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PubMed2002

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

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.

Bacteria↗

PubMed1992

'In vitro' action of histamine and cimetidine on amylase secretion in the rat pancreas.

We report here the effect of histamine on amylase secretion in isolated lobules from the rat pancreas. Different concentrations of histamine increased amylase secretion, while the stimulatory effect was reduced by cimetidine although not by chlorpheniramine. These findings suggest that pancreatic lobules have H2 receptors. On the other hand, bethanechol induced a stimulatory effect on pancreatic lobules that was inhibited by cimetidine. Vibrio cholerae toxin stimulated amylase secretion, but this effect was not reduced by cimetidine. Cimetidine may thus block the stimulated amylase secretion interfering with the Ca2+ messenger system.

Amylases↗

PubMed2007

Stability and catalytic activity of alpha-amylase from barley malt at different pressure-temperature conditions.

The impact of high hydrostatic pressure and temperature on the stability and catalytic activity of alpha-amylase from barley malt has been investigated. Inactivation experiments with alpha-amylase in the presence and absence of calcium ions have been carried out under combined pressure-temperature treatments in the range of 0.1-800 MPa and 30-75 degrees C. A stabilizing effect of Ca(2+) ions on the enzyme was found at all pressure-temperature combinations investigated. Kinetic analysis showed deviations of simple first-order reactions which were attributed to the presence of isoenzyme fractions. Polynomial models were used to describe the pressure-temperature dependence of the inactivation rate constants. Derived from that, pressure-temperature isokinetic diagrams were constructed, indicating synergistic and antagonistic effects of pressure and temperature on the inactivation of alpha-amylase. Pressure up to 200 MPa significantly stabilized the enzyme against temperature-induced inactivation. On the other hand, pressure also hampers the catalytic activity of alpha-amylase and a progressive deceleration of the conversion rate was detected at all temperatures investigated. However, for the overall reaction of blocked p-nitrophenyl maltoheptaoside cleavage and simultaneous occurring enzyme inactivation in ACES buffer (0.1 M, pH 5.6, 3.8 mM CaCl(2)), a maximum of substrate cleavage was identified at 152 MPa and 64 degrees C, yielding approximately 25% higher substrate conversion after 30 min, as compared to the maximum at ambient pressure and 59 degrees C.

Calcium↗

PubMed2004

Adaptation of class-13 alpha-amylases to diverse living conditions.

There are currently 35 available nonredundant molecular structures of class-13 alpha-amylases (EC 3.2.1.1), mostly from microbial organisms living under a wide range of environmental conditions. One of the most recent additions has been the first alpha-amylase structure of a hyperthermophilic archaeon [Linden et al., J. Biol. Chem. 2003, 278, 9875-9884]. The structure has been used for comparative analyses with a representative set of three alpha-amylases from thermophilic, mesophilic and psychrophilic sources to identify molecular parameters for environmental adaptation. Our analysis supports generally observed trends such as an increase in structural compactness as well as an increase in salt bridges in order to cope with high-temperature conditions. The two representative thermophilic structures used in this comparative study have independently evolved di-metal centres--not present in the mesophilic and psychrophilic structures--in the vicinity of the active site. These observations may provide impetus for the design of alpha-amylases with improved molecular properties to enhance their utility in biotechnological processes.

Adaptation, Physiological↗

PubMed1986

The protein inhibitors of amylases and peptidases isolated from cereal grains.

The peptidase and amylase inhibitors were isolated from rye and preliminarily purified to investigate some of their properties. It was shown, that both are of albumin character despite the acetic acid solubility of the former. They are not active against the native cereal grain peptidases or amylases (rye, wheat, triticale) but the latter inhibited the animal (hog pancreas, man salivary) and bacterial (Thermamyl, NOVO) alpha-amylases. In the long-term experiments on various rye genotypes there was shown, that the peptidase inhibitor level was rather differentiated between them, but more stable as concerns particular ones. On the other hand the level differentiation of alpha-amylase inhibitor between the genotypes was rather small, but that of climate much more significant.

Amylases↗

PubMed1993

Extrahepatic peribiliary glands express alpha-amylase isozymes, trypsin and pancreatic lipase: an immunohistochemical analysis.

