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PubMed2005

Purification and characterization of an extracellular alpha-amylase from Bacillus subtilis AX20.

A Bacillus subtilis AX20 from soil with ability to produce extracellular alpha-amylases was isolated. The characterization of microorganism was performed by biochemical tests as well as 16S rDNA sequencing. Maximum amylase activity (38 U/ml) was obtained at stationery phase when the culture was grown at 37 degrees C. The enzyme was purified to homogeneity with an overall recovery of 24.2% and specific activity of 4133 U/mg. The native protein showed a molecular mass of 149 kDa composed of a homodimer of 78 kDa polypeptide by SDS-PAGE. The optimum pH and temperature of the amylase were 6 and 55 degrees C, respectively. The enzyme was inhibited by Hg(2+), Ag(2+), and Cu(2+) and it did not show an obligate requirement of metal ions. The enzyme was not inhibited by EDTA or EGTA, suggesting that this enzyme is not a metalloenzyme. The end products of corn starch and soluble starch were glucose (70-75%) and maltose (20-25%). Rapid reduction of blue value and the end products suggest an endo mode of action for the amylase. The purified amylase shows interesting properties useful for industrial applications.

Bacillus subtilis↗
PubMed2002

Comparison of amylase mRNAs from rat parotid gland, pancreas and liver using reverse transcriptase-polymerase chain reaction.

The expression of mRNA for amylase was examined using the reverse transcriptase-polymerase chain reaction (RT-PCR). An amylase product was strongly detected in parotid and pancreas, but less strongly in liver. The degree of identity between the PCR products was assessed by restriction-enzyme mapping using two restriction enzymes, EcoRI and ScaI, and DNA sequencing. The PCR product from pancreas was cut by both EcoRI and ScaI, while the products from parotid and liver were cut by EcoRI but not by ScaI. The sequence of the parotid product was 90.4% homologous to that of the pancreas, and 100% homologous to that of the liver. These results indicate that the same amylase mRNA may be expressed in parotid and liver. In addition, the expression of amylase mRNAs in other rat tissues was investigated using RT-PCR, and the sensitivity of each PCR product to ScaI was tested. A weak single band was detected in submandibular gland, sublingual gland and stomach. ScaI digestion cut the stomach product into two fragments, but had no effect on the submandibular and sublingual products. Thus, it may be possible to classify amylase isoenzymes into pancreatic and parotid types based on the sensitivity of their PCR products to ScaI.

Amylases↗
PubMed2001

Increased serum amylase and lipase in fructose malabsorbers.

BACKGROUND: Fructose malabsorption is frequently seen in the general population and is characterised by the inability to absorb fructose efficiently. Due to fructose malabsorption, fructose reaches the colon where it is broken down by bacteria to short fatty acids, CO(2) and H(2). Bloating, cramps, osmotic diarrhea and other symptoms of irritable bowel syndrome are the consequence. We recently found that fructose malabsorption is associated with low plasma folic acid concentrations and low serum tryptophan and zinc. Because fructose malabsorption apparently is associated not only with malabsorption of other nutrients, but also with abdominal discomfort, it was of interest to examine whether mild pancreatitis may be involved. METHODS: We retrospectively examined our data in 159 otherwise healthy adults (110 females, 49 males) aged 14-84 years (mean 45.6+/-14.4 S.D.) with gastrointestinal complaints for serum amylase and serum lipase concentrations. The patients have been tested earlier for fructose malabsorption and lactose maldigestion by measuring breath H(2) concentrations after an oral dose of 25 g fructose and 50 g lactose, respectively, 1 week apart. RESULTS: Fructose malabsorption (H(2) concentrations > or =20 ppm over baseline values) was detected in 107 of 159 individuals (67.3%). These subjects with fructose malabsorption presented with significantly higher serum amylase concentrations (73.1 U/l+/-25.7 S.D.) compared to individuals with normal fructose absorption (59.6 U/l+17.9 S.D; p=0.0009). Fructose malabsorbers also presented with higher serum lipase concentrations (122.0 U/l+/-100.3 S.D.) compared to normals (89.5 U/l+/-46.5 S.D.; p<0.05). To determine whether this finding is a consequence of any sort of malabsorption syndrome or whether it is specific for fructose malabsorption, all subjects were screened for lactose maldigestion. Lactose maldigestion (H(2) concentrations>20 ppm over baseline after lactose loading) was found in 50 of 159 individuals (31.4%). There were no significant differences in either amylase or lipase concentrations in lactose maldigestors. CONCLUSION: Serum amylase and lipase concentrations are higher in subjects with fructose malabsorption compared to normals. Therefore, fructose malabsorption should be considered as a differential diagnosis in moderately elevated serum amylase.

Adolescent↗
PubMed2002

A restriction endonuclease assay for expression of human alpha-amylase isozymes.

