Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “AMYLASES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,783 records · Page 99Linked to original sources
PubMed1999

Modeling and optimization of alpha-amylase production in a recombinant yeast fed-batch culture taking account of the cell cycle population distribution.

A simple mathematical model describing the cell cycle dependency of rice alpha-amylase production by a recombinant yeast was constructed to investigate the efficiency of cell cycle population control. First, the effects of the glucose concentration and cultivation temperature on the specific growth rate, the specific production rate of rice alpha-amylase, and the distribution of the cell cycle population were studied under balanced growth conditions. On the basis of the results, parameter values for the mathematical model were then estimated. The proposed model was shown to be applicable for unbalanced as well as balanced growth phases. The optimal control strategy in respect of temperature and glucose concentration for maximum rice alpha-amylase production, taking into account the cell cycle population, was determined and the result was compared with that obtained by a simple mathematical model in which cell cycle distribution was not considered. Finally, the effect of the initial population of each cell cycle phase on the final amount of the product under optimal operational conditions was investigated. The simulation and experimental data coincided well with each other, and the model was used to optimize the control strategy for maximum alpha-amylase production.

Cell Cycle↗
PubMed1999

Cross-linked amylose tablets containing alpha-amylase: an enzymatically-controlled drug release system.

An oral controlled release system based on direct compression of cross-linked amylose (CLA) and drug powders was previously introduced. For drugs with limited solubility or for some drugs for which solubility can be influenced by variation of gastro-intestinal pH, a system is required to accelerate drug release. This paper describes a novel enzymatically-controlled drug release (ECDR) system based on the addition of alpha-amylase to CLA tablets, which can modulate the release kinetics of drugs. The alpha-amylase within the tablets is able to hydrolyze alpha-1-4-glucosidic bonds present in the CLA semisynthetic substrate. Increasing amounts of alpha-amylase (5 to 25 EU) within the tablets induced a significant decrease in release time from 24 to 6 h. High amounts of external alpha-amylase (300-6000 EU/l) had a slight effect on the release rate. Drug release from the ECDR system seems to be controlled by two sequential mechanisms: (a) hydration and swelling of CLA tablets followed by (b) internal enzymatic hydrolysis of the hydrated gel phase.

Amylose↗
PubMed2001

An investigation of the action of porcine pancreatic alpha-amylase on native and gelatinised starches.

The action of pancreatic alpha-amylase (EC 3.2.1.1) on various starches has been studied in order to achieve better understanding of how starch structural properties influence enzyme kinetic parameters. Such studies are important in seeking explanations for the wide differences reported in postprandial glycaemic and insulinaemic indices associated with different starchy foodstuffs. Using starches from a number of different sources, in both native and gelatinised forms, as substrates for porcine alpha-amylase, we showed by enzyme kinetic studies that adsorption of amylase to starch is of kinetic importance in the reaction mechanism, so that the relationship between reaction velocity and enzyme concentration [E0] is logarithmic and described by the Freundlich equation. Estimations of catalytic efficiencies were derived from measurements of kcat/Km performed with constant enzyme concentration so that comparisons between different starches were not complicated by the logarithmic relationship between E0 and reaction velocity. Such studies reveal that native starches from normal and waxy rice are slightly better substrates than those from wheat and potato. After gelatinisation at 100 degrees C, kcat/Km values increased by 13-fold (waxy rice) to 239-fold (potato). Phosphate present in potato starch may aid the swelling process during heating of suspensions; this seems to produce a very favourable substrate for the enzyme. Investigation of pre-heat treatment effects on wheat starch shows that the relationship between treatment and kcat/Km is not a simple one. The value of kcat/Km rises to reach a maximum at a pre-treatment temperature of 75 degrees C and then falls sharply if the treatment is conducted at higher temperatures. It is known that amylose is leached from starch granules during heating and dissolves. On cooling, the dissolved starch is likely to retrograde and become resistant to amylolysis. Thus the catalytic efficiency tends to fall. In addition, we find that the catalytic efficiency on the different starches varies inversely with their solubility and we interpret this finding on the assumption that the greater the solubility, the greater is the likelihood of retrogradation. We conclude that although alpha-amylase is present in high activity in digestive fluid, the enzymic hydrolysis of starch may be a limiting factor in carbohydrate digestion because of factors related to the physico-chemical properties of starchy foods.

Animals↗
PubMed1978

Utilization of industrial and agricultural by-products for fungal amylase production.