We examined expression of alpha-amylase isozymes (pancreatic and salivary), trypsin and pancreatic lipase on the epithelium of extrahepatic peribiliary glands immunohistochemically using 53 autopsied normal extrahepatic bile ducts. Three parts of the extrahepatic bile duct (common bile duct, intrapancreatic bile duct and bile duct at the ampulla of Vater) were examined in each case. Histologically, the extrahepatic bile duct harbored branched tubular glands (extrahepatic peribiliary glands). Extrahepatic peribiliary glands were few in the common bile duct and intrapancreatic bile duct and numerous in the bile duct at the ampulla of Vater. Immunohistochemically, pancreatic alpha-amylase was expressed in the epithelium of extrahepatic peribiliary glands in 42 cases (79%). Salivary alpha-amylase was expressed in the epithelium of the glands in 38 cases (72%). Trypsin was expressed in the epithelium of the glands in 32 cases (60%). Pancreatic lipase was expressed in the epithelium of the glands in 45 cases (85%). The immunoreactivity of these enzymes was granular and located in the supranuclear cytoplasm (corresponding to the Golgi apparatus) of the epithelium of the glands. We confirmed the specificity of the immunoreactivity of these enzymes with various methods. These results suggest that extrahepatic peribiliary glands produce alpha-amylase isozymes, trypsin and pancreatic lipase and secrete these enzymes into lumens of the extrahepatic bile duct. The secreted enzymes may play an important role in the physiology of the extrahepatic bile duct and bile.

Adult↗

PubMed1991

New method for the determination of pancreatic amylase evaluated.

We have carried out a multicenter evaluation of a new reagent carrier for Reflotron, specific for pancreatic amylase where the salivary isoenzyme is inhibited by two specific monoclonal antibodies. This new procedure combines easy handling with low imprecision (median CV less than 3%) in control material, serum, heparinized blood, and plasma) and close correlation (r = 0.991 to 0.999) with established manual and automated methods. The same close correlation was found with values obtained from either venous or capillary finger-stick blood. Salivary amylase up to 54 kU/L (37 degrees C) was inhibited to about 97%. Endogenous interference by hemoglobin, bilirubin, triglycerides, cholesterol, or hematocrit was found to be negligible within a wide range of interferent concentrations. Out of a panel of 28 commonly used drugs it was shown that only two (ascorbic acid and paracetamol), and then only at toxic concentrations, caused a deviation in amylase activity of greater than 10%. From the results of this study we conclude that this new method is suitable for highly precise and accurate measurements of pancreatic amylase in emergency and routine laboratories.

Amylases↗

PubMed2001

Effect of substrate size on immunoinhibition of amylase activity.

Immunoinhibition assays are hypothesized to work by antibodies blocking substrate access to enzyme active sites. To test this hypothesis, the inhibition of amylase isoenzymes by monoclonal and polyclonal antisera was assessed using substrates of varying sizes: chromogenic sustrates 3, 5, or 7 glucose units in length, novel synthetic macromolecular substrates, and starch. The synthetic macromolecular substrates consisted of small oligosaccharide substrates linked to an inert polymer that conferred a large size to substrate molecules as determined by gel filtration chromatography. When substrate size increased, amylase activity could be inhibited equivalently by antibody concentrations that are 10-fold lower. Progressively less polyclonal serum was required to inhibit amylase activity as substrate length increased from 3 to 5 to 7 glucose units and as size was increased by linkage to a polymer. Different effects of substrate size were observed with two monoclonal antibodies. One monoclonal antibody blocked amylase activity independent of substrate size, while another monoclonal antibody had little inhibitory effect except using starch as substrate. We conclude that use of larger substrates can expand the repertoire of inhibitory epitopes on enzymes and convert a noninhibitory antibody into an inhibitory one.

Amylases↗

PubMed1996

Crystallization and preliminary X-ray diffraction studies of alpha-amylase from the antarctic psychrophile Alteromonas haloplanctis A23.

A cold-active alpha-amylase was purified from culture supernatants of the antarctic psychrophile Alteromonas haloplanctis A23 grown at 4 degrees C. In order to contribute to the understanding of the molecular basis of cold adaptations, crystallographic studies of this cold-adapted enzyme have been initiated because a three-dimensional structure of a mesophilic counterpart, pig pancreatic alpha-amylase, already exists. alpha-Amylase from A. haloplanctis, which shares 53% sequence identity with pig pancreatic alpha-amylase, has been crystallized and data to 1.85 A have been collected. The space group is found to be C222(1) with a = 71.40 A, b = 138.88 A, and c = 115.66 A. Until now, a three-dimensional structure of a psychrophilic enzyme is lacking.