BACKGROUND: The alpha-amylase isozymes can be detected separately by electrophoresis; however, sometimes the identification is difficult because of their microheterogeneity. In the present study, we tried to establish a convenient method for the detection of alpha-amylase isozyme expression. METHODS: The procedure is based on three different restriction sites presented in those genes; a PstI site in both AMY 2A and 2B genes, a HaeII site in both AMY 1 and 2A genes, and a BamHI site in AMY 2B gene. After amplification from total tissue RNAs by RT-PCR with primers that were able to cover each exon, the products were cleaved with corresponding restriction endonucleases. RESULTS: This method was applied to human samples from the parotid gland, liver (non-hepatoma), hepatoma and white blood cells (WBCs). The results indicated that the parotid gland and hepatoma (also liver) clearly expressed AMY 1 and AMY 2B genes, respectively. However, AMY 2B gene was also expressed apparently in WBCs, which produced salivary-type isozyme of the alpha-amylase, although the amylase protein was not able to identify for the hepatic isozyme. CONCLUSIONS: The method presented here might be convenient and useful for the determination of alpha-amylase isozyme expression in humans.

Carcinoma, Hepatocellular↗
PubMed1998

Determination of alpha-amylase using 4-O-beta-D-galactopyranosylmaltotetraose (Gal-G4) as a substrate.

A new substrate, 4-O-beta-D-galactopyranosylmaltotetraose (Gal-G4) is applied for the determination of alpha-amylase in serum and urine in a coupled assay with alpha-glucosidase (EC 3.2.1.20), glucokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) as auxiliary enzymes. Gal-G4 having a 4-position of the non-reducing-end glucose residue modified by a beta-galactopyranose group is resistant for degradation by alpha-glucosidase as auxiliary enzyme. Moreover, this substrate is hydrolyzed at just one position by alpha-amylase in serum and urine. More than 99% of the products generated from Gal-G4 by alpha-amylase are identified 4-O-beta-D-galactopyranosylmaltose (Gal-G2), maltose, respectively. Glucose and maltose do not interfere the value of alpha-amylase activity at least up to 0.056 mmol/l (1 g/dl) glucose and 0.027 mmol/l (1 g/dl) maltose, respectively. We are now carrying out this work under the authority of The Enzyme committee of Japanese Society of Clinical Chemistry (JSCC) as a standard method for determination of alpha-amylase in clinical chemistry.

Carbohydrate Conformation↗
PubMed2001

Purification and characterization of two alpha-amylase inhibitors from seeds of tepary bean (Phaseolus acutifolius A. Gray).

Two proteinaceous alpha-amylase inhibitors termed alphaAI-Pa1 and alphaAI-Pa2 were purified from seeds of a cultivated tepary bean (Phaseolus acutifolius A. Gray, cv. PI311897). The two inhibitors differed in their specificity towards alpha-amylases of insect pests such as bruchids, although neither showed any inhibitory activity against alpha-amylases of mammalian, bacterial or fungal origin. AlphaAI-Pa2 resembles two common bean inhibitors, alphaAI-1 and alphaAI-2, in several characteristics such as N-terminal amino acid sequences and oligomeric structure being composed of alpha and beta subunits. In contrast alphaAI-Pa1 is composed of a single glycopolypeptide with a molecular mass of 35 kDa, and its N-terminal amino acid sequence resembled that of seed lectins in tepary bean and common bean. The information on the two tepary bean alpha-amylase inhibitors may be useful not only for providing insight into critical structure for the specificity towards different alpha-amylase enzymes but also for enhancing insect resistance in crops.

Amino Acid Sequence↗
PubMed1996

Pituitary adenylate cyclase-activating peptide stimulates amylase release and cyclic adenosine monophosphate production in pancreatic acinar cells.

BACKGROUND: Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide member of the secretin/glucagon family of peptides, which also includes vasoactive intestinal peptide (VIP). This study was designed to examine the effects of PACAP-38 on pancreatic exocrine function in vitro. METHODS: Amylase release and signal transduction pathways were examined by using dispersed guinea pig acinar cells. RESULTS: PACAP-38 produced dose-dependent increases in amylase release. Coincubation of PACAP-38 (1 nmol/L) additively augmented amylase release stimulated by cholecystokinin, carbachol, or bombesin. Coexposure with PACAP-38 did not affect amylase release in response to maximally stimulatory concentrations of VIP (1 nmol/L). The VIP antagonist, [N-Ac-Tyr1,D-Phe2]-GRF(1-29)-NH2, abolished amylase release in response to either PACAP-38 or VIP. Dose-dependent increases in cyclic adenosine monophosphate production were noted on exposure to PACAP-38, with a 70-fold increment relative to the control value at 1 nmol/L PACAP-38. Inositol phosphate turnover and intracellular Ca2+ levels were not affected by PACAP-38 exposure. CONCLUSIONS: In guinea pig pancreatic acini, VIP preferring receptors for PACAP-38 are functionally linked to adenylate cyclase.

Amylases↗
PubMed1977

On the cause of raised serum-amylase in diabetic ketoacidosis.

