylase and α-glucosidase inhibiting compounds. In the current study, the protein extracts from the fruits of M. charantia var. charantia (MCC) and M. charantia var. muricata (MCM) were tested for α-amylase and α-glucosidase inhibiting activities in vitro, and glucose lowering activity after oral administration in vivo. The protein extract from both MCC and MCM inhibited the activity of α-amylase and α-glucosidase through competitive inhibition, which was on par with Acarbose as indicated by in vitro percentage of inhibition (66 to 69 %) and IC50 (0.26 to 0.29 mg/ml). Both the protein extracts significantly reduced peak blood glucose and area under the curve in Streptozotocin-induced diabetic rats, which were orally challenged with starch and sucrose. Protein extracts from the fruits of the two varieties of bitter gourd inhibited α-amylase and α-glucosidase in vitro and lowered the blood glucose level in vivo on par with Acarbose when orally administrated to Streptozotocin-induced diabetic rats. Further studies on mechanism of action and methods of safe and biologically active delivery will help to develop an anti-diabetic oral protein drug from these plants.

Amylase, Lipase, and Acute Pancreatitis in People With Type 2 Diabetes Treated With Liraglutide: Results From the LEADER Randomized Trial.

PubMed

Steinberg, William M; Buse, John B; Ghorbani, Marie Louise Muus; Ørsted, David D; Nauck, Michael A

2017-07-01

To evaluate serum amylase and lipase levels and the rate of acute pancreatitis in patients with type 2 diabetes and high cardiovascular risk randomized to liraglutide or placebo and observed for 3.5-5.0 years. A total of 9,340 patients with type 2 diabetes were randomized to either liraglutide or placebo (median observation time 3.84 years). Fasting serum lipase and amylase were monitored. Acute pancreatitis was adjudicated in a blinded manner. Compared with the placebo group, liraglutide-treated patients had increases in serum lipase and amylase of 28.0% and 7.0%, respectively. Levels were increased at 6 months and then remained stable. During the study, 18 (0.4% [1.1 events/1,000 patient-years of observation] [PYO]) liraglutide-treated and 23 (0.5% [1.7 events/1,000 PYO]) placebo patients had acute pancreatitis confirmed by adjudication. Most acute pancreatitis cases occurred ≥12 months after randomization. Liraglutide-treated patients with prior history of pancreatitis ( n = 147) were not more likely to develop acute pancreatitis than similar patients in the placebo group ( n = 120). Elevations of amylase and lipase levels did not predict future risk of acute pancreatitis (positive predictive value <1.0%) in patients treated with liraglutide. In a population with type 2 diabetes at high cardiovascular risk, there were numerically fewer events of acute pancreatitis among liraglutide-treated patients (regardless of previous history of pancreatitis) compared with the placebo group. Liraglutide was associated with increases in serum lipase and amylase, which were not predictive of an event of subsequent acute pancreatitis. © 2017 by the American Diabetes Association.

Enzyme exposure in the British baking industry.

PubMed

Elms, J; Robinson, E; Mason, H; Iqbal, S; Garrod, A; Evans, G S

2006-06-01

Enzymes are commonly used in the baking industry, as they can improve dough quality and texture and lengthen the shelf life of the final product. There is little published information highlighting exposure to enzymes (other than fungal alpha-amylase) in the baking industry, therefore the purpose of this study was to identify antibodies and develop assays for the measurement of a variety of such enzymes in samples of airborne flour dust. Polyclonal antibodies to bacterial amylase, glucose oxidase and amyloglucosidase were identified and developed into ELISA assays. The assays showed limited cross-reactivity with other enzymes commonly used in the baking industry. We measured levels of airborne enzymes in 195 personal air samples taken from a sample of 55 craft baking establishments. We were able to detect amyloglucosidase in 9% (16/184) of the samples, fungal alpha-amylase in 6% (11/171), bacterial alpha-amylase in 7% (13/195). However, we were unable to detect glucose oxidase in any of the samples. Measurements for protease enzymes were not carried out. Median levels in detectable samples of amyloglucosidase, fungal alpha-amylase and bacterial amylase were similar at 10.3, 5.3 and 5.9 ng/m(3), respectively. These figures represent the total enzyme protein (active and inactive) measured. There are few data in the literature regarding sensitization and exposure-response relationships to these enzymes, and indeed there is often a lack of information within the industry as to the precise enzyme content of particular baking ingredients. As a precautionary measure, all enzymes are regarded as having the potential to cause respiratory sensitization. Consequently, exposures need to be controlled to as low a level as reasonably practicable, and future investigation may highlight the importance of measuring a variety of enzyme exposures and standardizing these methodologies to inform approaches to adequate control.

Remarkable evolutionary relatedness among the enzymes and proteins from the α-amylase family.

PubMed

Janeček, Štefan; Gabriško, Marek

2016-07-01

The α-amylase is a ubiquitous starch hydrolase catalyzing the cleavage of the α-1,4-glucosidic bonds in an endo-fashion. Various α-amylases originating from different taxonomic sources may differ from each other significantly in their exact substrate preference and product profile. Moreover, it also seems to be clear that at least two different amino acid sequences utilizing two different catalytic machineries have evolved to execute the same α-amylolytic specificity. The two have been classified in the Cabohydrate-Active enZyme database, the CAZy, in the glycoside hydrolase (GH) families GH13 and GH57. While the former and the larger α-amylase family GH13 evidently forms the clan GH-H with the families GH70 and GH77, the latter and the smaller α-amylase family GH57 has only been predicted to maybe define a future clan with the family GH119. Sequences and several tens of enzyme specificities found throughout all three kingdoms in many taxa provide an interesting material for evolutionarily oriented studies that have demonstrated remarkable observations. This review emphasizes just the three of them: (1) a close relatedness between the plant and archaeal α-amylases from the family GH13; (2) a common ancestry in the family GH13 of animal heavy chains of heteromeric amino acid transporter rBAT and 4F2 with the microbial α-glucosidases; and (3) the unique sequence features in the primary structures of amylomaltases from the genus Borrelia from the family GH77. Although the three examples cannot represent an exhaustive list of exceptional topics worth to be interested in, they may demonstrate the importance these enzymes possess in the overall scientific context.

Cortisol and alpha-amylase as stress response indicators during pre-hospital emergency medicine training with repetitive high-fidelity simulation and scenarios with standardized patients.

PubMed

Valentin, Bernd; Grottke, Oliver; Skorning, Max; Bergrath, Sebastian; Fischermann, Harold; Rörtgen, Daniel; Mennig, Marie-Therese; Fitzner, Christina; Müller, Michael P; Kirschbaum, Clemens; Rossaint, Rolf; Beckers, Stefan K

2015-04-08

In emergency medicine, the benefits of high-fidelity simulation (SIM) are widely accepted and standardized patients (SP) are known to mimic real patients accurately. However, only limited data are available concerning physicians' stress markers within these training environments. The aim of this pilot study was to investigate repetitive stress among healthcare professionals in simulated pre-hospital emergency scenarios using either SIM or SPs. Teams with one emergency medical services (EMS) physician and two paramedics completed three SIM scenarios and two SP scenarios consecutively. To evaluate stress, salivary cortisol and alpha-amylase were measured in saliva samples taken before, during and after the scenarios. A total of 14 EMS physicians (29% female; mean age: 36.8 ± 5.0 years; mean duration of EMS-experience: 9.1 ± 5.8 years) and 27 paramedics (11% female; age: 30.9 ± 6.9 years; EMS experience: 8.1 ± 6.0 years) completed the study. Alpha-amylase and cortisol levels did not differ significantly between the two professions. Cortisol values showed a gradual and statistically significant reduction over time but little change was observed in response to each scenario. In contrast, alpha-amylase activity increased significantly in response to every SIM and SP scenario, but there was no clear trend towards an overall increase or decrease over time. Increases in salivary alpha-amylase activity suggest that both SIM and SP training produce stress among emergency healthcare professionals. Corresponding increases in salivary cortisol levels were not observed. Among physicians in the emergency setting, it appears that alpha-amylase provides a more sensitive measure of stress levels than cortisol.

Use of enzymes to minimize the rheological dough problems caused by high levels of damaged starch in starch-gluten systems.

PubMed

Barrera, Gabriela N; León, Alberto E; Ribotta, Pablo D

2016-05-01

During wheat milling, starch granules can experience mechanical damage, producing damaged starch. High levels of damaged starch modify the physicochemical properties of wheat flour, negatively affecting the dough behavior as well as the flour quality and cookie and bread making quality. The aim of this work was to evaluate the effect of α-amylase, maltogenic amylase and amyloglucosidase on dough rheology in order to propose alternatives to reduce the issues related to high levels of damaged starch. The dough with a high level of damaged starch became more viscous and resistant to deformations as well as less elastic and extensible. The soluble fraction of the doughs influenced the rheological behavior of the systems. The α-amylase and amyloglucosidase reduced the negative effects of high damaged starch contents, improving the dough rheological properties modified by damaged starch. The rheological behavior of dough with the higher damaged-starch content was related to a more open gluten network arrangement as a result of the large size of the swollen damaged starch granules. We can conclude that the dough rheological properties of systems with high damaged starch content changed positively as a result of enzyme action, particularly α-amylase and amyloglucosidase additions, allowing the use of these amylases and mixtures of them as corrective additives. Little information was reported about amyloglucosidase activity alone or combined with α-amylase. The combinations of these two enzymes are promising to minimize the negative effects caused by high levels of damaged starch on product quality. More research needs to be done on bread quality combining these two enzymes. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Evaluation of the relationship between passive smoking and salivary electrolytes, protein, secretory IgA, sialic acid and amylase in young children.

PubMed

Avşar, Aysun; Darka, Ozge; Bodrumlu, Ebru Hazar; Bek, Yüksel

2009-05-01

To evaluate the relationship between passive smoking as determined by salivary cotinine levels and salivary electrolytes, protein, secretory IgA, sialic acid and amylase in children. Saliva was collected from 90 passive smoker (PS) subjects (the study group) and 90 healthy age-matched children (the control group). The study group was divided into three subgroups according the number of cigarettes smoked. Socio-economic status, dental and dietary habits were recorded by questionnaire. Stimulated salivary calcium (Ca), phosphate (P), sodium (Na), potassium (P), total protein, amylase activity, sialic acid level, secretory IgA concentration and cotinine level were analysed. All data were analysed using SPSS, version 13.0. Socio-economic status, dental and dietary habits were similar between the two groups. The salivary electrolytes concentrations did not reveal significant difference between the two groups (p>0.05). The mean cotinine levels of PS children were 1.58+/-4.3 ng/mL. The salivary concentrations of protein were similar between the two groups (p>0.05). The salivary secretory IgA concentration was significantly lower in the PS group than controls. The sialic acid level and amylase activity in PS group were found significantly higher compared with the controls (p<0.05). No difference was observed for all these parameters with sex (p>0.05). When saliva samples were analysed for output, the sialic acid level and amylase activity increased significantly in PS subjects (p<0.05). Further, the output of secretory IgA concentration was found significantly lower compared with the controls (p<0.05). In conclusion, we show that passive smoking was associated with a decrease in secretory IgA concentration, whereas with increase in amylase activity and sialic acid level of stimulated whole saliva in young children.

Utilization of α-amylase enzyme from Bacillus stearothermophilus RSAII1B for maltodextrin production from sago starch

NASA Astrophysics Data System (ADS)

Arfah, R. A.; Ahmad, A.; Dali, S.; Djide, M. N.; Mahdalia; Arif, A. R.

2018-03-01

The dried sago flour derived from Palopo contains 28.80% amylose and 91.23% total carbohydrate. Based on the data, sago starch has the potential to become an alternative raw material for themaltodextrin production. Maltodextrin is one of the starch derivative products produced by hydrolysis process using the α-amylase enzyme with amaximum DE (dextrose equivalent) value of 20. The use of maltodextrin for food and pharmaceutical industries is increasing because of maltodextrin is widely used as thickener filler, surfactant and sugar substitute in milk powder. The aims of this study are to optimize the addition of enzyme concentration and hydrolysis time of α -amylase enzyme to obtain high quality ofmaltodextrin This study also aimed to characterization the obtained maltodextrin. The first step was isolation and purification α-amylase from the isolate of Bacillus stearothermophilus RSAII1B, followed by determination of the α-amylase concentration (0.05%, 0.07% and 0.09%) in 2.0% starch substrate, and the hydrolysis time ofα-amilase (60, 90, 120, 240 minutes). Maltodextrin characters observed were dextrose equivalent (DE), reducing sugar, moisture content, pH changes, color, solubility, viscosity, and total plate count (TPC). The results showed that the value of DE was 12.31, reducing sugar was 11.4%; water content was 10.92%; pH was 4.85; The color of maltodextrin powder was white bone color; solubility was 153.2 g/L; Viscositywas 210-240 cps, TPCwas 380 cfu/g. Maltodextrins produced from sago starch using the α-amylase enzyme from B.stearothermophillus RSAIIm met the quality requirements of SNI 7599: 2010.

The effect of exercise mode and intensity of sub-maximal physical activities on salivary testosterone to cortisol ratio and α-amylase in young active males

PubMed Central

AZARBAYJANI, MOHAMMAD ALI; FATOLAHI, HOSEYN; RASAEE, MOHAMMAD JAVAD; PEERI, MAGHSOD; BABAEI, ROHOLAH

2011-01-01

We examined the effect of exercise intensity and mode on the acute responses of free testosterone to cortisol ratio and salivary α-amylase. We also evaluated the relationship between cortisol and salivary α-amylase. Ten healthy young active males participated voluntarily in this study in six single sessions. They exercised on a cycle ergo meter, treadmill, and elliptical instrument at intensities of 70% and 85% maximum heart rate for 25 minutes. Saliva samples were collected 5 minutes before and 5 minutes after each exercise session. No significant changes were observed for cortisol. Free testosterone to cortisol ratio increased during each exercise session (F5, 45=3.15, P=0.02). However, these changes are only significant after exercise on the treadmill at 70% maximum heart rate (t=2.94, P=0.02) and 85% maximum heart rate (t=0.53, P=0.03). Salivary α-amylase significantly varied among exercise sessions (F5, 45=3.97, P=0.005), and a significant decline was observed after exercise on the elliptical instrument (t=2.38, P=0.04) and treadmill (t=3.55, P=0.006) at 85% maximum heart rate. We found that the free testosterone to cortisol ratio is dependent on the exercise mode, while the salivary α-amylase response is dependent on the intensity of exercise. The increase of free testosterone to cortisol ratio in this study may indicate lower physiological stress in response to performing these exercises. Applying muscular strength with moderate intensity weight-bearing exercises possibly activates the anabolic pathways. Although the cortisol and salivary α-amylase responses were opposite in the majority of the exercise sessions, no significant inverse relationship was observed. PMID:27182369

The effect of exercise mode and intensity of sub-maximal physical activities on salivary testosterone to cortisol ratio and α-amylase in young active males.