Bacterial Proteins↗

PubMed2006

Screening of traditional antidiabetic medicinal plants of Mauritius for possible alpha-amylase inhibitory effects in vitro.

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.

Animals↗

PubMed1994

Efficient expression and secretion of recombinant alpha amylase in Pichia pastoris using two different signal sequences.

We have cloned and expressed a bacterial thermostable alpha amylase gene in Pichia pastoris using the methanol-controlled alcohol oxidase (AOX1) promoter. Two integrative vectors were constructed with two different secretion signal sequences in order to obtain efficient secretion of the protein. One vector contains the structural gene encoding the mature alpha amylase fused to the SUC2 gene signal sequence from Saccharomyces cerevisiae. In the other vector, the alpha amylase is expressed with its own signal sequence. In both cases, the alpha amylase were secreted into the culture medium with high efficiency, around 2.5 and 0.9 g/l respectively.

Alcohol Oxidoreductases↗

PubMed1999

Comparative study of the inhibition of alpha-glucosidase, alpha-amylase, and cyclomaltodextrin glucanosyltransferase by acarbose, isoacarbose, and acarviosine-glucose.

Bacillus stearothermophilus maltogenic amylase hydrolyzes the first glycosidic linkage of acarbose to give acarviosine-glucose. In the presence of carbohydrate acceptors, acarviosine-glucose is primarily transferred to the C-6 position of the acceptor. When d-glucose is the acceptor, isoacarbose is formed. Acarbose, acarviosine-glucose, and isoacarbose were compared as inhibitors of alpha-glucosidase, alpha-amylase, and cyclomaltodextrin glucanosyltransferase. The three inhibitors were found to be competitive inhibitors for alpha-glucosidase and mixed noncompetitive inhibitors for alpha-amylase and cyclomaltodextrin glucanosyltransferase. The K(i) values were dependent on the type of enzyme and their source. Acarviosine-glucose was a potent inhibitor for baker's yeast alpha-glucosidase, inhibiting 430 times more than acarbose, and was an excellent inhibitor for cyclomaltodextrin glucanosyltransferase, inhibiting 6 times more than acarbose. Isoacarbose was the most effective inhibitor of alpha-amylase and cyclomaltodextrin glucanosyltransferase, inhibiting 15.2 and 2.0 times more than acarbose, respectively.

Acarbose↗

PubMed1997

Inhibitory action of sphingosine or ceramide on amylase secretion from isolated rat pancreatic acini.

Sphingosine and ceramide, products of sphingomyelin hydrosis by sphingomyelinase, have recently been regarded as second messengers for cell biological actions. On the other hand, exocrine pancreas is a typical organ to perform regulatory secretion of digestive enzymes, depending upon extracellular signals. We investigated the effects of sphingosine or ceramide on amylase secretion from isolated rat pancreatic acini. Either sphingosine or cell-permeable ceramide inhibits CCK8- or carbachol-induced enzyme secretion from isolated rat pancreatic acini in a dose-dependent manner. Sphingosine or ceramide itself does not affect basal amylase secretion from the acini. Ceramide also inhibits NaF-induced amylase secretion, indicating that it acts post the activation of receptor-linked GTP-binding protein. In our experiments, ceramide inhibited Ca2+ ionophore-induced amylase secretion, but not phorbol ester-induced secretion. These results indicate that ceramide affects secretory processes post intracellular Ca2+ mobilization in the exocrine pancreas.

Amylases↗

PubMed1997

Inhibition of expression of PKC-alpha by antisense mRNA is associated with diminished cell growth and inhibition of amylase secretion by AR4-2J cells.

AR4-2J pancreatoma cells were stably transfected with an expression vector containing the cDNA for PKC-alpha in the antisense orientation. Transfectants designated antisense-alpha AA1, AA2, and AA3 exhibited marked reductions in PKC-alpha expression and decrements in cell growth. The magnitude of the decrement in cell growth paralleled the reduction in PKC-alpha expression, i.e., AA3 > AA1 > AA2. The ability of dexamethasone to induce cell differentiation as assessed by a rise in cellular amylase levels was not markedly affected by the reduction in PKC-alpha expression. Unstimulated amylase release was attenuated in AA1 cells and almost completely blocked in AA2 transfectants. The AA2 transfectant cell line failed to elicit a secretory response to caerulein, and the AA1 transfectant exhibited a lack of the secondary phase of stimulated amylase secretion. These findings demonstrate that PKC-alpha is involved in the mechanisms regulating growth and secretion in AR4-2J cells, but is not necessary for the induction of amylase stores following differentiation.