Attempts were made for using industrial and agricultural by-products and wastes as carbon and nitrogen sources in fermentation medium for alpha-amylase production by Aspergillus niger NRRL-337. The original carbon source of the basal medium was replaced by one of the following materials: rice bran, wheat bran, corn bran, corn starch, cane molasses, and glucose syrup. Rice bran proved to be the best carbon source that secured the highest amylase activity. The nitrogen source of the basal medium was then replaced by different cheap materials, viz: dried yeast, corn steep liquor, gluten-30, gluten-50, and corn steep precipitate. Corn steep precipitate proved to be superior in amylase production. In consideration of these results an economical medium that secured high activity, containing the following ingredients, was suggested: 2.5% corn steep precipitate, 7.2% rice bran, 0.1% MgSO4, 0.1% KH2PO4, and 0.1% CaCO3. From this medium fungal amylase was precipitated and purified. The pure enzyme gave the highest activity at 40 degrees C and pH 4.3.

Amylases↗
PubMed2000

Rapid, high-level expression of glycosylated rice alpha-amylase in transfected plants by an RNA viral vector.

Tobamoviral vectors have been developed for the heterologous expression of glycoproteins in plants. The rice alpha-amylase gene (OS103) was placed under the transcriptional control of a tobamovirus subgenomic promoter in a RNA viral vector. One to two weeks after inoculation, transfected Nicotiana benthamiana plants accumulated glycosylated alpha-amylase to levels of at least 5% total soluble protein. The 46kDa recombinant enzyme was purified, and its structural and biological properties were analyzed. Post-translational modifications of the secreted protein were compared to rice alpha-amylase expressed in amylolytic strains of Pichia pastoris and Saccharomyces cerevisiae. Endo-H analysis revealed that the alpha-amylase was moderately glycosylated in transfected plants and hyperglycosylated in yeast.

Amino Acid Sequence↗
PubMed2000

Structural similarities and evolutionary relationships in chloride-dependent alpha-amylases.

The alpha-amylase sequences contained in databanks were screened for the presence of amino acid residues Arg195, Asn298 and Arg/Lys337 forming the chloride-binding site of several specialized alpha-amylases allosterically activated by this anion. This search provides 38 alpha-amylases potentially binding a chloride ion. All belong to animals, including mammals, birds, insects, acari, nematodes, molluscs, crustaceans and are also found in three extremophilic Gram-negative bacteria. An evolutionary distance tree based on complete amino acid sequences was constructed, revealing four distinct clusters of species. On the basis of multiple sequence alignment and homology modeling, invariable structural elements were defined, corresponding to the active site, the substrate binding site, the accessory binding sites, the Ca(2+) and Cl(-) binding sites, a protease-like catalytic triad and disulfide bonds. The sequence variations within functional elements allowed engineering strategies to be proposed, aimed at identifying and modifying the specificity, activity and stability of chloride-dependent alpha-amylases.

Amino Acid Sequence↗
PubMed2002

SAMY, a novel mammalian reporter gene derived from Bacillus stearothermophilus alpha-amylase.

The Bacillus stearothermophilus alpha-amylase (amyS) is a heat-stable monomeric exoenzyme which catalyses random hydrolysis of 1,4-alpha-glucosidic linkages in polyglucosans. The Bacillus alpha-amylase was engineered for use as an intracellular (AmyS(Delta S)) as well as a secreted reporter protein (SAMY; secreted alpha-amylase) in mammalian cells. The 5' end of amyS containing the prokaryotic secretion signal was either deleted (amyS(Delta S)) or replaced by a murine immunoglobulin secretion signal. SAMY was cloned under control of the cytomegalovirus promoter (P(CMV)) in a mammalian expression vector or the promoter of the human elongation factor 1 alpha (P(EF1 alpha)) in a lentiviral expression context. A variety of mammalian and human cell lines growing as monolayers, in suspension or as three-dimensional spheroids were transfected/transduced with SAMY- or amyS(Delta S)-encoding expression/lentiviral vectors and alpha-amylase activity was measured in cell lysates and culture supernatants. These experiments showed that SAMY and AmyS(Delta S) were either secreted or remained intracellular as highly sensitive reporter enzymes. SAMY expression and detection was fully compatible with established SEAP (human secreted alkaline phosphatase) and u-PA(LMW) (low molecular weight urokinase-type plasminogen activator) reporter systems and could be used to quantify expression of up to three independent genes in one culture supernatant.

3T3 Cells↗
PubMed2000

New approach for separating Bacillus subtilis metalloprotease and alpha-amylase by affinity chromatography and for purifying neutral protease by hydrophobic chromatography.