PubMed

Azarbayjani, Mohammad Ali; Fatolahi, Hoseyn; Rasaee, Mohammad Javad; Peeri, Maghsod; Babaei, Roholah

We examined the effect of exercise intensity and mode on the acute responses of free testosterone to cortisol ratio and salivary α-amylase. We also evaluated the relationship between cortisol and salivary α-amylase. Ten healthy young active males participated voluntarily in this study in six single sessions. They exercised on a cycle ergo meter, treadmill, and elliptical instrument at intensities of 70% and 85% maximum heart rate for 25 minutes. Saliva samples were collected 5 minutes before and 5 minutes after each exercise session. No significant changes were observed for cortisol. Free testosterone to cortisol ratio increased during each exercise session (F 5, 45 =3.15, P=0.02) . However, these changes are only significant after exercise on the treadmill at 70% maximum heart rate ( t=2.94, P=0.02 ) and 85% maximum heart rate ( t=0.53, P=0.03 ). Salivary α-amylase significantly varied among exercise sessions (F 5, 45 =3.97, P=0.005), and a significant decline was observed after exercise on the elliptical instrument (t=2.38, P=0.04) and treadmill ( t=3.55, P=0.006 ) at 85% maximum heart rate. We found that the free testosterone to cortisol ratio is dependent on the exercise mode, while the salivary α-amylase response is dependent on the intensity of exercise. The increase of free testosterone to cortisol ratio in this study may indicate lower physiological stress in response to performing these exercises. Applying muscular strength with moderate intensity weight-bearing exercises possibly activates the anabolic pathways. Although the cortisol and salivary α-amylase responses were opposite in the majority of the exercise sessions, no significant inverse relationship was observed.

Gedunin and Azadiradione: Human Pancreatic Alpha-Amylase Inhibiting Limonoids from Neem (Azadirachta indica) as Anti-Diabetic Agents.

PubMed

Ponnusamy, Sudha; Haldar, Saikat; Mulani, Fayaj; Zinjarde, Smita; Thulasiram, Hirekodathakallu; RaviKumar, Ameeta

2015-01-01

Human pancreatic α-amylase (HPA) inhibitors offer an effective strategy to lower postprandial hyperglycemia via control of starch breakdown. Limonoids from Azadirachta indica known for their therapeutic potential were screened for pancreatic α-amylase inhibition, a known anti-diabetic target. Studies were carried out to reveal their mode of action so as to justify their hypoglycemic potential. Of the nine limonoids isolated/semi-synthesized from A.indica and screened for α-amylase inhibition, azadiradione and exhibited potential inhibition with an IC50 value of 74.17 and 68.38 μM, respectively against HPA under in vitro conditions. Further screening on AR42J α-amylase secretory cell line for cytotoxicity and bioactivity revealed that azadiradione and gedunin exhibited cytotoxicity with IC50 of 11.1 and 13.4μM. Maximal secreted α-amylase inhibition of 41.8% and 53.4% was seen at 3.5 and 3.3μM, respectively. Michaelis-Menten kinetics suggested a mixed mode of inhibition with maltopentaose (Ki 42.2, 18.6 μM) and starch (Ki' 75.8, 37.4 μM) as substrate with a stiochiometry of 1:1 for both azadiradione and gedunin, respectively. The molecular docking simulation indicated plausible π-alkyl and alkyl-alkyl interactions between the aromatic amino acids and inhibitors. Fluorescence and CD confirmed the involvement of tryptophan and tyrosine in ligand binding to HPA. Thermodynamic parameters suggested that binding is enthalpically and entropically driven with ΔG° of -21.25 kJ mol-1 and -21.16 kJ mol-1 for azadiradione and gedunin, respectively. Thus, the limonoids azadiradione and gedunin could bind and inactivate HPA (anti-diabetic target) and may prove to be lead drug candidates to reduce/control post-prandial hyperglycemia.

The α-Amylase Induction in Endosperm during Rice Seed Germination Is Caused by Gibberellin Synthesized in Epithelium1

PubMed Central

Kaneko, Miyuki; Itoh, Hironori; Ueguchi-Tanaka, Miyako; Ashikari, Motoyuki; Matsuoka, Makoto

2002-01-01

We recently isolated two genes (OsGA3ox1 and OsGA3ox2) from rice (Oryza sativa) encoding 3β-hydroxylase, which catalyzes the final step of active gibberellin (GA) biosynthesis (H. Itoh, M. Ueguchi-Tanaka, N. Sentoku, H. Kitano, M. Matsuoka, M. Kobayashi [2001] Proc Natl Acad Sci USA 98: 8909–8914). Using these cloned cDNAs, we analyzed the temporal and spatial expression patterns of the 3β-hydroxylase genes and also an α-amylase gene (RAmy1A) during rice seed germination to investigate the relationship between GA biosynthesis and α-amylase expression. Northern-blot analyses revealed that RAmy1A expression in the embryo occurs before the induction of 3β-hydroxylase expression, whereas in the endosperm, a high level of RAmy1A expression occurs 1 to 2 d after the peak of OsGA3ox2 expression and only in the absence of uniconazol. Based on the analysis of an OsGA3ox2 null mutant (d18-Akibare dwarf), we determined that 3β-hydroxylase produced by OsGA3ox2 is important for the induction of RAmy1A expression and that the OsGA3ox1 product is not essential for α-amylase induction. The expression of OsGA3ox2 was localized to the shoot region and epithelium of the embryo, strongly suggesting that active GA biosynthesis occurs in these two regions. The synthesis of active GA in the epithelium is important for α-amylase expression in the endosperm, because an embryonic mutant defective in shoot formation, but which developed epithelium cells, induced α-amylase expression in the endosperm, whereas a mutant defective in epithelium development did not. PMID:11950975

Effect of pH on Cleavage of Glycogen by Vaginal Enzymes

PubMed Central

Spear, Greg T.; McKenna, Mary; Landay, Alan L.; Makinde, Hadijat; Hamaker, Bruce; French, Audrey L.; Lee, Byung-Hoo

2015-01-01

Glycogen expressed by the lower genital tract epithelium is believed to support Lactobacillus growth in vivo, although most genital isolates of Lactobacillus are not able to use glycogen as an energy source in vitro. We recently reported that α-amylase is present in the genital fluid of women and that it breaks down glycogen into small carbohydrates that support growth of lactobacilli. Since the pH of the lower genital tract can be very low, we determined how low pH affects glycogen processing by α-amylase. α-amylase in saliva degraded glycogen similarly at pH 6 and 7, but activity was reduced by 52% at pH 4. The glycogen degrading activity in nine genital samples from seven women showed a similar profile with an average reduction of more than 50% at pH 4. However, two samples collected from one woman at different times had a strikingly different pH profile with increased glycogen degradation at pH 4, 5 and 6 compared to pH 7. This second pH profile did not correlate with levels of human α-acid glucosidase or human intestinal maltase glucoamylase. High-performance anion-exchange chromatography showed that mostly maltose was produced from glycogen by samples with the second pH profile in contrast to genital α-amylase that yielded maltose, maltotriose and maltotetraose. These studies show that at low pH, α-amylase activity is reduced to low but detectable levels, which we speculate helps maintain Lactobacillus growth at a limited but sustained rate. Additionally, some women have a genital enzyme distinct from α-amylase with higher activity at low pH. Further studies are needed to determine the identity and distribution of this second enzyme, and whether its presence influences the makeup of genital microbiota. PMID:26171967

Effects of sorghum (Sorghum bicolor (L.) Moench) tannins on α-amylase activity and in vitro digestibility of starch in raw and processed flours.

PubMed

Mkandawire, Nyambe L; Kaufman, Rhett C; Bean, Scott R; Weller, Curtis L; Jackson, David S; Rose, Devin J

2013-05-08

The purpose of this study was to investigate the effects of tannins on starch digestion in tannin-containing sorghum extracts and wholegrain flours from 12 sorghum varieties. Extracts reduced amylase activity in a tannin concentration-dependent manner when the extract was mixed with the enzyme before substrate (amylopectin) addition, with higher molecular weight tannins showing greater reduction. Conversely, when the extract and substrate were combined before enzyme addition an enhancement in amylase activity was experienced. In uncooked, cooked, and cooked and stored wholegrain sorghum flours, rapidly digestible, slowly digestible, and resistant starches were not correlated with tannin content or molecular weight distribution. Resistant starch increased from 6.5% to 22-26% when tannins were added to starch up to 50% (starch weight). Tannin extracts both reduced and enhanced amylase activity depending on conditions, and, while these trends were clear in extracts, the effects on starch digestion in wholegrain flours was more complex.

2-Aryl benzimidazoles: Synthesis, In vitro α-amylase inhibitory activity, and molecular docking study.

PubMed

Adegboye, Akande Akinsola; Khan, Khalid Mohammed; Salar, Uzma; Aboaba, Sherifat Adeyinka; Kanwal; Chigurupati, Sridevi; Fatima, Itrat; Taha, Mohammad; Wadood, Abdul; Mohammad, Jahidul Isalm; Khan, Huma; Perveen, Shahnaz

2018-04-25

Despite of many diverse biological activities exhibited by benzimidazole scaffold, it is rarely explored for the α-amylase inhibitory activity. For that purpose, 2-aryl benzimidazole derivatives 1-45 were synthesized and screened for in vitro α-amylase inhibitory activity. Structures of all synthetic compounds were deduced by various spectroscopic techniques. All compounds revealed inhibition potential with IC 50 values of 1.48 ± 0.38-2.99 ± 0.14 μM, when compared to the standard acarbose (IC 50  = 1.46 ± 0.26 μM). Limited SAR suggested that the variation in the inhibitory activities of the compounds are the result of different substitutions on aryl ring. In order to rationalize the binding interactions of most active compounds with the active site of α-amylase enzyme, in silico study was conducted. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Impact of amylases on biopolymer dynamics during storage of straight-dough wheat bread.

PubMed

Bosmans, Geertrui M; Lagrain, Bert; Fierens, Ellen; Delcour, Jan A

2013-07-03

When Bacillus stearothermophilus α-amylase (BStA), Pseudomonas saccharophila α-amylase (PSA), or Bacillus subtilis α-amylase (BSuA) was added to a bread recipe to impact bread firming, amylose crystal formation was facilitated, leading to lower initial crumb resilience. Bread loaves that best retained their quality were those obtained when BStA was used. The enzyme hindered formation of an extended starch network, resulting in less water immobilization and smaller changes in crumb firmness and resilience. BSuA led to extensive degradation of the starch network during bread storage with release of immobilized water, eventually resulting in partial structure collapse and poor crumb resilience. The most important effect of PSA was an increased bread volume, resulting in smaller changes in crumb firmness and resilience. A negative linear relation was found between NMR proton mobilities of water and biopolymers in the crumb and crumb firmness. The slope of that relation gave an indication of the strength of the starch network.

Purification and characterization of an alpha-amylase of Pichia burtonii isolated from the traditional starter "murcha" in Nepal.

PubMed

Takeuchi, Akiko; Shimizu-Ibuka, Akiko; Nishiyama, Yoshitaka; Mura, Kiyoshi; Okada, Sanae; Tokue, Chiyoko; Arai, Soichi

2006-12-01

Among more than 20 yeast strains isolated from the traditional starter "murcha" in Nepal, we characterized a yeast that might be involved in saccharification. This strain, identified as Pichia burtonii, produced an extracellular amylolytic enzyme when cultured in the presence of starch in the medium. Since no amylase secreted by P. burtonii has yet been reported, we purified the enzyme and determined its N-terminal amino acid sequence. Together with the results of a hydrolyzing activity assay toward various substrates, it was found to be an alpha-amylase. The purified enzyme, named Pichia burtonii alpha-amylase (PBA), was a glycoprotein with an apparent molecular mass of 51 kDa. Enzyme activity was optimal at pH 5.0 at 40 degrees C. The enzyme retained 80% of its original activity after incubation under the optimal pH condition at 50 degrees C for 30 min. The activity was inhibited by metal ions such as Cd(2+), Cu(2+), Hg(2+), Al(3+), and Zn(2+).

Function of the parotid gland in juvenile recurrent parotitis: a case series.

PubMed

Xie, Li-song; Pu, Yi-ping; Zheng, Ling-yan; Yu, Chuang-qi; Wang, Zhi-jun; Shi, Huan

2016-04-01

Our aim was to find out how the parotid gland functions in 44 patients with juvenile recurrent parotitis, and to assess the value of measuring the serum amylase activity. Clinical and personal details were recorded, and all patients had their serum amylase activity measured together with sialography during the chronic phase. The function of the gland was classified by sialographic images. The chi square test and Spearman's rank correlation coefficient were used in the statistical analyses. There was a significant association between the degree of glandular function and serum amylase activity (p=0.014). The patients with unilateral and bilateral disease differed significantly in their degree of glandular function (p=0.020), those with bilateral disease having poorer function. There were no significant correlations between other clinical variables and glandular function. Serum amylase activity is an important diagnostic variable in juvenile recurrent parotitis, and poor parotid function reflects the severity of the disease. Copyright © 2016 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Sociodemographic Risk, Parenting, and Effortful Control: Relations to Salivary Alpha-amylase and Cortisol in Early Childhood

PubMed Central

Taylor, Zoe E.; Spinrad, Tracy L.; VanSchyndel, Sarah K.; Eisenberg, Nancy; Huynh, Jacqueline; Sulik, Michael J.; Granger, Douglas A.