Amylases↗

PubMed1994

Refined molecular structure of pig pancreatic alpha-amylase at 2.1 A resolution.

The structure of pig pancreatic alpha-amylase has been determined by X-ray diffraction analysis using multiple isomorphous replacement in a crystal of space group P2(1)2(1)2(1) (a = 70.6 A, b = 114.8 A, c = 118.8 A) containing nearly 75% solvent. The structure was refined by simulated annealing and Powell minimization, as monitored by 2Fo-Fc difference Fourier syntheses, to a conventional R of 0.168 at 2.1 A resolution. The final model consists of all 496 amino acid residues, a chloride and a calcium ion, 145 water molecules and an endogenous disaccharide molecule that contiguously links protein molecules related by the 2(1) crystallographic operator along x. The protein is composed of a large domain (amino acid residues 1 to 403) featuring a central alpha ta-barrel of eight parallel strands and connecting helices with a prominent excursion between strand beta 3 and helix alpha 3 (amino acid residues 100 to 168). The final 93 amino acid residues at the carboxyl terminus form a second small domain consisting of a compact Greek key beta-barrel. The domains are tightly associated through hydrophobic interfaces. The beta 3/alpha 3 excursion and portions of the central alpha/beta-barrel provide four protein ligands to the tightly bound Ca ion; three water molecules complete the coordination. The Cl- ion is bound within one end of the alpha/beta-barrel by two arginine residues in a manner suggesting a plausible mechanism for its allosteric activation of the enzyme. A crystalline complex of the pancreatic alpha-amylase with alpha-cyclodextrin, a cyclic substrate analog of six glucose residues, reveals, in difference Fourier maps, three unique binding sites. One of the alpha-cyclodextrin sites is near the center of the long polysaccharide binding cleft that traverses one end of the alpha/beta-barrel, another is at the extreme of this cleft. By symmetry this can also be considered as two half sites located at the extremes of the active site cleft. This latter alpha-cyclodextrin displaces the endogenous disaccharide when it binds and, along with the first sugar ring, delineates the extended starch binding site. The third alpha-cyclodextrin binds at an "accessory site" near the edge of the protein and is quite distant from the polysaccharide binding cleft. Its presence explains the multivalency of alpha-amylase binding to dextrins in solution. The extended active site cleft is formed by large, sweeping, connecting loops at one end of the alpha/beta-barrel. These include three sequence segments that are highly conserved among alpha-amylases.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1998

Molecular structure of a barley alpha-amylase-inhibitor complex: implications for starch binding and catalysis.

alpha-Amylases are widely occurring, multidomain proteins with a catalytic (beta/alpha)8-barrel. In barley alpha-amylase, insight into the catalytic mechanism is gained from the X-ray crystal structure of its molecular complex with acarbose, a pseudotetrasaccharide that acts like a transition-state analogue and which is shown to bind at two specific regions of the enzyme. The structure of the complex has been refined to an R-factor of 15.1% for all observations with Fo>sigma(Fo) between 10 and 2.8 A resolution. A difference Fourier map produced after refinement of the native structure against the data of the acarbose complex clearly revealed density corresponding to two oligosaccharide-binding sites. One of these is defined as the surface-located starch granule-binding site characteristic of cereal alpha-amylases. It involves stacking of two acarbose rings on Trp276 and Trp277. The other binding region is the active site covering subsites -1, +1 and +2. Here, Glu204 is positioned to act in general acid/base catalysis protonating the glucosidic oxygen atom assisted by Asp289. A water molecule that bridges Glu204 and Asp289 is found at the entrance cavity containing a total of five water molecules. This water molecule is proposed to reprotonate Glu204 and supply the hydroxyl ion for nucleophilic attack on the glucosyl C1 atom. Asp 179 acts as the nucleophile that can bind covalently to the substrate intermediate after bond cleavage. The present complex structure together with the conservation of active-site residues among alpha-amylases and related enzymes, are consistent with a common catalytic mechanism for this class of retaining carbohydrases.