Proteases are commonly used in the biscuit and cracker industry as processing aids. They cause moderate hydrolysis of gluten proteins and improve dough rheology to better control product texture and crunchiness. Commercial bacterial proteases are derived from Bacillus fermentation broth. As filtration and ultrafiltration are carried out as the only recovery steps, these preparations contain also alpha-amylase and beta-glucanase as the main side activities. The aim of this study is to purify and characterize the Bacillus subtilis metalloprotease from a commercial preparation, in order to study separately the impact of the protease activity with regards to its functionality on biscuit properties. Purification was achieved by means of affinity chromatography on Cibacron Blue and HIC as a polishing step. Affinity appeared to be the most appropriate matrix for large scale purification while ion exchange chromatography was inefficient in terms of recovery yields. The crude product was first loaded on a Hi Trap Blue column (34 microm, Pharmacia Biotech); elution was carried out with a gradient of NaCl in the presence of 1 mM ZnCl2. This step was only efficient in the presence of Zn cations, because this salt promoted both protease stabilization resulting in high recovery yields and also complexation of amylase units into dimers resulting in amylase retention on the column and a better separation of the 3 activities. Beta-glucanase was mostly non retained on the column and a part was coeluted with the protease. This protease fraction was then loaded on a Resource Phe column (15 microm, Pharmacia Biotech) in a last step of polishing. Elution was carried out with a linear gradient of 100-0% ammonium sulfate 1.3 M; protease was eluted at the beginning of the gradient and well separated from amylase and glucanase trace impurities. The homogeneity of the purified protease was confirmed by SDS-PAGE, which showed that its MW was about 38. pH and temperature optima were also determined on the fraction.

Bacillus subtilis↗
PubMed1999

Phenylarsine oxide evokes intracellular calcium increases and amylase secretion in isolated rat pancreatic acinar cells.

The effects of the thiol reagent, phenylarsine oxide (PAO, 10(-5)-10(-3) M ), a membrane-permeable trivalent arsenical compound that specifically complexes vicinal sulfhydryl groups of proteins to form stable ring structures, were studied by monitoring intracellular free calcium concentration ([Ca2+]i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. PAO increased [Ca2+]i by mobilizing calcium from intracellular stores, since this increase was observed in the absence of extracellular calcium. PAO also prevented the CCK-8-induced signal of [Ca2+]i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF-4). In addition to the effects of PAO on calcium mobilization, it caused a significant increase in amylase secretion and reduced the secretory response to either CCK-8 or AlF-4. The effects of PAO on both [Ca2+]i and amylase release were reversed by the sulfhydryl reducing agent, dithiothreitol (2 mM). Pretreatment of acinar cells with high concentration of ryanodine (50 microM) reduced the PAO-evoked calcium release. However, PAO was still able to release a small fraction of Ca2+ from acinar cells in which agonist-releasable Ca2+ pools had been previously depleted by thapsigargin (0.5 microM) and ryanodine receptors were blocked by 50 microM ryanodine. We conclude that, in pancreatic acinar cells, PAO mainly releases Ca2+ from the ryanodine-sensitive calcium pool and consequently induces amylase secretion. These effects are likely to be due to the oxidizing effects of this compound.

Amylases↗
PubMed2001

Digestion of legume starch granules by larvae of Zabrotes subfasciatus (Coleoptera: bruchidae) and the induction of alpha-amylases in response to different diets.

Zabrotes subfasciatus larvae possess three alpha-amylase isoforms as determined by in gel assays following SDS-PAGE. The two minor isoforms present lower electrophoretic mobility than the major form, and seem to occur as a heterodimer. When developed inside Vigna unguiculata (cowpea) seeds, fourth instar larvae have minor quantities of the slow-migrating forms, but when reared on seeds of Phaseolus vulgaris (common bean) or Phaseolus lunatus, the two slow-migrating forms are expressed in higher amounts, while activity of the major form was independent of the host seed. Larvae developing inside cowpea seeds at the beginning of the fourth instar were fed on flour from cotyledons of cowpea or common bean. Larvae fed on the common bean flour started to express the dimer in higher amounts when compared with the control larvae fed on cowpea flour. In an attempt to correlate differences between starch granules and the induction of alpha-amylases, a detailed study on the digestive process of the granules was conducted. Incorporation of purified starch granules into artificial diets did not induce the two minor alpha-amylases. The in vitro hydrolysis rates of purified granules and the pattern of dextrins liberated by the different alpha-amylases were similar for the two legume species. The starch granules enter the midgut extensively damaged, which may facilitate the access to the more susceptible parts of the granules to enzymatic attack.