2012-01-01

Early sociodemographic risk, parenting, and temperament were examined as predictors of the activity of children’s (N = 148; 81 boys, 67 girls) hypothalamic-pituitary-adrenal axis and autonomic nervous system. Demographic risk was assessed at 18 months (T1), intrusive-overcontrolling parenting and effortful control were assessed at 30 months (T2), and salivary cortisol and alpha-amylase were collected at 72 (T3) months of age. Demographic risk at T1 predicted lower levels of children’s effortful control and higher levels of mothers’ intrusive-overcontrolling parenting at T2. Intrusive-overcontrolling parenting at T2 predicted higher levels of children’s cortisol and alpha-amylase at T3, but effortful control did not uniquely predict children’s cortisol or alpha-amylase. Findings support the open nature of stress responsive physiological systems to influence by features of the early caregiving environment and underscore the utility of including measures of these systems in prevention trials designed to influence child outcomes by modifying parenting behavior. PMID:22949301

Characteristic hydrolyzing of megalosaccharide by human salivary α-amylase and small intestinal enzymes, and its bioavailability in healthy subjects.

PubMed

Nakamura, Sadako; Takami, Masayuki; Tanabe, Kenichi; Oku, Tsuneyuki

2014-09-01

The digestibility of Megalosaccharide® (newly developed carbohydrate comprising α-1,4-glucosaccharide) was investigated in vitro and in vivo. Isomaltosyl-megalosaccharide® (IMS) and nigerosyl-megalosaccharide® (NMS) contain 20% and 50% of the megalosaccharide fraction (degree of polymerization (DP) 10-35), respectively. IMS was hydrolyzed readily by α-amylase to oligosaccharides (DP ≤ 7), and a small amount of glucose was produced from oligosaccharides by small intestinal enzymes (SIEs). NMS was partially hydrolyzed by α-amylase to oligosaccharides, and a small amount of glucose produced by SIEs. When IMS and NMS were treated by SIEs after treatment with human saliva α-amylase for a few minutes, IMS and NMS were hydrolyzed readily to glucose. Plasma levels of glucose and insulin upon ingestion of 50 g of IMS or NMS were elevated the same as those for 50 g of glucose, and breath hydrogen was not excreted. These results suggest that IMS and NMS are digestible carbohydrates.

The important role of salivary α-amylase in the gastric digestion of wheat bread starch.

PubMed

Freitas, Daniela; Le Feunteun, Steven; Panouillé, Maud; Souchon, Isabelle

2018-01-24

The role of salivary α-amylase (HSA) in starch digestion is often overlooked in favour of that of pancreatic α-amylase due to the short duration of the oral phase. Although it is generally accepted that the amylase of salivary origin can continue to be active in the stomach, studies ascertaining its contribution are lacking. This study aimed to address this issue by coupling in vitro oral processing with an in vitro dynamic system that mimicked different postprandial gastric pH reduction kinetics observed in vivo following a snack- or lunch-type meal. The digestion of both starch and protein from wheat bread as well as the interplay between the two processes were studied. We have observed that the amylolytic activity of saliva plays a preponderant role hydrolysing up to 80% of bread starch in the first 30 min of gastric digestion. Amylolysis evolved exponentially and nearly superimposing curves were obtained regardless of the acidification profiles, revealing its high efficiency.

A novel raw starch digesting alpha-amylase from a newly isolated Bacillus sp. YX-1: purification and characterization.

PubMed

Liu, Xu Dong; Xu, Yan

2008-07-01

This study reports the purification and characterization of a novel raw starch digesting alpha-amylase from a newly isolated Bacillus sp. YX-1. Maximum alpha-amylase activity (53 U mL(-1)) was obtained at 45 degrees C after 44 h of incubation. The enzyme was purified using ammonium sulfate precipitation, ion exchange and gel filtration chromatography, and showed a molecular weight of 56 kDa by SDS-PAGE. This enzyme exhibited maximum activity at pH 5.0, performed stability over a broad range of pH 4.5-11.0, and was optimally active at 40-50 degrees C. The enzyme preparation had a strong digesting ability towards various raw starches and efficiently hydrolyzed raw corn starch at a concentration of 20% and pH 5.0, which were normally used in the starch industries, in a period of 12h. By analyzing its partial amino acid sequences, the enzyme was proposed to be a novel alpha-amylase.

[The primary structure of the alpha-amylase inhibitor Hoe 467A from Streptomyces tendae 4158. A new class of inhibitors].

PubMed

Aschauer, H; Vértesy, L; Nesemann, G; Braunitzer, G

1983-10-01

The native or modified alpha-amylase inhibitor Hoe 467A - isolated from the culture medium of Streptomyces tendae 4158 - and overlapping peptides were degraded by the automatic Edman technique. The oxidized or aminoethylated or oxidized and maleoylated inhibitor was digested with trypsin and the native inhibitor with pepsin. Further digestion with Staphylococcus aureus proteinase was also carried out. After peptic digestion two cystin peptides were isolated, which allowed the establishment of the disulfide bonds. The alpha-amylase inhibitor is a polypeptid consisting of 74 amino-acid residues with a molecular mass of 7958 Da. The inhibitor is composed of all naturally occurring amino acids except methionine and phenylalanine and shows no sequence homology to known inhibitors. The clinical and pharmacological importance in respect to the inhibitors ability for inactivation of human salivary and pancreatic alpha-amylase is discussed. Especially the proteinase resistance of the inhibitor enables a clinical application in human (e.g. Diabetes mellitus) per os.

FT-Raman spectroscopic analysis of enhanced activity of supercritical carbon dioxide treated bacterial alpha-amylase.

PubMed

Paul, Kaninika; Dutta, Sayantani; Bhattacharjee, Paramita

2017-09-01

Our previous investigation on high pressure supercritical carbon dioxide treatment of a bacterial α-amylase had revealed enhanced activity of the same. 1 H NMR analysis of the activity enhanced enzyme led the authors to hypothesize that the enhancement was possibly owing to alterations in the active site of the enzyme. In the present study, the changes in the active site of the treated enzyme was analysed by Fourier-transform Raman (FT-Raman) spectroscopy. The spectra obtained revealed shifting of bands in the active site of α-amylase indicating a nudging effect of the bonds in this region consequent to high pressure treatment. Also, shifts in bands in the OH stretching vibration of water were observed in the enzyme spectra. These variations in the spectra confirmed changes in the active site as well as in the water associated with the same that perhaps had a concerted effect on the increased activity of α-amylase. Copyright © 2017 Elsevier Inc. All rights reserved.

ANTIOXIDANT ACTIVITY AND A-AMYLASE INHIBITORY POTENTIAL OF ROSA CANINA L.

PubMed

Jemaa, Houda Ben; Jemia, Amani Ben; Khlifi, Sarra; Ahmed, Halima Ben; Slama, Fethi Ben; Benzarti, Anis; Elati, Jalila; Aouidet, Abdallah

2017-01-01

Diabetes mellitus is one of the most common endocrinal disorders and medicinal plants continue to play an important role in the management of this disease. In this study, Rosa canina was investigated for the antioxidant and α-amylase inhibition activities. Methanolic extract of Rosa canina was investigated for its potential antioxidant activity. The extracts' total phenolic and flavonoid contents and scavenging capacity for free radicals were evaluated. The α-amylase inhibition assay was also carried. Rosa canina extract exhibits a total Phenolic and flavonoid levels respectively (21.918 mg GAE/g and 2.647mg ER/g). The free radical scavenging activity was found to be prominent against DPPH with an IC50 of 0.668 mg/ml and against ABTS with an IC50 of 0.467 mg/ml. Extract showed a significant ferric ion reducing activities with an IC50 of4.962 mg/ml. Rosa canina exerted a higher inhibitory activity against α-amylase. The obtained results support the antidiabetic use of rosa canina .

Kinetic study of the thermal denaturation of a hyperthermostable extracellular α-amylase from Pyrococcus furiosus.

PubMed

Brown, I; Dafforn, T R; Fryer, P J; Cox, P W

2013-12-01

Hyperthermophilic enzymes are of industrial importance and interest, especially due to their denaturation kinetics at commercial sterilisation temperatures inside safety indicating time-temperature integrators (TTIs). The thermal stability and irreversible thermal inactivation of native extracellular Pyrococcus furiosus α-amylase were investigated using differential scanning calorimetry, circular dichroism and Fourier transform infrared spectroscopy. Denaturation of the amylase was irreversible above a Tm of approximately 106°C and could be described by a one-step irreversible model. The activation energy at 121°C was found to be 316kJ/mol. Using CD and FT-IR spectroscopy it was shown that folding and stability greatly increase with temperature. Under an isothermal holding temperature of 121°C, the structure of the PFA changes during denaturation from an α-helical structure, through a β-sheet structure to an aggregated protein. Such data reinforces the use of P. furiosus α-amylase as a labile species in TTIs. © 2013.

Genetic, Hormonal, and Physiological Analysis of Late Maturity α-Amylase in Wheat1[W][OA

PubMed Central

Barrero, Jose M.; Mrva, Kolumbina; Talbot, Mark J.; White, Rosemary G.; Taylor, Jennifer; Gubler, Frank; Mares, Daryl J.

2013-01-01

Late maturity α-amylase (LMA) is a genetic defect that is commonly found in bread wheat (Triticum aestivum) cultivars and can result in commercially unacceptably high levels of α-amylase in harvest-ripe grain in the absence of rain or preharvest sprouting. This defect represents a serious problem for wheat farmers, and apart from the circumstantial evidence that gibberellins are somehow involved in the expression of LMA, the mechanisms or genes underlying LMA are unknown. In this work, we use a doubled haploid population segregating for constitutive LMA to physiologically analyze the appearance of LMA during grain development and to profile the transcriptomic and hormonal changes associated with this phenomenon. Our results show that LMA is a consequence of a very narrow and transitory peak of expression of genes encoding high-isoelectric point α-amylase during grain development and that the LMA phenotype seems to be a partial or incomplete gibberellin response emerging from a strongly altered hormonal environment. PMID:23321420

Protoplast transformation in coryneform bacteria and introduction of an alpha-amylase gene from Bacillus amyloliquefaciens into Brevibacterium lactofermentum.

PubMed Central

Smith, M D; Flickinger, J L; Lineberger, D W; Schmidt, B

1986-01-01

The goal of this study was to investigate the likelihood of developing useful transformation systems for coryneform bacteria. Two species of coryneform bacteria, Brevibacterium lactofermentum and Corynebacterium lilium, were transformed with chimeras constructed from pUB110 and a cryptic coryneform plasmid (pGX1901). C. lilium protoplasts were also efficiently transfected with phage CS1 DNA. High transformation and transfection frequencies were obtained after only 2 min of lysozyme treatment of lysozyme-sensitive mutants. A series of experiments was also conducted to determine whether DNA from other species of important industrial microbes from the genus Bacillus could be expressed in coryneform bacteria. Evidence of restriction of Bacillus subtilis DNA by B. lactofermentum was observed but could be overcome. A Bacillus amyloliquefaciens alpha-amylase gene (amyEBamP) was subcloned onto a plasmid able to replicate in B. lactofermentum. B. lactofermentum transformants for this plasmid expressed amylase activity and produced material cross-reactive to amylase antibody. Images PMID:3008649

Drain Management after Pancreatoduodenectomy: Reappraisal of a Prospective Randomized Trial Using Risk Stratification.

PubMed

McMillan, Matthew T; Malleo, Giuseppe; Bassi, Claudio; Butturini, Giovanni; Salvia, Roberto; Roses, Robert E; Lee, Major K; Fraker, Douglas L; Drebin, Jeffrey A; Vollmer, Charles M

2015-10-01

A recent randomized trial used the Fistula Risk Score (FRS) to develop guidelines for selective drainage based on clinically relevant fistula (CR-POPF) risk. Additionally, postoperative day (POD) 1 drain and serum amylase have been identified as accurate postoperative predictors of CR-POPF. This study sought to identify patients who may benefit from selective drainage, as well as the optimal timing for drain removal after pancreatoduodenectomy. One hundred six pancreatoduodenectomies from a previously reported RCT were assessed using risk-adjustment. The incidence of CR-POPF was compared between FRS risk cohorts. Drain and serum amylase values from POD 1 were evaluated using receiver operating characteristic (ROC) analysis to establish cut-offs predictive of CR-POPF occurrence. A regression analysis compared drain removal randomizations (POD 3 vs POD 5). Three-quarters of patients had moderate/high CR-POPF risk. This group had a CR-POPF rate of 36.3% vs 7.7% among negligible/low risk patients (p = 0.005). The areas under the ROC curve for CR-POPF prediction using POD 1 drain and serum amylase values were 0.800 (p = 0.000001; 95% CI 0.70-0.90) and 0.655 (p = 0.012; 95% CI 0.55-0.77), respectively. No significant serum amylase cut-offs were identified. Moderate/high risk patients with POD 1 drain amylase ≤ 5,000 U/L had significantly lower rates of CR-POPF when randomized to POD 3 drain removal (4.2% vs 38.5%; p = 0.003); moreover, these patients experienced fewer complications and shorter hospital stays. A clinical care protocol is proposed whereby drains are recommended for moderate/high FRS risk patients, but may be omitted in patients with negligible/low risk. Drain amylase values in moderate/high risk patients should then be evaluated on POD 1 to determine the optimal timing for drain removal. Moderate/high risk patients with POD 1 drain amylase ≤ 5,000 U/L have lower rates of CR-POPF with POD 3 (vs POD ≥ 5) drain removal; early drain removal is recommended for these patients. Copyright © 2015 American College of Surgeons. Published by Elsevier Inc. All rights reserved.

Energy and nutrient intake increased by 47–67% when amylase was added to fortified blended foods—a study among 12‐ to 35‐month‐old Burkinabe children

PubMed Central

Kampstra, Nynke A.; Van Hoan, Nguyen; Koenders, Damiet J.P.C.; Schoop, Rotraut; Broersen, Britt C.; Mouquet‐Rivier, Claire; Traoré, Tahirou; Bruins, Maaike J.