Acarbose↗

PubMed2000

Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase.

Bacillus licheniformis alpha-amylase (BLA) is a starch-degrading enzyme that is highly thermostable although it is produced by a rather mesophilic organism. Over the last decade, the origin of BLA thermal properties has been extensively investigated in both academic and industrial laboratories, yet it is poorly understood. Here, we have used structure-based mutagenesis in order to probe the role of amino acid residues previously proposed as being important for BLA thermostability. Residues involved in salt-bridges, calcium binding or potential deamidation processes have been selected and replaced with various amino acids using a site-directed mutagenesis method, based on informational suppression. A total of 175 amylase variants were created and analysed in vitro. Active amylase variants were tested for thermostability by measuring residual activities after incubation at high temperature. Out of the 15 target residues, seven (Asp121, Asn126, Asp164, Asn192, Asp200, Asp204 and Ala269) were found to be particularly intolerant to any amino acid substitutions, some of which lead to very unstable mutant enzymes. By contrast, three asparagine residues (Asn172, Asn188 and Asn190) could be replaced with amino acid residues that significantly increase the thermostability compared to the wild-type enzyme. The highest stabilization event resulted from the substitution of phenylalanine in place of asparagine at position 190, leading to a sixfold increase of the enzyme's half-life at 80 degrees C (pH 5.6, 0.1 mM CaCl(2)). These results, combined with those of previous mutational analyses, show that the structural determinants contributing to the overall thermostability of BLA concentrate in domain B and at its interface with the central A domain. This region contains a triadic Ca-Na-Ca metal-binding site that appears extremely sensitive to any modification that may alter or reinforce the network of electrostatic interactions entrapping the metal ions. In particular, a loop spanning from residue 178 to 199, which undergoes pronounced conformational changes upon removal of calcium, appears to be the key feature for maintaining the enzyme structural integrity. Outside this region, most salt-bridges that were destroyed by mutations were found to be dispensable, except for an Asp121-Arg127 salt-bridge that contributes to the enhanced thermostability of BLA compared to other homologous bacterial alpha-amylases. Finally, our studies demonstrate that the natural resistance of BLA against high temperature is not optimized and can be enhanced further through various means, including the removal of possibly deamidating residues.

Alanine↗

PubMed1996

Overexpression, purification, and characterization of recombinant barley alpha-amylases 1 and 2 secreted by the methylotrophic yeast Pichia pastoris.

Recombinant barley alpha-amylase isozymes 1 and 2 were secreted by Pichia pastoris at up to 50 and 1 mg/liter, respectively, representing approximately a 50-fold increase compared to the levels of the heterologous expression by Saccharomyces cerevisiae. The cDNA clones E or pM/C encoding isozymes 1 and 2, respectively, were placed under the control of regulatory sequences from the Pichia AOX1 gene in the vector pHIL-D2. Both isozymes were effectively secreted to the medium as directed by their own signal sequences and easily purified to homogeneity in quantitative yield by affinity chromatography on beta-cyclodextrin-Sepharose. The N-terminal sequence, pI, and Mr indicated that native-like processing took place. Electrospray ionization mass spectrometry, however, revealed microheterogeneity for recombinant isozyme 1. While Mr of one recombinant isozyme 1 form of 45,452 was in excellent agreement with a value of 45,447 calculated from the sequence, liquid chromatography/mass spectrometry of endo Lys C-generated peptides followed by tandem mass spectrometry on a nanoelectrospray ionization/mass spectrometry/mass spectrometry system identified additional recombinant isozyme 1 forms to be glycosylated on Thr410, N-acetylated on His1, S-glutathionylated on Cys95, or C-terminally truncated of -412RS, -411QRS, and -410LQRS. The recombinant enzymes and the alpha-amylases from barley malt closely resembled each other in enzymatic activity on insoluble Blue Starch, amylose of degree of polymerization 17, and 2-chloro-4-nitrophenyl beta-D-maltoheptaoside as well as in Ca2+ dependency of activity. Pichia pastoris thus produced in high yields recombinant alpha-amylase that is similar with respect to structure and function to the enzyme purified from malt extracts. This greatly facilitates future mutational analysis of barley alpha-amylase in order to probe structure/function relationships.

Amylases↗