Animals↗
PubMed2000

Salivary amylase activity of the phlebotomine sand fly, Lutzomyia longipalpis.

Both male and female adult stages of the sand fly Lutzomyia longipalpis have detectable amylase activity in their salivary glands, as indicated by formation of p-nitrophenyl-alpha-D-maltoside from p-nitrophenyl-alpha-D-octoside and by hydrolysis of 4-nitrophenyl-alpha-D-maltoheptaoside-4,6,-O-ethylidene. No salivary alpha-glucosidase was detected. Amylase activity was also found in the crop and midgut of female flies, although in a smaller amount. Salivary amylase is significantly reduced from the salivary glands immediately after a blood meal, as is the case with salivary alpha-glucosidases in mosquitoes. Presence of salivary gland amylase in these sand flies, and absence of salivary alpha-glucosidase, indicates that in nature these insects may have a significant intake of carbohydrates in the form of starch, as suggested by their plant-feeding behavior, previously demonstrated by Schlein and Warburg (Schlein, Y., Warburg, A., 1986. Phytophagy and the feeding cycle of Phlebotomus papatasi (Diptera: Psychodidae) under experimental conditions. Journal of Medical Entomology 23, 11-15), and Alexander and Usma (Alexander, B., Usma, M.C., 1994. Potential sources of sugar for the phlebotomine sandfly Lutzomyia youngi (Diptera: Psychodidae) in a Columbia coffee plantation. Ann. Trop. Med. Parasitol. 88, 543-549).

Animals↗
PubMed2000

Comparison of alpha-amylase and protease activities of a zoophytophagous and two phytozoophagous Heteroptera.

To better understand the nature of facultative phytophagy in the zoophytophagous Geocoris punctipes (Say), and facultative zoophagy in phytozoophagous Lygus hesperus (Knight) and Lygus lineolaris (Palisot de Beauvois), we compared the activities of both the starch digesting enzyme alpha-amylase and of general proteases in these species. The alpha-amylases and proteases were demonstrated in L. hesperus, L. lineolaris and G. punctipes. The presence of alpha-amylase in the salivary gland complexes of G. punctipes indicates a disposition of this species toward utilization of nutrients that can be derived only from plants, either directly from ingestion of plant macromolecules or from second-hand ingestion of plant material from the digestive system of their prey. The alpha-amylase activity in G. punctipes was much less than those of phytozoophagous L. hesperus and L. lineolaris. The relative importance of amylolytic activity and proteolytic activity is also discussed.

Adaptation, Physiological↗
PubMed2003

Hibernation reduces pancreatic amylase levels in ground squirrels.

Pancreatic enzyme levels in mammals are influenced by food intake and dietary composition. In this study, we examined the activity and expression of pancreatic amylase in a hibernating mammal, a natural model for long-term fasting. Pancreatic tissues were obtained from summer-active 13-lined ground squirrels and hibernating squirrels that had not eaten for at least 6 weeks. Amylase specific activity was reduced by approximately 50% in the torpid hibernators compared with summer squirrels, and immunoblot analysis revealed that amylase protein expression was reduced by approximately 40% in the hibernators. Similar reductions in amylase specific activity were observed in interbout euthermic hibernators. These results support a strong influence of food intake on pancreatic enzyme expression in hibernating mammals. The maintenance of basal levels of this key digestive enzyme at approximately 50% of summer values despite the extended winter fast likely facilitates the rapid resumption of digestive function after terminal arousal in the spring.

Amylases↗
PubMed2001

Parasympathetic modulation of amylase secretion by IFN gamma in murine submandibular glands.

IFN gamma is a pleiotropic cytokine that exerts immunologic and non-immunologic functions. We show here that at low doses (10 U/ml), it stimulates amylase secretion in murine submandibular glands (SMG) "via" muscarinic receptor activation, comparable to that produced by the muscarinic agonist carbachol. Both effects are blocked by atropine. NG-monomethyl-L-arginine (L-NMMA) and EGTA inhibited the cytokine effect on amylase secretion, involving the participation of a calcium-dependent isoform of nitric oxide synthase (NOS). We confirm NOS activation because IFN gamma stimulates nitrite production and enzyme activity in SMG. Carbachol (10(-7) M) did not modify basal nitric oxide production. In addition, both IFN gamma and carbachol increase prostaglandin E2 production in SMG, but while indomethacin potentiates IFN gamma effect on amylase secretion, it blunted amylase secretion exerted by carbachol. Thus, IFN gamma and carbachol stimulate IFN gamma secretion on SMG in a dose-dependent manner. Our results are pointing to neuroregulatory functions of IFN gamma in murine SMG, because it regulates its own levels in oral cavity, perhaps to exert a local immuno-surveillance.