2017-01-01

Abstract Adding amylase to fortified blended foods can improve energy density, and increase child's energy and nutrient intake. The efficacy of this strategy is unknown for the World Food Programme's Super Cereal Plus (SC+) and Super Cereal (SC) blends. The primary goal of this study was to investigate the increased energy intake from amylase‐containing SC+ and SC compared to control porridges in Burkinabe children. Secondly, energy intake from amylase‐containing porridges compared to CERELAC®, Vitazom, and eeZeeBAR™ was studied. Thirdly, caregivers' (n = 100) porridge acceptability was investigated. The design was a randomized double‐blind controlled cross‐over trial studying the effect of amylase addition to SC+ and SC flours on porridge energy and nutrient intake in healthy Burkinabe children aged 12–23 (n = 80) and 24–35 months (n = 40). Amylase added to porridges increased energy density from 0.68 to 1.16 kcal/g for SC+ and from 0.66 to 1.03 kcal/g for SC porridges. Among children aged 12–23 months, mean energy intake from all porridges with amylase (135–164 kcal/meal) was significantly higher compared to control SC+ porridges (84–98 kcal/meal; model‐based average). Among children aged 24–35 months, mean energy intakes were also significantly higher from all porridges with amylase added (245–288 kcal/meal) compared to control SC porridges (175–183 kcal/meal). Acceptability of the porridges among caregivers was rated neutral to good, both for amylase‐added and non‐amylase‐containing porridges. These findings suggest that, among 12–35‐month‐old, adding amylase to fortified blended foods significantly increased energy and consequently nutrient intake per meal by 67% for SC+ and 47% for SC. Moreover, amylase‐containing porridges were well accepted by the caregivers. PMID:28466569

The spectrum of low molecular weight alpha-amylase/protease inhibitor genes expressed in the US bread wheat cultivar Butte 86

PubMed Central

2011-01-01

Background Wheat grains accumulate a variety of low molecular weight proteins that are inhibitors of alpha-amylases and proteases and play an important protective role in the grain. These proteins have more balanced amino acid compositions than the major wheat gluten proteins and contribute important reserves for both seedling growth and human nutrition. The alpha-amylase/protease inhibitors also are of interest because they cause IgE-mediated occupational and food allergies and thereby impact human health. Results The complement of genes encoding alpha-amylase/protease inhibitors expressed in the US bread wheat Butte 86 was characterized by analysis of expressed sequence tags (ESTs). Coding sequences for 19 distinct proteins were identified. These included two monomeric (WMAI), four dimeric (WDAI), and six tetrameric (WTAI) inhibitors of exogenous alpha-amylases, two inhibitors of endogenous alpha-amylases (WASI), four putative trypsin inhibitors (CMx and WTI), and one putative chymotrypsin inhibitor (WCI). A number of the encoded proteins were identical or very similar to proteins in the NCBI database. Sequences not reported previously included variants of WTAI-CM3, three CMx inhibitors and WTI. Within the WDAI group, two different genes encoded the same mature protein. Based on numbers of ESTs, transcripts for WTAI-CM3 Bu-1, WMAI Bu-1 and WTAI-CM16 Bu-1 were most abundant in Butte 86 developing grain. Coding sequences for 16 of the inhibitors were unequivocally associated with specific proteins identified by tandem mass spectrometry (MS/MS) in a previous proteomic analysis of milled white flour from Butte 86. Proteins corresponding to WDAI Bu-1/Bu-2, WMAI Bu-1 and the WTAI subunits CM2 Bu-1, CM3 Bu-1 and CM16 Bu-1 were accumulated to the highest levels in flour. Conclusions Information on the spectrum of alpha-amylase/protease inhibitor genes and proteins expressed in a single wheat cultivar is central to understanding the importance of these proteins in both plant defense mechanisms and human allergies and facilitates both breeding and biotechnology approaches for manipulating the composition of these proteins in plants. PMID:21774824

Isolation, purification and characterization of β-amylase from Dioscorea hispida Dennst

NASA Astrophysics Data System (ADS)

Oktiarni, Dwita; Lusiana, Simamora, Febri Yanti; Gaol, Jusni M. Lumban

2015-09-01

β-amylase (E.C 3.2.1.2) is an enzyme commonly found in plants and bacteria. The enzyme is an exo-acting carbohydrolase which hydrolyzes α-1.4-glucosidic linkages of starch, removing maltose units from the non-reducing end of the polysaccharide chain, producing β-maltose and β-limit dextrin as the final product. β-amylase is widely distributed in the higher plants such as sweet potato. Besides the use in starch hydrolysis, starch-converting enzymes are also used in a number of other industrial applications, such as laundry and porcelain detergents or as anti-stalling agents in baking. This enzyme was extracted from Dioscorea hispida Dennst in 0.05 M acetate buffer pH 4.8 and followed by ammonium sulfate fractionation at cold temperature (10°C). Ammonium sulfate fractionation was shared into fraction of 0-60%, 60-70%, 70-80% and 80-100%. The fraction containing high of specific activity (determined by Somogyi-Nelson and Lowry methods) was futher purified by dialysis. Fraction with high enzyme activity of β-amylase were fraction 60-70% and 70-80%, with specific activity of Dioscorea hispida Dennst were 1.32 and 1.55 mg sugar.mg protein-1.minute-1, whereas specific activity of crude extract enzyme was 0.21 mg sugar.mg protein-1.minute-1. After purified with dialysis, fraction with high enzyme activity of β-amylase were fraction of 60-70% and 70-80%, with specific activity of Dioscorea hispida Dennst was 2.72 and 4.24 mg sugar.mg protein-1.minute-1. The purified Dioscorea hispida Dennst β-amylase from dialysis showed increasing in spesific activity the crude enzyme as much as 24 folds. The characterization of enzyme showed that Dioscorea hispida Dennst derived enzyme had optimum pH of 5.5 and temperature of 70°C. The kinetic parameters of purified Dioscorea hispida Dennst β-amylase showed that the KMapp, Vmaxapp value and Hill constant were 0.0211 mg/ml, 9.63 mg sugar.minute-1 and 1.34, respectively.

Study of fluorescence quenching of Barley α-amylase

NASA Astrophysics Data System (ADS)

Bakkialakshmi, S.; Shanthi, B.; Bhuvanapriya, T.

2012-05-01

The fluorescence quenching of Barley α-amylase by acrylamide and succinimide has been studied in water using steady-state and time-resolved fluorescence techniques. The steady-state fluorescence quenching technique has been performed in three different pHs (i.e., 6, 7 and 8) of water. Ground state and excited state binding constants (Kg &Ke) have been calculated. From the calculated binding constants (Kg &Ke) the free energy changes for the ground (ΔGg) and excited (ΔGe) states have been calculated and are presented in tables. UV and FTIR spectra have also been recorded to prove the binding of Barley α-amylase with acrylamide and succinimide.

Componential profile and amylase inhibiting activity of phenolic compounds from Calendula officinalis L. leaves.

PubMed

Olennikov, Daniil N; Kashchenko, Nina I

2014-01-01

An ethanolic extract and its ethyl acetate-soluble fraction from leaves of Calendula officinalis L. (Asteraceae) were found to show an inhibitory effect on amylase. From the crude extract fractions, one new phenolic acid glucoside, 6'-O-vanilloyl-β-D-glucopyranose, was isolated, together with twenty-four known compounds including five phenolic acid glucosides, five phenylpropanoids, five coumarins, and nine flavonoids. Their structures were elucidated based on chemical and spectral data. The main components, isoquercitrin, isorhamnetin-3-O-β-D-glucopyranoside, 3,5-di-O-caffeoylquinic acid, and quercetin-3-O-(6''-acetyl)-β-D-glucopyranoside, exhibited potent inhibitory effects on amylase.

Local entropy difference upon a substrate binding of a psychrophilic α-amylase and a mesophilic homologue

NASA Astrophysics Data System (ADS)

Kosugi, Takahiro; Hayashi, Shigehiko

2011-01-01

Psychrophilic α-amylase from the antarctic bacterium pseudoalteromonashaloplanktis (AHA) and its mesophilic homologue, porcine pancreatic α-amylase (PPA) are theoretically investigated with molecular dynamics (MD) simulations. We carried out 240-ns MD simulations for four systems, AHA and PPA with/without the bound substrate, and examined protein conformational entropy changes upon the substrate binding. We developed an analysis that decomposes the entropy changes into contributions of individual amino acids, and successfully identified protein regions responsible for the entropy changes. The results provide a molecular insight into the structural flexibilities of those enzymes related to the temperature dependences of the enzymatic activity.

Determination of the effects of initial glucose on the production of α-amylase from Penicillium sp. under solid-state and submerged fermentation.

PubMed

Ertan İnceoğlu, Figen; Balkan, Bilal; Yarkın, Zehra

2014-01-02

The effects of catabolite repression of initial glucose on the synthesis of α-amylase from Penicillium chrysogenum and Penicillium griseofulvum were investigated under solid-state fermentation (SSF) and submerged fermentation (SmF) systems. The results obtained from either fermentation were compared with each other. In the SmF system, initial glucose concentration above 10 mg/mL completely repressed the production of α-amylase from P. chrysogenum and P . griseofulvum . However, the repression in the SSF system was not complete, even when the glucose level was raised to 160 mg/g.

Cloning and characterization of a novel α-amylase from a fecal microbial metagenome.

PubMed

Xu, Bo; Yang, Fuya; Xiong, Caiyun; Li, Junjun; Tang, Xianghua; Zhou, Junpei; Xie, Zhenrong; Ding, Junmei; Yang, Yunjuan; Huang, Zunxi

2014-04-01

To isolate novel and useful microbial enzymes from uncultured gastrointestinal microorganisms, a fecal microbial metagenomic library of the pygmy loris was constructed. The library was screened for amylolytic activity, and 8 of 50,000 recombinant clones showed amylolytic activity. Subcloning and sequence analysis of a positive clone led to the identification a novel gene (amyPL) coding for α-amylase. AmyPL was expressed in Escherichia coli BL21 (DE3) and the purified AmyPL was enzymatically characterized. This study is the first to report the molecular and biochemical characterization of a novel α-amylase from a gastrointestinal metagenomic library.

Abdominal pain and hyperamylasaemia—not always pancreatitis

PubMed Central

Slack, Sally; Abbey, Ianthe; Smith, Dominic

2010-01-01

A raised serum amylase concentration, at least four times the upper limit of normal (ULN), is used to support the diagnosis of acute pancreatitis in a patient presenting with abdominal pain. The authors report a case of toxic shock syndrome complicated by a raised serum amylase concentration that peaked at 50 times the ULN in a patient with recurrent abdominal pain. The commonest cause of hyperamylasaemia is pancreatic; however, further investigation of serum lipase and amylase isoenzyme studies found this to be of salivary origin and attributable to soft tissue inflammation of the salivary gland. This case highlights the need to consider non-pancreatic causes of hyperamylasaemia. PMID:22767564

Abdominal pain and hyperamylasaemia--not always pancreatitis.

PubMed

Slack, Sally; Abbey, Ianthe; Smith, Dominic

2010-07-21

A raised serum amylase concentration, at least four times the upper limit of normal (ULN), is used to support the diagnosis of acute pancreatitis in a patient presenting with abdominal pain. The authors report a case of toxic shock syndrome complicated by a raised serum amylase concentration that peaked at 50 times the ULN in a patient with recurrent abdominal pain. The commonest cause of hyperamylasaemia is pancreatic; however, further investigation of serum lipase and amylase isoenzyme studies found this to be of salivary origin and attributable to soft tissue inflammation of the salivary gland. This case highlights the need to consider non-pancreatic causes of hyperamylasaemia.

Raw Starch Degrading Amylase Production by Various Fungal Cultures Grown on Cassava Waste

PubMed Central

Balaji, P.; Eyini, M.

2006-01-01

The solid waste of sago industry using cassava was fermented by Aspergillus niger, Aspergillus terreus and Rhizopus stolonifer in solid state fermentation. Cassava waste contained 52 per cent starch and 2.9 per cent protein by dry weight. The amylase activity was maintained at a high level and the highest amylase activity was observed on the 8th day in R. stolonifer mediated fermentation. R. stolonifer was more efficient than Aspergillus niger and Aspergillus terreus in bioconverting cassava waste into fungal protein (90.24 mg/g) by saccharifying 70% starch and releasing 44.5% reducing sugars in eight days of solid state fermentation. PMID:24039485

21 CFR 862.1070 - Amylase test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Amylase test system. 862.1070 Section 862.1070 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862...

21 CFR 862.1070 - Amylase test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Amylase test system. 862.1070 Section 862.1070 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862...

21 CFR 862.1070 - Amylase test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Amylase test system. 862.1070 Section 862.1070 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862...

21 CFR 862.1070 - Amylase test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Amylase test system. 862.1070 Section 862.1070 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862...

Relatedness of amylase-producing, endospore-forming bacteria from the alimentary tract of commercially processed broilers

USDA-ARS?s Scientific Manuscript database

Introduction: Competitive exclusion (CE) by bacteria from adult poultry reduces colonization of young chicks by Salmonella. CE might include the ability of these bacteria to breakdown complex carbohydrates to produce metabolites that inhibit Salmonella growth. Purpose: To isolate amylase producing, ...

21 CFR 862.1070 - Amylase test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Amylase test system. 862.1070 Section 862.1070 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862...

The effect of alpha amylase enzyme on quality of sweet sorghum juice for chrystal sugar

NASA Astrophysics Data System (ADS)

Marwati, T.; Cahyaningrum, N.; Widodo, S.; Astiati, U. T.; Budiyanto, A.; Wahyudiono; Arif, A. B.; Richana, N.

2018-01-01

Sweet sorghum juice (Sorghum bicolor L. Moench) has characteristics similar to sugar cane juice and potentially used for sugar substitutes that can support food security. Nevertheless the sweet sorghum juicecontain starch which impede sorghum sugar crystallization. Therefore, research on the enzymatic process is needed to convert starch into reducing sugar. The experimental design used was the Factorial Randomized Design with the first factor was alpha amylase enzyme concentration (0, 20, 40, 60, 80, 100, 120 μL/100 mL) and second factor was incubation time (0, 30, 60, 90 minute) at temperature 100°C. The experiment was conducted on fresh sweet sorghum. The results showed that the addition of the alpha amylase enzyme increased the content of reducing sugar and decreased levels of starch. Elevating concentration of alpha amylase enzyme will increase the reducing sugar content in sweet sorghum juice. The optimum alpha amylase enzyme concentration to produce the highest total sugar was 80 μL/100 mL of sweet sorghum juice with the optimum incubation time was 90 minutes. The results of this study are expected to create a new sweetener for sugar substitution. From the economic prospective aspect, sorghum is a potential crop and can be relied upon to support the success of the food diversification program which further leads to the world food security

Inhibitory Activities of Cyanidin and Its Glycosides and Synergistic Effect with Acarbose against Intestinal α-Glucosidase and Pancreatic α-Amylase

PubMed Central

Akkarachiyasit, Sarinya; Charoenlertkul, Piyawan; Yibchok-anun, Sirintorn; Adisakwattana, Sirichai

2010-01-01

Cyanidin and its glycosides are naturally dietary pigments which have been indicated as promising candidates to have potential benefits to humans, especially in the prevention and treatment of diabetes mellitus. We investigated the structure activity relationships of cyanidin and its glycosides to inhibit intestinal α-glucosidases and pancreatic α-amylase in vitro. The results found that cyanidin and its glycosides are more specific inhibitors of intestinal sucrase than intestinal maltase. Cyanidin-3-galactoside and cyanidin-3-glucoside were the most potent inhibitors against intestinal sucrase and pancreatic α-amylase with IC50 values of 0.50 ± 0.05 and 0.30 ± 0.01 mM, respectively. Our findings indicate that the structural difference between glucose and galactose at the 3-O-position of cyanidin was an important factor for modulating the inhibition of intestinal sucrase and pancreatic α-amylase. The combination of cyandin-3-glucoside, cyanidin-3- galactoside or cyanidin-3,5-diglucosides with a low concentration of acarbose showed synergistic inhibition on intestinal maltase and sucrase. The synergistic inhibition was also found for a combination of cyanidin or cyanidin-3-glucoside with a low concentration of acarbose. The findings could provide a new insight into a use for the naturally occurring intestinal α-glucosidase and pancreatic α-amylase inhibitors for the prevention and treatment of diabetes and its complications. PMID:20957102

Higher levels of salivary alpha-amylase predict failure of cessation efforts in male smokers.

PubMed

Dušková, M; Simůnková, K; Hill, M; Hruškovičová, H; Hoskovcová, P; Králíková, E; Stárka, L

2010-01-01

The ability to predict the success or failure of smoking cessation efforts will be useful for clinical practice. Stress response is regulated by two primary neuroendocrine systems. Salivary cortisol has been used as a marker for the hypothalamus-pituitary-adrenocortical axis and salivary alpha-amylase as a marker for the sympathetic adrenomedullary system. We studied 62 chronic smokers (34 women and 28 men with an average age of 45.2+/-12.9 years). The levels of salivary cortisol and salivary alpha-amylase were measured during the period of active smoking, and 6 weeks and 24 weeks after quitting. We analyzed the men separately from the women. The men who were unsuccessful in cessation showed significantly higher levels of salivary alpha-amylase over the entire course of the cessation attempt. Before stopping smoking, salivary cortisol levels were higher among the men who were unsuccessful in smoking cessation. After quitting, there were no differences between this group and the men who were successful in cessation. In women we found no differences between groups of successful and unsuccessful ex-smokers during cessation. In conclusions, increased levels of salivary alpha-amylase before and during smoking cessation may predict failure to quit in men. On the other hand, no advantage was found in predicting the failure to quit in women. The results of our study support previously described gender differences in smoking cessation.

Serum Amylase in Bulimia Nervosa and Purging Disorder: Differentiating the Association with Binge Eating versus Purging Behavior

PubMed Central

Wolfe, Barbara E.; Jimerson, David C.; Smith, Adrian; Keel, Pamela K.

2011-01-01

Objective Elevated serum amylase levels in bulimia nervosa (BN), associated with increased salivary gland size and self-induced vomiting in some patients, provide a possible marker of symptom severity. The goal of this study was to assess whether serum hyperamylasemia in BN is more closely associated with binge eating episodes involving consumption of large amounts of food or with purging behavior. Method Participants included women with BN (n=26); women with “purging disorder” (PD), a subtype of EDNOS characterized by recurrent purging in the absence of objectively large binge eating episodes (n=14); and healthy non-eating disorder female controls (n=32). There were no significant differences in age or body mass index (BMI) across groups. The clinical groups reported similar frequency of self-induced vomiting behavior and were free of psychotropic medications. Serum samples were obtained after overnight fast and were assayed for alpha-amylase by enzymatic method. Results Serum amylase levels were significantly elevated in BN (60.7 ± 25.4 international units [IU]/liter, mean ± sd) in comparison to PD (44.7 ± 17.1 IU/L, p < 02) and to Controls (49.3 ± 15.8, p < .05). Conclusion These findings provide evidence to suggest that it is recurrent binge eating involving large amounts of food, rather than self-induced vomiting, which contributes to elevated serum amylase values in BN. PMID:21781981

Physical structure and absorption properties of tailor-made porous starch granules produced by selected amylolytic enzymes

PubMed Central

Jung, Yi-seul; Lee, Byung-Hoo

2017-01-01

Porous starch granules (PSGs) with various pores and cavity sizes were prepared by amylolysis enzymes. The greatest hydrolysis rate on corn starch granule was observed with α-amylase, followed by gluco- and β-amylases. Temperature increase enhanced glucoamylase reaction rate more drastically than other enzyme treatments. Final hydrolysis level with glucoamylase reached to 66.9%, close to 67.5% of α-amylolysis. The α-amylase-treated PSGs displayed the greatest pore size and ratio of cavity-to-granule diameters. Gelatinization onset temperatures of PSGs increased to 72.1 (α-), 68.7 (β-), and 68.1°C (gluco-amylolysis) after 8 h; enthalpy changes of β- and gluco-amylase-treated PSGs increased to 13.4, and 13.1 J/g but α-amylase-treated one showed slightly reduced value of 8.5 J/g. Water holding capacities of PSGs were 209.7 (α-), 94.6 (β-), and 133.8% (gluco-amylolysis), and the untreated control had 89.1%; oil holding capacities of them showed 304.5, 182.7, and 211.5%, respectively, while the untreated control had 161.8%. Thus, enzyme types and their reaction conditions can be applied to generate desirable cavity and pore sizes in starch granules. This biocatalytic approach could contribute to develop tailor-made PSGs with distinct internal structure for specific uses in wide range of food, pharmaceutical and other industrial applications. PMID:28727742

Effect of domestic cooking on the starch digestibility, predicted glycemic indices, polyphenol contents and alpha amylase inhibitory properties of beans (Phaseolis vulgaris) and breadfruit (Treculia africana).

PubMed

Chinedum, E; Sanni, S; Theressa, N; Ebere, A

2018-01-01

The effect of processing on starch digestibility, predicted glycemic indices (pGI), polyphenol contents and alpha amylase inhibitory properties of beans (Phaseolis vulgaris) and breadfruit (Treculia africana) was studied. Total starch ranged from 4.3 to 68.3g/100g, digestible starch ranged from 4.3 to 59.2 to 65.7g/100g for the raw and processed legumes; Resistance starch was not detected in most of the legumes except in fried breadfruit and the starches in both the raw and processed breadfruit were more rapidly digested than those from raw and cooked beans. Raw and processed breadfruit had higher hydrolysis curves than raw and processed beans with the amylolysis level in raw breadfruit close to that of white bread. Raw beans had a low glycemic index (GI); boiled beans and breadfruit had intermediate glycemic indices respectively while raw and fried breadfruit had high glycemic indices. Aqueous extracts of the food samples had weak α-amylase inhibition compared to acarbose. The raw and processed legumes contained considerable amounts of dietary phenols and flavonoids. The significant correlation (r=0.626) between α-amylase inhibitory actions of the legumes versus their total phenolic contents suggests the contribution of the phenolic compounds in these legumes to their α-amylase inhibitory properties. Copyright © 2017 Elsevier B.V. All rights reserved.

Screening of traditional antidiabetic medicinal plants of Mauritius for possible alpha-amylase inhibitory effects in vitro.

PubMed

Kotowaroo, M I; Mahomoodally, M F; Gurib-Fakim, A; Subratty, A H

2006-03-01

In this study, seven exotic/indigenous medicinal plants of Mauritius, namely Coix lacryma-jobi (Poaceae), Aegle marmelos (Rutaceae), Artocarpus heterophyllus (Moraceae), Vangueria madagascariensis (Rubiaceae), Azadirachta indica (Meliaceae), Eriobotrya japonica (Rosaceae) and Syzigium cumini (Myrtaceae) were studied for possible effects on starch breakdown by alpha-amylase in vitro. The results showed that only Artocarpus heterophyllus significantly (p < 0.05) inhibited alpha-amylase activity in vitro. To confirm the observed effects, a further biochemical assay was undertaken to investigate the effects of Artocarpus heterophyllus on alpha-amylase activity using rat plasma in vitro. It was found that the aqueous leaf extract significantly (p < 0.05) inhibited alpha-amylase activity in rat plasma. The highest inhibitory activity (27.20 +/- 5.00%) was observed at a concentration of 1000 microg/mL. However, in both cases dose dependency was not observed. Enzyme kinetic studies using the Michaelis-Menten and Lineweaver-Burk equations were performed to establish the type of inhibition involved. In the presence of the plant extract the maximal velocity (Vmax) remained constant (1/150 g / L/s) whereas the Michaelis-Menten constant (Km) increased by 5.79 g / L, indicating that the aqueous leaf extract of Artocarpus heterophyllus behaved as a competitive inhibitor. Results from the present study tend to indicate that Artocarpus heterophyllus could act as a 'starch blocker' thereby reducing post-prandial glucose peaks. Copyright 2006 John Wiley & Sons, Ltd.

Improved Production of a Heterologous Amylase in Saccharomyces cerevisiae by Inverse Metabolic Engineering

PubMed Central

Liu, Zihe; Liu, Lifang; Österlund, Tobias; Hou, Jin; Huang, Mingtao; Fagerberg, Linn; Petranovic, Dina; Uhlén, Mathias

2014-01-01

The increasing demand for industrial enzymes and biopharmaceutical proteins relies on robust production hosts with high protein yield and productivity. Being one of the best-studied model organisms and capable of performing posttranslational modifications, the yeast Saccharomyces cerevisiae is widely used as a cell factory for recombinant protein production. However, many recombinant proteins are produced at only 1% (or less) of the theoretical capacity due to the complexity of the secretory pathway, which has not been fully exploited. In this study, we applied the concept of inverse metabolic engineering to identify novel targets for improving protein secretion. Screening that combined UV-random mutagenesis and selection for growth on starch was performed to find mutant strains producing heterologous amylase 5-fold above the level produced by the reference strain. Genomic mutations that could be associated with higher amylase secretion were identified through whole-genome sequencing. Several single-point mutations, including an S196I point mutation in the VTA1 gene coding for a protein involved in vacuolar sorting, were evaluated by introducing these to the starting strain. By applying this modification alone, the amylase secretion could be improved by 35%. As a complement to the identification of genomic variants, transcriptome analysis was also performed in order to understand on a global level the transcriptional changes associated with the improved amylase production caused by UV mutagenesis. PMID:24973076

Extension Facts: Managing the risk of low falling numbers in wheat

USDA-ARS?s Scientific Manuscript database

This article reviews the Hagberg-Perten Falling Number test, and the environmental conditions that result in reduced falling numbers. Preharvest sprouting and Late Maturity Alpha-amylase can both result in reduced falling number as a consequence of starch digestion by the enzyme alpha-amylase. Pre...

Influence of α-amylase template concentration on systematic entrapment of highly stable and monodispersed colloidal gold nanoparticles

NASA Astrophysics Data System (ADS)

Ananth, A. Nitthin; Ananth, A. Nimrodh; Jose, Sujin P.; Umapathy, S.; Mathavan, T.

2016-01-01

Nano gold / α-amylase colloidal dispersions of profound stability were made using simple procedure with a conventional reducing agent. The surface plasmon resonance of the gold nanocrystals was used to quantify the extent of the dispersion stability and functionalization. It is found that the reduced gold nanoparticles were trapped into the protein network without denaturation the structure of α-amylase protein. This kind of entrapment of particles into the protein network prevents clustering of individual gold nanoparticles (6.42 nm ± 0.92 nm) by acting as a natural spacer. Systematic entrapment was facilitated by the affinity of gold to the sulfur moieties (Au-S) in the protein structure.

Componential Profile and Amylase Inhibiting Activity of Phenolic Compounds from Calendula officinalis L. Leaves

PubMed Central

Olennikov, Daniil N.; Kashchenko, Nina I.

2014-01-01

An ethanolic extract and its ethyl acetate-soluble fraction from leaves of Calendula officinalis L. (Asteraceae) were found to show an inhibitory effect on amylase. From the crude extract fractions, one new phenolic acid glucoside, 6′-O-vanilloyl-β-D-glucopyranose, was isolated, together with twenty-four known compounds including five phenolic acid glucosides, five phenylpropanoids, five coumarins, and nine flavonoids. Their structures were elucidated based on chemical and spectral data. The main components, isoquercitrin, isorhamnetin-3-O-β-D-glucopyranoside, 3,5-di-O-caffeoylquinic acid, and quercetin-3-O-(6′′-acetyl)-β-D-glucopyranoside, exhibited potent inhibitory effects on amylase. PMID:24683352

Leaf starch degradation comes out of the shadows.