Amylases↗
PubMed1999

Structure of raw starch-digesting Bacillus cereus beta-amylase complexed with maltose.

The crystals of beta-amylase from Bacillus cereus belong to space group P21 with the following cell dimensions: a = 57.70 A, b = 92.87 A, c = 65.93 A, and beta =101.95 degrees. The structures of free and maltose-bound beta-amylases were determined by X-ray crystallography at 2.1 and 2.5 A with R-factors of 0.170 and 0.164, respectively. The final model of the maltose-bound form comprises 516 amino acid residues, four maltose molecules, 275 water molecules, one Ca2+, one acetate, and one sulfate ion. The enzyme consists of a core (beta/alpha)8-barrel domain (residues 5-434) and a C-terminal starch-binding domain (residues 435-613). Besides the active site in the core where two maltose molecules are bound in tandem, two novel maltose-binding sites were found in the core L4 region and in the C-terminal domain. The structure of the core domain is similar to that of soybean beta-amylase except for the L4 maltose-binding site, whereas the C-terminal domain has the same secondary structure as domain E of cyclodextrin glucosyltransferase. These two maltose-binding sites are 32-36 A apart from the active site. These results indicate that the ability of B. cereus beta-amylase to digest raw starch can be attributed to the additional two maltose-binding sites.

Acetates↗
PubMedDate not supplied

Macroaffinity ligand-facilitated three-phase partitioning (MLFTPP) of alpha-amylases using a modified alginate.

The crude extracts of alpha-amylases when mixed with alginate, tert-butyl alcohol, and ammonium sulfate resulted in an interfacial precipitate containing polymer-bound amylase. The precipitate was dissolved in 1 M maltose to recover alpha-amylase activity. The recovery of alpha-amylases were 74%, 77%, and 92% in the case of Bacillus amyloliquefaciens, wheat germ, and porcine pancreas, respectively. All purified preparations showed a single band on SDS-PAGE.

Adsorption↗
PubMedDate not supplied

Stable high-copy-number integration of Aspergillus oryzae alpha-AMYLASE cDNA in an industrial baker's yeast strain.

The Aspergillus oryzae alpha-amylase cDNA was placed under the control of the Saccharomyces cerevisiae actin promoter (pACT1) and introduced into the ribosomal DNA locus of an industrial baker's yeast strain. To obtain a strain eligible for commercial use, we constructed an integrative cassette lacking bacterial DNA sequences but containing the alpha-amylase cDNA and ribosomal DNA sequences to target the integration to this locus. High-copy-number integrants were obtained including a defective TRP1d promoter in the integrative cassette. We selected one transformant, Rib-AMY (CECT10872), in which the multi-integrated sequences were stable even after 200 generations of growth in nonselective medium. This transformant also expressed and secreted high levels of alpha-amylase. Bread made with this strain had a higher volume, lower density, and softer crumbs than bread made with a control strain. The Rib-AMY transformant also was useful in retarding bread firming. This new strain fulfills all the requirements for commercial utilization and should reduce or eliminate the requirement for addition of exogenous alpha-amylase to the flour, reducing allergenic work-related symptoms due to this enzyme.

Aspergillus oryzae↗
PubMed2003

Purification and characterization of amylases from small abalone (Sulculus diversicolor aquatilis).

Amylases II-1 and II-2 with molecular weights of 55.7 and 65 kDa, respectively, were purified to electrophoretical homogeneity from small abalone (Sulculus diversicolor aquatilis) by ammonium sulfate fractionation, Sepharose CL-6B, CM-Sepharose CL-6B, and Sephacryl S-100 chromatographs. They had optimal temperatures of 45 and 50 degrees C and an optimal pH of 6.0. The purified amylases were stable at pH 5.0-8.0 and 6.0-8.0, respectively. They were completely or partially inhibited by Hg(2+), Cu(2+), Cd(2+), Zn(2+), iodoacetamide, phenylmethanesulfonyl fluoride, and N-ethylmaleimide, suggesting the existence of cysteine at their active sites. Digestion tests against various polysaccharides suggested that the purified amylases II-1 and II-2 are neoamylases which can hydrolyze both alpha-1,4 and alpha-1,6 glucosidic bonds. Amylase II-2 might be an exo- and II-1 an endo-/exo-amylase.

Amylases↗