PubMed

Lloyd, James R; Kossmann, Jens; Ritte, Gerhard

2005-03-01

During the day, plants accumulate starch in their leaves as an energy source for the coming night. Based on recent findings, the prevailing view of how the transitory starch is remobilized needs considerable revision. Analyses of transgenic and mutant plants demonstrate that plastidic glucan phosphorylase is not required for normal starch breakdown and cast doubt on the presumed essential role of alpha-amylase but do show that beta-amylase is important. Repression of the activity of a plastidic beta-amylase, the export of its product (maltose) or further metabolism of maltose by a newly identified transglucosidase impairs starch degradation. Breakdown of particulate starch also depends on the activity of glucan-water dikinase, which phosphorylates glucosyl residues within the polymer.

Comparative expression analysis of hordein and beta-amylase in developing barley grains

USDA-ARS?s Scientific Manuscript database

Hordeins are the major seed storage proteins (SSP) in the barley grain. They account for the majority of all proteins in the mature grain. Hordeins accumulate and are stored during grain development. Their primary function is to act as nitrogen, carbon, and sulfur reserves. Beta-amylase is a starch ...

Relationships between falling number, a-Amylase activity, milling, and sponge cake quality of soft white wheat

USDA-ARS?s Scientific Manuscript database

Falling Number of wheat is an important quality predictor and carries with it significant economic impact. Lower Falling Numbers are associated with higher a-amylase activity and poorer soft wheat end-use quality, especially sponge cake. In the present study two sample sets were examined, the first ...

Re-examining the applications of amylase in the sugar industry: Conquering the control of insoluble and soluble starch

USDA-ARS?s Scientific Manuscript database

The new knowledge that there is markedly more insoluble starch than previously considered in products across both the sugarcane factory and refinery has processing implications. Processing parameters such as viscosity and filtration are implicated, as well as the application of '-amylases in the fa...

Inhibitory activity on type 2 diabetes and hypertension key-enzymes, and antioxidant capacity of Veronica persica phenolic-rich extracts.

PubMed

Sharifi-Rad, M; Tayeboon, G S; Sharifi-Rad, J; Iriti, M; Varoni, E M; Razazi, S

2016-05-30

Veronica genus (Plantaginaceae) is broadly distributed in different habitats. In this study, the inhibitory activity of free soluble and conjugated phenolic extracts of Veronica persica on key enzymes associated to type 2 diabetes (α-glucosidase and α-amylase) and hypertension (angiotensin I converting enzyme, ACE) was assessed, as well as their antioxidant power. Our results showed that both the extracts inhibited α-amylase, α-glucosidase and ACE in a dose-dependent manner. In particular, free phenolic extract significantly (P<0.05) inhibited α-glucosidase (IC50 532.97 µg/mL), whereas conjugated phenolic extract significantly (P<0.05) inhibited α-amylase (IC50 489.73 µg/mL) and ACE (290.06 µg/mL). The enzyme inhibitory activities of the extracts were not associated with their phenolic content. Anyway, the inhibition of α-amylase, α-glucosidase and ACE, along with the antioxidant capacity of the phenolic-rich extracts, could represent a putative mechanism through which V. persica exerts its antidiabetes and antihypertension effects.

Hypoglycemic activity of Pyrus biossieriana Buhse leaf extract and arbutin: Inhibitory effects on alpha amylase and alpha glucosidase

PubMed Central

Yousefi, Fatemeh; Mahjoub, Soleiman; Pouramir, Mahdi; Khadir, Fatemeh

2013-01-01

Background: The mechanism of hypoglycemic and hypolipidemic activities of Pyrus biossieriana Buhse leaf extract (PbBLE) and its phytochemical component arbutin, have not been well determined. The present study was performed to understand the hypoglycemic activity mechanisms of pbBLE and arbutin more clearly. Methods: In vitro enzymatic carbohydrate digestion with PbBLE and arbutin was assessed using α-amylase and α-glucosidase powders. The enzyme solutions were premixed with PbBLE and arbutin at different concentrations (0.1, 1, 10 and 100 mg/ml). Substrate solutions and colorimetric reagents were added to the reaction. The release of glucose was determined by spectrophotometric method. Acarbose was used as the positive control. Results: The extract (10, 100 mg/ ml) completely inhibit α- amylase and α- glucosidase activities. The extract produced higher reduction of α-amylase and α-glucosidase activity than arbutin. Inhibition at various concentrations (0.1, 1, 10, 100 mg/ml) were significantly different (p<0.05). Conclusion: Our results exhibited that both the extract and arbutin were able to suppress the enzymes strongly. PMID:24294470

A chimeric α-amylase engineered from Bacillus acidicola and Geobacillus thermoleovorans with improved thermostability and catalytic efficiency.

PubMed

Parashar, Deepak; Satyanarayana, T

2016-04-01

The α-amylase (Ba-amy) of Bacillus acidicola was fused with DNA fragments encoding partial N- and C-terminal region of thermostable α-amylase gene of Geobacillus thermoleovorans (Gt-amy). The chimeric enzyme (Ba-Gt-amy) expressed in Escherichia coli displays marked increase in catalytic efficiency [K cat: 4 × 10(4) s(-1) and K cat/K m: 5 × 10(4) mL(-1) mg(-1) s(-1)] and higher thermostability than Ba-amy. The melting temperature (T m) of Ba-Gt-amy (73.8 °C) is also higher than Ba-amy (62 °C), and the CD spectrum analysis revealed the stability of the former, despite minor alteration in secondary structure. Langmuir-Hinshelwood kinetic analysis suggests that the adsorption of Ba-Gt-amy onto raw starch is more favourable than Ba-amy. Ba-Gt-amy is thus a suitable biocatalyst for raw starch saccharification at sub-gelatinization temperatures because of its acid stability, thermostability and Ca(2+) independence, and better than the other known bacterial acidic α-amylases.

Natural Prenylchalconaringenins and Prenylnaringenins as Antidiabetic Agents: α-Glucosidase and α-Amylase Inhibition and in Vivo Antihyperglycemic and Antihyperlipidemic Effects.

PubMed

Sun, Hua; Wang, Dong; Song, Xiaotong; Zhang, Yazhou; Ding, Weina; Peng, Xiaolin; Zhang, Xiaoting; Li, Yashan; Ma, Ying; Wang, Runling; Yu, Peng

2017-03-01

Inhibition of α-glucosidase and α-amylase decreases postprandial blood glucose levels and delays glucose absorption, making it a treatment strategy for type 2 diabetes. This study examined in vivo and in vitro antidiabetic activities of natural prenylchalconaringenins 1 and 2 and prenylnaringenins 3 and 4, found in hops and beer. 3'-Geranylchalconaringenin (2) competitively and irreversibly inhibited α-glucosidase (IC 50 = 1.08 μM) with activity 50-fold higher than that of acarbose (IC 50 = 51.30 μM) and showed moderate inhibitory activity against α-amylase (IC 50 = 20.46 μM). Docking analysis substantiated these findings. In addition, compound 2 suppressed the increase in postprandial blood glucose levels and serum levels of total cholesterol and triglycerides in streptozotocin-induced diabetic mice. Taken together, these results suggest that 2 has dual inhibitory activity against α-glucosidase and α-amylase and alleviates diabetic hyperglycemia and hyperlipidemia, making it a potential functional food ingredient and drug candidate for management of type 2 diabetes.

Economic Analysis of the Production of Amylases and Other Hydrolases by Aspergillus awamori in Solid-State Fermentation of Babassu Cake

PubMed Central

de Castro, Aline Machado; Carvalho, Daniele Fernandes; Freire, Denise Maria Guimarães; Castilho, Leda dos Reis

2010-01-01

Amylases are one of the most important industrial enzymes produced worldwide, with their major application being in ethanol manufacturing. This work investigated the production of amylases by solid-state fermentation of babassu cake, using the filamentous fungus Aspergillus awamori IOC-3914. Lab-scale experiments were carried out to generate input data for simulations of an industrial plant for amylase production. Additionally to the target enzymes, other hydrolases (cellulases, xylanases, and proteases) were also produced, enriching the final product. The most suitable fermentation time was 144 hours, when exoamylase and endoamylase activities of 40.5 and 42.7 U g−1 were achieved, respectively. A first evaluation showed a large impact of the inoculum propagation medium on production costs. Therefore, five propagation media were compared, and PDA medium presented the best cost-benefit ratio. The credits obtained from sales of fermented cake as a coproduct enabled a significant decrease in the production cost of the enzyme product, down to 10.40 USD kg−1. PMID:21048867

Application of decolourized and partially purified polygalacturonase and α-amylase in apple juice clarification.

PubMed

Dey, Tapati Bhanja; Banerjee, Rintu

2014-01-01

Polygalacturonase and α-amylase play vital role in fruit juice industry. In the present study, polygalacturonase was produced by Aspergillus awamori Nakazawa MTCC 6652 utilizing apple pomace and mosambi orange (Citrus sinensis var mosambi) peels as solid substrate whereas, α-amylase was produced from A. oryzae (IFO-30103) using wheat bran by solid state fermentation (SSF) process. These carbohydrases were decolourized and purified 8.6-fold, 34.8-fold and 3.5-fold, respectively by activated charcoal powder in a single step with 65.1%, 69.8% and 60% recoveries, respectively. Apple juice was clarified by these decolourized and partially purified enzymes. In presence of 1% polygalacturonase from mosambi peels (9.87 U/mL) and 0.4% α-amylase (899 U/mL), maximum clarity (%T(660 nm) = 97.0%) of juice was attained after 2 h of incubation at 50 °C in presence of 10 mM CaCl2. Total phenolic content of juice was reduced by 19.8% after clarification, yet with slightly higher %DPPH radical scavenging property.

Cysteine Protease-Binding Protein Family 6 Mediates the Trafficking of Amylases to Phagosomes in the Enteric Protozoan Entamoeba histolytica

PubMed Central

Furukawa, Atsushi; Nakada-Tsukui, Kumiko

2013-01-01

Phagocytosis plays a pivotal role in nutrient acquisition and evasion from the host defense systems in Entamoeba histolytica, the intestinal protozoan parasite that causes amoebiasis. We previously reported that E. histolytica possesses a unique class of a hydrolase receptor family, designated the cysteine protease-binding protein family (CPBF), that is involved in trafficking of hydrolases to lysosomes and phagosomes, and we have also reported that CPBF1 and CPBF8 bind to cysteine proteases or β-hexosaminidase α-subunit and lysozymes, respectively. In this study, we showed by immunoprecipitation that CPBF6, one of the most highly expressed CPBF proteins, specifically binds to α-amylase and γ-amylase. We also found that CPBF6 is localized in lysosomes, based on immunofluorescence imaging. Immunoblot and proteome analyses of the isolated phagosomes showed that CPBF6 mediates transport of amylases to phagosomes. We also demonstrated that the carboxyl-terminal cytosolic region of CPBF6 is engaged in the regulation of the trafficking of CPBF6 to phagosomes. Our proteome analysis of phagosomes also revealed new potential phagosomal proteins. PMID:23509141

Structural forms of the human amylase locus and their relationships to SNPs, haplotypes, and obesity

PubMed Central

Usher, Christina L; Handsaker, Robert E; Esko, Tõnu; Tuke, Marcus A; Weedon, Michael N; Hastie, Alex R; Cao, Han; Moon, Jennifer E; Kashin, Seva; Fuchsberger, Christian; Metspalu, Andres; Pato, Carlos N; Pato, Michele T; McCarthy, Mark I; Boehnke, Michael; Altshuler, David M; Frayling, Timothy M; Hirschhorn, Joel N; McCarroll, Steven A

2016-01-01

Hundreds of genes reside in structurally complex, poorly understood regions of the human genome1-3. One such region contains the three amylase genes (AMY2B, AMY2A, and AMY1) responsible for digesting starch into sugar. The copy number of AMY1 is reported to be the genome’s largest influence on obesity4, though genome-wide association studies for obesity have found this locus unremarkable. Using whole genome sequence analysis3,5, droplet digital PCR6, and genome mapping7, we identified eight common structural haplotypes of the amylase locus that suggest its mutational history. We found that AMY1 copy number in individuals’ genomes is generally even (rather than odd) and partially correlates to nearby SNPs, which do not associate with BMI. We measured amylase gene copy number in 1,000 obese or lean Estonians and in two other cohorts totaling ~3,500 individuals. We had 99% power to detect the lower bound of the reported effects on BMI4, yet found no association. PMID:26098870

Restoration of Traditional Korean Nuruk and Analysis of the Brewing Characteristics.

PubMed

Lee, Jang-Eun; Lee, Ae Ran; Kim, HyeRyun; Lee, Eunjung; Kim, Tae Wan; Shin, Woo Chang; Kim, Jae Ho

2017-05-28

In this study, a total of 58 different kinds of nuruk (a traditional Korean fermentation starter) were prepared, including 46 kinds of restored nuruk from ancient documents. Each nuruk was evaluated by analysis of its saccharification power, and the enzyme activities of glucoamylase, α-amylase, β-amylase, protease, and β-glucanase. The range of saccharification power (sp) of the restored nuruk ranged between 85 and 565 sp. The diastatic enzymes, α-amylase, β-amylase, and glucoamylase, were significantly correlated to the saccharification power value; conversely, β-glucanase and protease did not have a correlative relationship with saccarification power. In addition, their brewing properties on chemical and organoleptic aspects of traditional alcoholic beverage production were compared. Each raw and supplementary material contained in nuruk showed its own unique characteristics on Korean alcoholic beverage brewing. For the first time, in this study, the traditional Korean nuruk types mentioned in ancient documents were restored using modernized production methods, and also characterized based on their brewing properties. Our results could be utilized as a basis for further study of traditional alcoholic beverages and their valuable microorganisms.

ANTIOXIDANT ACTIVITY AND A-AMYLASE INHIBITORY POTENTIAL OF ROSA CANINA L

PubMed Central

Jemaa, Houda Ben; Jemia, Amani Ben; Khlifi, Sarra; Ahmed, Halima Ben; Slama, Fethi Ben; Benzarti, Anis; Elati, Jalila; Aouidet, Abdallah

2017-01-01

Background: Diabetes mellitus is one of the most common endocrinal disorders and medicinal plants continue to play an important role in the management of this disease. In this study, Rosa canina was investigated for the antioxidant and α-amylase inhibition activities. Materials and Methods: Methanolic extract of Rosa canina was investigated for its potential antioxidant activity. The extracts’ total phenolic and flavonoid contents and scavenging capacity for free radicals were evaluated. The α-amylase inhibition assay was also carried. Results: Rosa canina extract exhibits a total Phenolic and flavonoid levels respectively (21.918 mg GAE/g and 2.647mg ER/g). The free radical scavenging activity was found to be prominent against DPPH with an IC50 of 0.668 mg/ml and against ABTS with an IC50 of 0.467 mg/ml. Extract showed a significant ferric ion reducing activities with an IC50 of4.962 mg/ml. Conclusion: Rosa canina exerted a higher inhibitory activity against α-amylase. The obtained results support the antidiabetic use of rosa canina. PMID:28573216

Unraveling the Dynamics of the Human Vaginal Microbiome.

PubMed

Nunn, Kenetta L; Forney, Larry J

2016-09-01

Four Lactobacillus species, namely L. crispatus , L. iners , L. gasseri , and L. jensenii , commonly dominate the vaginal communities of most reproductive-age women. It is unclear why these particular species, and not others, are so prevalent. Historically, estrogen-induced glycogen production by the vaginal epithelium has been proffered as being key to supporting the proliferation of vaginal lactobacilli. However, the 'fly in the ointment' (that has been largely ignored) is that the species of Lactobacillus commonly found in the human vagina cannot directly metabolize glycogen. It would appear that this riddle has been solved as studies have demonstrated that vaginal lactobacilli can metabolize the products of glycogen depolymerization by α-amylase, and fortunately, amylase activity is found in vaginal secretions. These amylases are presumed to be host-derived, but we suggest that other bacterial populations in vaginal communities could also be sources of amylase in addition to (or instead of) the host. Here we briefly review what is known about human vaginal bacterial communities and discuss how glycogen-derived resources and resource competition might shape the composition and structure of these communities.

Unraveling the Dynamics of the Human Vaginal Microbiome

PubMed Central

Nunn, Kenetta L.; Forney, Larry J.

2016-01-01

Four Lactobacillus species, namely L. crispatus, L. iners, L. gasseri, and L. jensenii, commonly dominate the vaginal communities of most reproductive-age women. It is unclear why these particular species, and not others, are so prevalent. Historically, estrogen-induced glycogen production by the vaginal epithelium has been proffered as being key to supporting the proliferation of vaginal lactobacilli. However, the ‘fly in the ointment’ (that has been largely ignored) is that the species of Lactobacillus commonly found in the human vagina cannot directly metabolize glycogen. It would appear that this riddle has been solved as studies have demonstrated that vaginal lactobacilli can metabolize the products of glycogen depolymerization by α-amylase, and fortunately, amylase activity is found in vaginal secretions. These amylases are presumed to be host-derived, but we suggest that other bacterial populations in vaginal communities could also be sources of amylase in addition to (or instead of) the host. Here we briefly review what is known about human vaginal bacterial communities and discuss how glycogen-derived resources and resource competition might shape the composition and structure of these communities. PMID:27698617

Saliva amylase as a measure of sympathetic change elicited by autogenic training in patients with functional somatic syndromes.

PubMed

Kiba, Tadashi; Kanbara, Kenji; Ban, Ikumi; Kato, Fumie; Kawashima, Sadanobu; Saka, Yukie; Yamamoto, Kazumi; Nishiyama, Junji; Mizuno, Yasuyuki; Abe, Tetsuya; Fukunaga, Mikihiko

2015-12-01

The aim of this study was to discuss the effect of autogenic training (AT) on patients with functional somatic syndrome (FSS) using salivary amylase, the skin temperature of the finger, subjective severity of symptoms, and psychological characteristics as measures. We assessed 20 patients with FSS and 23 healthy controls before and after AT. Baseline levels of salivary amylase prior to an AT session were significantly higher in the FSS group than in the control group. However, this difference was not significant after AT. The skin temperature of the finger increased after AT in both the FSS and control groups. AT contributed to the improvement of somatic symptoms in patients with FSS. Our results regarding psychological characteristics suggest that mood disturbances are deeply involved in the pathology of FSS. Individuals with FSS exhibited elevated levels of sympathetic activity compared with healthy controls. Our data indicates that AT eased dysregulation of the autonomic nervous system in patients with FSS. Thus, salivary amylase may be a useful index of change induced by AT in patients with FSS.

Application of decolourized and partially purified polygalacturonase and α-amylase in apple juice clarification

PubMed Central

Dey, Tapati Bhanja; Banerjee, Rintu

2014-01-01

Polygalacturonase and α-amylase play vital role in fruit juice industry. In the present study, polygalacturonase was produced by Aspergillus awamori Nakazawa MTCC 6652 utilizing apple pomace and mosambi orange (Citrus sinensis var mosambi) peels as solid substrate whereas, α-amylase was produced from A. oryzae (IFO-30103) using wheat bran by solid state fermentation (SSF) process. These carbohydrases were decolourized and purified 8.6-fold, 34.8-fold and 3.5-fold, respectively by activated charcoal powder in a single step with 65.1%, 69.8% and 60% recoveries, respectively. Apple juice was clarified by these decolourized and partially purified enzymes. In presence of 1% polygalacturonase from mosambi peels (9.87 U/mL) and 0.4% α-amylase (899 U/mL), maximum clarity (%T660nm = 97.0%) of juice was attained after 2 h of incubation at 50 °C in presence of 10 mM CaCl2. Total phenolic content of juice was reduced by 19.8% after clarification, yet with slightly higher %DPPH radical scavenging property. PMID:24948919

Micrococcus luteus mediated dual mode synthesis of gold nanoparticles: involvement of extracellular α-amylase and cell wall teichuronic acid.

PubMed

Arunkumar, Pichaimani; Thanalakshmi, Muthukrishnan; Kumar, Priyadarsini; Premkumar, Kumpati

2013-03-01

In the present study we have utilized the bioreductive potential of Micrococcus luteus for the synthesis of gold nanoparticles. Biochemical and physiological analysis indicate that the biosynthesized GNPs were achieved by dual mode, involving extracellular α-amylase and cell wall teichuronic acid (TUA) of M. luteus. The biosynthetic potential of both α-amylase and TUA, after isolation from bacterium, was examined. Under optimum conditions, these biomolecules reduces Au(3+) into Au(0) and the resulting GNPs were found to be stable for 1 month. The synthesized GNPs were characterized by UV-VIS spectrometry, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR) and dynamic light scattering (DLS). Results demonstrated that the synthesized GNPs were found to be monodispersive and spherical in shape with an average size of ∼6 nm and ∼50 nm for α-amylase and teichuronic acid, respectively. These findings suggest that M. luteus can be exploited as a potential biosource for the eco-friendly synthesis of gold nanoparticles. Copyright © 2012 Elsevier B.V. All rights reserved.

Isolation and characterisation of a novel alpha-amylase from the extreme haloarchaeon Haloterrigena turkmenica.

PubMed

Santorelli, Marco; Maurelli, Luisa; Pocsfalvi, Gabriella; Fiume, Immacolata; Squillaci, Giuseppe; La Cara, Francesco; Del Monaco, Giovanni; Morana, Alessandra

2016-11-01

An extracellular halophilic alpha-amylase (AmyA) was produced by the haloarchaeon Haloterrigena turkmenica grown in medium enriched with 0.2% (w/v) starch. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) analyses showed a major band at 66.0kDa and a peak of 54.0kDa, respectively. Analysis of tryptic fragments of the protein present in the major SDS-PAGE band by nano-LC-ESI-MS/MS led to identification of the alpha-amylase catalytic region, encoded by the htur2110 gene, as the protein possessing the described activity. Optimal values for activity were 55°C, pH 8.5 and 2M NaCl, and high thermostability was showed at 55°C and 3M NaCl. AmyA activity was enhanced by Triton X-100 and was not influenced by n-hexane and chloroform. Starch hydrolysis produced different oligomers with maltose as the smallest end-product. The efficiency of AmyA in degrading starch contained in agronomic residues was tested in grape cane chosen as model substrate. Preliminary results showed that starch was degraded making the enzyme a potential candidate for utilization of agro-industrial waste in fuel and chemicals production. AmyA is one of the few investigated amylases produced by haloarchaea, and the first alpha-amylase described among microorganisms belonging to the genus Haloterrigena. Copyright © 2016 Elsevier B.V. All rights reserved.

Hyperactive α-amylase production by Aspergillus oryzae IFO 30103 in a new bioreactor.

PubMed

Dey, T B; Banerjee, R

2012-02-01

  To improve the α-amylase production in solid-state fermentation (SSF) condition utilizing a new bioreactor (NB) system.   In NB system, 20 g of wheat bran moistened with liquid medium in 1:1 ratio (w/v) was taken on the tray present inside the upper vessel and an additional 80 ml medium was supplemented into the lower vessel. Oxygen uptake rate was improved by supplying compressed air that lifted the liquid medium into the upper vessel and touched the substrate bed. This condition probably facilitated the heat transfer to liquid medium, reduce water loss and catabolite repression. With 1% glucose supplementation, maximum α-amylase activity of 22 317 Ugds(-1) was produced by Aspergillus oryzae IFO 30103 within a very short incubation period (48 h) at 2-cm bed height with air flow rate of 0·1 l min(-1)  g(-1) wheat bran at 32°C and initial medium pH of 6.   Within a short incubation period, significantly high α-amylase activity was obtained and it is higher than those reported to date at bioreactor scale operating with a fungal strain.   The reactor is novel and can overcome some of the major problems associated with SSF process. A. oryzae IFO 30103 is reported as the best fungal source for α-amylase production. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

Evaluation of Traditional Indian Antidiabetic Medicinal Plants for Human Pancreatic Amylase Inhibitory Effect In Vitro

PubMed Central

Ponnusamy, Sudha; Ravindran, Remya; Zinjarde, Smita; Bhargava, Shobha; Ravi Kumar, Ameeta

2011-01-01

Pancreatic α-amylase inhibitors offer an effective strategy to lower the levels of post prandial hyperglycemia via control of starch breakdown. Eleven Ayurvedic Indian medicinal plants with known hypoglycemic properties were subjected to sequential solvent extraction and tested for α-amylase inhibition, in order to assess and evaluate their inhibitory potential on pancreatic α-amylase. Analysis of 91 extracts, showed that 10 exhibited strong Human Pancreatic Amylase (HPA) inhibitory potential. Of these, 6 extracts showed concentration dependent inhibition with IC50 values, namely, cold and hot water extracts from Ficus bengalensis bark (4.4 and 125 μgmL−1), Syzygium cumini seeds (42.1 and 4.1 μgmL−1), isopropanol extracts of Cinnamomum verum leaves (1.0 μgmL−1) and Curcuma longa rhizome (0.16 μgmL−1). The other 4 extracts exhibited concentration independent inhibition, namely, methanol extract of Bixa orellana leaves (49 μgmL−1), isopropanol extract from Murraya koenigii leaves (127 μgmL−1), acetone extracts from C. longa rhizome (7.4 μgmL−1) and Tribulus terrestris seeds (511 μgmL−1). Thus, the probable mechanism of action of the above fractions is due to their inhibitory action on HPA, thereby reducing the rate of starch hydrolysis leading to lowered glucose levels. Phytochemical analysis revealed the presence of alkaloids, proteins, tannins, cardiac glycosides, flavonoids, saponins and steroids as probable inhibitory compounds. PMID:20953430

Utility of hepatic transaminases in children with concern for abuse.

PubMed

Lindberg, Daniel M; Shapiro, Robert A; Blood, Emily A; Steiner, R Daryl; Berger, Rachel P

2013-02-01

Routine testing of hepatic transaminases, amylase, and lipase has been recommended for all children evaluated for physical abuse, but rates of screening are widely variable, even among abuse specialists, and data for amylase and lipase testing are lacking. A previous study of screening in centers that endorsed routine transaminase screening suggested that using a transaminase threshold of 80 IU/L could improve injury detection. Our objectives were to prospectively validate the test characteristics of the 80-IU/L threshold and to determine the utility of amylase and lipase to detect occult abdominal injury. This was a retrospective secondary analysis of the Examining Siblings To Recognize Abuse research network, a multicenter study in children younger than 10 years old who underwent subspecialty evaluation for physical abuse. We determined rates of identified abdominal injuries and results of transaminase, amylase, and lipase testing. Screening studies were compared by using basic test characteristics (sensitivity, specificity) and the area under the receiver operating characteristic curve. Abdominal injuries were identified in 82 of 2890 subjects (2.8%; 95% confidence interval: 2.3%-3.5%). Hepatic transaminases were obtained in 1538 (53%) subjects. Hepatic transaminases had an area under the receiver operating characteristic curve of 0.87. A threshold of 80 IU/L yielded sensitivity of 83.8% and specificity of 83.1%. The areas under the curve for amylase and lipase were 0.67 and 0.72, respectively. Children evaluated for physical abuse with transaminase levels >80 IU/L should undergo definitive testing for abdominal injury.

The Gastric/Pancreatic Amylase Ratio Predicts Postoperative Pancreatic Fistula With High Sensitivity and Specificity

PubMed Central

Jin, Shuo; Shi, Xiao-Ju; Sun, Xiao-Dong; Zhang, Ping; Lv, Guo-Yue; Du, Xiao-Hong; Wang, Si-Yuan; Wang, Guang-Yi

2015-01-01

Abstract This article aims to identify risk factors for postoperative pancreatic fistula (POPF) and evaluate the gastric/pancreatic amylase ratio (GPAR) on postoperative day (POD) 3 as a POPF predictor in patients who undergo pancreaticoduodenectomy (PD). POPF significantly contributes to mortality and morbidity in patients who undergo PD. Previously identified predictors for POPF often have low predictive accuracy. Therefore, accurate POPF predictors are needed. In this prospective cohort study, we measured the clinical and biochemical factors of 61 patients who underwent PD and diagnosed POPF according to the definition of the International Study Group of Pancreatic Fistula. We analyzed the association between POPF and various factors, identified POPF risk factors, and evaluated the predictive power of the GPAR on POD3 and the levels of serum and ascites amylase. Of the 61 patients, 21 developed POPF. The color of the pancreatic drain fluid, POD1 serum, POD1 median output of pancreatic drain fluid volume, and GPAR were significantly associated with POPF. The color of the pancreatic drain fluid and high GPAR were independent risk factors. Although serum and ascites amylase did not predict POPF accurately, the cutoff value was 1.24, and GPAR predicted POPF with high sensitivity and specificity. This is the first report demonstrating that high GPAR on POD3 is a risk factor for POPF and showing that GPAR is a more accurate predictor of POPF than the previously reported amylase markers. PMID:25621676

Daily Social Interactions and the Biological Stress Response: Are There Age Differences in Links Between Social Interactions and Alpha-Amylase?

PubMed

Birditt, Kira S; Tighe, Lauren A; Nevitt, Michael R; Zarit, Steven H

2017-12-12

According to the strength and vulnerability integration (SAVI) model, older people are better able to avoid negative social interactions than younger people, but when they do experience negative interactions, they are equally or more emotionally and physiologically reactive than younger people. Less is known about the links between daily negative and positive social encounters and the sympathetic adrenal medullary system (a key stress pathway) and whether there are age differences in these links. This study considers whether negative and positive social interactions are associated with diurnal alpha-amylase (a measure of the sympathetic adrenal medullary system) and whether there are differences in these links by age. Participants were from the Daily Health, Stress, and Relationship Study, which includes a random sample of 89 individuals (aged 40-95) who completed 14 days of daily diary interviews and provided saliva samples four times a day (wake, 30 min after wake, lunch, and bedtime) for four of those days that were assayed for alpha-amylase. Days in which people reported more negative interactions were associated with flatter morning declines in alpha-amylase, indicating greater stress. Links between positive interactions and diurnal alpha-amylase varied by age group. Findings are consistent with the SAVI model indicating that older adults respond differently to social stimuli than younger people. © The Author(s) 2017. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

[The activity of thermolability amylase in serum nonsmoking and smoking healthy persons and patients with pancreatitis].

PubMed

Sliwińska-Mossoń, Mariola; Milnerowicz, Halina

2008-01-01

The aim of this study is to prove the influence of tobacco smoking on total and thermolability amylase activity in the serum of non-smoking and smoking health persons and patients with diagnosed acute (AP), chronic exaggerated (CEP) and chronic pancreatitis (CP) and patients with diabetes. The blood has been collected from 28 healthy persons and 52 patients. The enzyme total activity has been determined using the colorimetric method with substrate 1,2-odilauryl-rac-glycero-3-glutaric acid -(6-methylresorufin) ester. The thermolability activity has been determined using the thermolability test. The tobacco smoke has been examined on the basic of concentration of cotinine in the serum of health persons and patients. The highest amylase total activity and her thermolability form have been found in smoking patients with diabetes. It has been noted that the serum amylase activity is significantly higher in smoking and healthy persons (p < 0.0002; p < 0.002) then in non-smoking and healthy patients. However no significant differences have been found between the thermolability total activity, however it has been noted higher thermolability thermolability activity in smoking patients with CP and nonsmoking patients with CP. Smoking patients with AP and CEP have been found to have a significantly increased enzyme and her form thermolability activity (p > 0.001; p > 0.005 respectively) when compared to non-smoking patients. Results of examination indicate that tobacco smoking has a significant influence on pancreatic amylase activity.

Relationship Between Meditation Depth and Waking Salivary Alpha-Amylase Secretion Among Long-Term MBSR Instructors.

PubMed

Haslam, Alyson; Wirth, Michael D; Robb, Sara Wagner

2017-08-01

The purpose of this study was to characterize sympathetic activity by using waking salivary alpha-amylase (sAA) concentrations in a group of long-term meditation instructors and to examine the association between meditation (depth, dose and duration) and the waking alpha-amylase response. Salivary alpha-amylase samples were collected (immediately upon waking and at 15-min, 30-min and 45-min intervals after waking) from mindfulness-based stress reduction instructors to determine both the area under the curve and the awakening slope (difference in alpha-amylase concentrations between waking and 30-min post-waking). It was determined through general linear models that neither years of meditation nor meditation dose were associated with the awakening sAA slope, but higher scores for meditation depth (greater depth) was associated with a more negative (or steeper) awakening slope [Quartile (Q)1: -7 versus Q4: -21 U/mL; p = 0.06], in fully adjusted models. Older age (p = 0.04) and a later time of waking (p < 0.01) also were associated with less negative awakening slope values. Smoking was associated with lower area under the curve values (smokers: 1716 U/mL versus nonsmokers: 2107 U/mL; p = 0.05) in fully adjusted models. The results suggest a 'healthy' sAA waking slope among individuals who meditate more deeply. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Concomitant production of detergent compatible enzymes by Bacillus flexus XJU-1.

PubMed

Niyonzima, Francois N; More, Sunil S

2014-01-01

A soil screened Bacillus flexus XJU-1 was induced to simultaneously produce alkaline amylase, alkaline lipase and alkaline protease at their optimum levels on a common medium under submerged fermentation. The basal cultivation medium consisted of 0.5% casein, 0.5% starch and 0.5% cottonseed oil as an inducer for protease, amylase, and lipase, respectively. The casein also served as nitrogen source for all 3 enzymes. The starch was also found to act as carbon source additive for both lipase and protease. Maximum enzyme production occurred on fermentation medium with 1.5% casein, 1.5% soluble starch, 2% cottonseed oil, 2% inoculum size, initial pH of 11.0, incubation temperature of 37 °C and 1% soybean meal as a nitrogen source supplement. The analysis of time course study showed that 24 h was optimum incubation time for amylase whereas 48 h was the best time for both lipase and protease. After optimization, a 3.36-, 18.64-, and 27.33-fold increase in protease, amylase and lipase, respectively was recorded. The lipase was produced in higher amounts (37.72 U/mL) than amylase and protease about 1.27 and 5.85 times, respectively. As the 3 enzymes are used in detergent formulations, the bacterium can be commercially exploited to secrete the alkaline enzymes for use in detergent industry. This is the first report for concomitant production of 3 alkaline enzymes by a bacterium.

The gastric/pancreatic amylase ratio predicts postoperative pancreatic fistula with high sensitivity and specificity.

PubMed

Jin, Shuo; Shi, Xiao-Ju; Sun, Xiao-Dong; Zhang, Ping; Lv, Guo-Yue; Du, Xiao-Hong; Wang, Si-Yuan; Wang, Guang-Yi

2015-01-01

This article aims to identify risk factors for postoperative pancreatic fistula (POPF) and evaluate the gastric/pancreatic amylase ratio (GPAR) on postoperative day (POD) 3 as a POPF predictor in patients who undergo pancreaticoduodenectomy (PD).POPF significantly contributes to mortality and morbidity in patients who undergo PD. Previously identified predictors for POPF often have low predictive accuracy. Therefore, accurate POPF predictors are needed.In this prospective cohort study, we measured the clinical and biochemical factors of 61 patients who underwent PD and diagnosed POPF according to the definition of the International Study Group of Pancreatic Fistula. We analyzed the association between POPF and various factors, identified POPF risk factors, and evaluated the predictive power of the GPAR on POD3 and the levels of serum and ascites amylase.Of the 61 patients, 21 developed POPF. The color of the pancreatic drain fluid, POD1 serum, POD1 median output of pancreatic drain fluid volume, and GPAR were significantly associated with POPF. The color of the pancreatic drain fluid and high GPAR were independent risk factors. Although serum and ascites amylase did not predict POPF accurately, the cutoff value was 1.24, and GPAR predicted POPF with high sensitivity and specificity.This is the first report demonstrating that high GPAR on POD3 is a risk factor for POPF and showing that GPAR is a more accurate predictor of POPF than the previously reported amylase markers.

Engineering yeasts for raw starch conversion.

PubMed

van Zyl, W H; Bloom, M; Viktor, M J

2012-09-01

Next to cellulose, starch is the most abundant hexose polymer in plants, an import food and feed source and a preferred substrate for the production of many industrial products. Efficient starch hydrolysis requires the activities of both α-1,4 and α-1,6-debranching hydrolases, such as endo-amylases, exo-amylases, debranching enzymes, and transferases. Although amylases are widely distributed in nature, only about 10 % of amylolytic enzymes are able to hydrolyse raw or unmodified starch, with a combination of α-amylases and glucoamylases as minimum requirement for the complete hydrolysis of raw starch. The cost-effective conversion of raw starch for the production of biofuels and other important by-products requires the expression of starch-hydrolysing enzymes in a fermenting yeast strain to achieve liquefaction, hydrolysis, and fermentation (Consolidated Bioprocessing, CBP) by a single organism. The status of engineering amylolytic activities into Saccharomyces cerevisiae as fermentative host is highlighted and progress as well as challenges towards a true CBP organism for raw starch is discussed. Conversion of raw starch by yeast secreting or displaying α-amylases and glucoamylases on their surface has been demonstrated, although not at high starch loading or conversion rates that will be economically viable on industrial scale. Once efficient conversion of raw starch can be demonstrated at commercial level, engineering of yeast to utilize alternative substrates and produce alternative chemicals as part of a sustainable biorefinery can be pursued to ensure the rightful place of starch converting yeasts in the envisaged bio-economy of the future.

Green Tea Consumption after Intense Taekwondo Training Enhances Salivary Defense Factors and Antibacterial Capacity

PubMed Central

Lin, Shiuan-Pey; Li, Chia-Yang; Suzuki, Katsuhiko; Chang, Chen-Kang; Chou, Kuei-Ming; Fang, Shih-Hua

2014-01-01

The aim of this study was to investigate the short-term effects of green tea consumption on selected salivary defense proteins, antibacterial capacity and anti-oxidation activity in taekwondo (TKD) athletes, following intensive training. Twenty-two TKD athletes performed a 2-hr TKD training session. After training, participants ingested green tea (T, caffeine 6 mg/kg and catechins 22 mg/kg) or an equal volume of water (W). Saliva samples were collected at three time points: before training (BT-T; BT-W), immediately after training (AT-T; AT-W), and 30 min after drinking green tea or water (Rec-T; Rec-W). Salivary total protein, immunoglobulin A (SIgA), lactoferrin, α-amylase activity, free radical scavenger activity (FRSA) and antibacterial capacity were measured. Salivary total protein, lactoferrin, SIgA concentrations and α-amylase activity increased significantly immediately after intensive TKD training. After tea drinking and 30 min rest, α-amylase activity and the ratio of α-amylase to total protein were significantly higher than before and after training. In addition, salivary antibacterial capacity was not affected by intense training, but green tea consumption after training enhanced salivary antibacterial capacity. Additionally, we observed that salivary FRSA was markedly suppressed immediately after training and quickly returned to pre-exercise values, regardless of which fluid was consumed. Our results show that green tea consumption significantly enhances the activity of α-amylase and salivary antibacterial capacity. PMID:24498143

Enhancement of carbachol-induced amylase secretion in parotid glands from rats with experimental periodontitis.

PubMed

Miozza, Valeria; Sánchez, Gabriel; Sterin-Borda, Leonor; Busch, Lucila

2011-12-01

In a previous study we observed that parotid glands from rats with experimental periodontitis showed an increase in basal amylase release as a result of an increase in cAMP accumulation induced by PGE(2) production. The aim of this work was to study whether this change in amylase release influences the secretory effect of carbachol. Experimental periodontitis was induced through placing a black thread around the cervix of the two lower first molars. Experiments were done 22 days after ligature induced periodontitis. Amylase release was evaluated in vitro and determined using a colorimetric method which uses starch as substrate. The effect of carbachol was increased in parotid glands from periodontitis rats. The effect of 10(-6)M carbachol was inhibited by 4-DAMP (10(-6)M), U-73122 (5 × 10(-6)M) and trifluoperazine (5 × 10(-6)M) in both groups. No changes were observed in the binding sites and affinity in parotid membranes from rats with experimental periodontitis. The inhibition of the adenylyl cyclase and the cyclooxygenase induced a right shift of the carbachol concentration-response curve in periodontitis group whilst the opposite effect was observed in control group in the presence of db-cAMP and PGE(2). Parotid glands from rats with experimental periodontitis release more amylase in response to carbachol suggesting an interaction between Ca(2+) and cAMP in the fusion/exocytosis step of secretory vesicles. Copyright © 2011 Elsevier Ltd. All rights reserved.

Longitudinal and Immediate Effect of Kundalini Yoga on Salivary Levels of Cortisol and Activity of Alpha-Amylase and Its Effect on Perceived Stress.

PubMed

García-Sesnich, Jocelyn N; Flores, Mauricio Garrido; Ríos, Marcela Hernández; Aravena, Jorge Gamonal

2017-01-01

Stress is defined as an alteration of an organism's balance in response to a demand perceived from the environment. Diverse methods exist to evaluate physiological response. A noninvasive method is salivary measurement of cortisol and alpha-amylase. A growing body of evidence suggests that the regular practice of Yoga would be an effective treatment for stress. To determine the Kundalini Yoga (KY) effect, immediate and after 3 months of regular practice, on the perception of psychological stress and the salivary levels of cortisol and alpha-amylase activity. To determine the psychological perceived stress, levels of cortisol and alpha-amylase activity in saliva, and compare between the participants to KY classes performed for 3 months and a group that does not practice any type of yoga. The total sample consisted of 26 people between 18 and 45-year-old; 13 taking part in KY classes given at the Faculty of Dentistry, University of Chile and 13 controls. Salivary samples were collected, enzyme-linked immunosorbent assay was performed to quantify cortisol and kinetic reaction test was made to determine alpha-amylase activity. Perceived Stress Scale was applied at the beginning and at the end of the intervention. Statistical analysis was applied using Stata v11.1 software. Shapiro-Wilk test was used to determine data distribution. The paired analysis was fulfilled by t -test or Wilcoxon signed-rank test. T -test or Mann-Whitney's test was applied to compare longitudinal data. A statistical significance was considered when P < 0.05. KY practice had an immediate effect on salivary cortisol. The activity of alpha-amylase did not show significant changes. A significant decrease of perceived stress in the study group was found. KY practice shows an immediate effect on salivary cortisol levels and on perceived stress after 3 months of practice.