7 of 13 patients with diabetic ketoacidosis had raised serum-amylase concentrations. Isoenzymes analysis by polyacrylamide-gel electrophoresis showed that salivary-type amylases and not pancreatic-type amylases were responsible for the rise in 6 or the 7 cases. This demonstrates that the pancreas is usually not the origin of the increase in serum-amylase. Since salivary-type amylases are widely distributed in glandular epithelium, it is suggested that hyperamylasaemia in diabetic ketoacidosis is most often caused by systemic derangement of carbohydrate metabolism.

Adolescent↗
PubMed1997

alpha-Amylase and salivary albumin adsorption onto titanium, enamel and dentin: an in vivo study.

In vivo adsorption of salivary albumin and alpha-amylase onto titanium, enamel and dentin was analysed following their exposure to the oral cavity for 2h. Oral appliances in six adults served as a platform for carrying 4-mm discs of the three materials. Adherent proteins were eluted from the discs and the amounts of salivary albumin and alpha-amylase were measured by an enzyme-linked immunosorbent assay. While significant difference between the adsorption of albumin and alpha-amylase onto enamel as compared with dentin was observed, adsorption onto titanium was significantly lower. A sample of whole saliva was also collected from each participant. The mean total amounts of albumin and alpha-amylase in the participants' whole saliva were 0.03 and 0.54 mg ml-1, respectively. Titanium adsorbed significantly less (4.43%) of the total albumin than did enamel (14.30%) or dentin (18.80%). No significant difference was found in the relative amounts of alpha-amylase adsorbed by the three materials. This significantly selective adsorption of proteins may enable the attachment of specific bacteria and thus alter the composition of the dental plaque and its potential pathogenicity.

Adsorption↗
PubMed2000

Protein engineering of bacterial alpha-amylases.

alpha-Amylases constitute a very diverse family of glycosyl hydrolases that cleave alpha1-->4 linkages in amylose and related polymers. Recent structural and mutagenic studies of archeael, mammalian and bacterial alpha-amylases have resulted in a wealth of information on the catalytic mechanism and on the structural features of this enzyme class. Because of their high thermo-stability, the Bacillus alpha-amylases have found widespread use in industrial processes, and much attention has been devoted to optimising these enzymes for the very harsh conditions encountered there. Stability has been a major area of focus in this respect, and several remarkably stable bacterial alpha-amylases have been produced by bioengineering techniques. Protein engineering studies of pH-activity profiles and of substrate specificities have also been initiated, although without much success. In the coming years it is likely, however, that the focus of alpha-amylase engineering will shift from engineering stability to these new areas.

Bacillus↗
PubMed2002

Effects of guanidine hydrochloride and high pressure on subsite flexibility of beta-amylase.

We investigated the effects of guanidine hydrochloride (GuHCl) and high pressure on the conformational flexibility of the active site of sweet potato beta-amylase by monitoring the sulfhydryl reaction and the enzymatic activity. The reactivity of Cys345 at the active site, one of six inert half cystine residues of this enzyme, was enhanced by GuHCl at concentrations below 0.5 M. A GuHCl-induced change of the active site was also observed through an intensity change in the near-UV circular dichroism (CD) spectrum. On the other hand, the native conformation of sweet potato beta-amylase observed through fluorescence polarization, far-UV CD spectrum and intrinsic fluorescence was not influenced by GuHCl at concentrations below 0.5 M. Therefore, Cys345 reaction caused by GuHCl was due to an alteration of the local conformation of the active site. GuHCl-induced reaction of Cys345, located in the vicinity of subsites 3 and 4, is attributed to enhanced subsite flexibility, which is responsible for substrate slipping in a single-chain attack mechanism. Due to the flexible conformation, the local region of the subsite is more susceptible to GuHCl perturbation than the molecule overall. The enzymatic activity of sweet potato beta-amylase was reversibly inhibited by GuHCl at concentrations below 0.5 M, and kinetic analysis of the enzymatic mechanism showed that GuHCl decreases the kcat value. High pressure below 400 MPa also inactivated sweet potato beta-amylase with an increase in Cys345 reactivity. These findings indicated that excessively enhanced subsite flexibility reduced the enzymatic activity of sweet potato beta-amylase.

Binding Sites↗
PubMed2003

Fermentation by Lactobacillus fermentum Ogi E1 of different combinations of carbohydrates occurring naturally in cereals: consequences on growth energetics and alpha-amylase production.

Glucose, fructose, sucrose and starch are naturally present in cereals. Fermentation of different combinations of these carbohydrates by Lactobacillus fermentum Ogi E1, a sourdough heterofermentative lactobacillus, was investigated to determine effects on fermentation kinetics, growth energetics and alpha-amylase production. Irrespective of the substrate combination, the strain was able to simultaneously produce alpha-amylase and consume starch, glucose, fructose and sucrose. In mixtures of starch with either sucrose or fructose or with both fructose and glucose, yields of alpha-amylase from biomass (Y(amy/x)) were similar to those observed for starch. However, for starch and glucose or starch, glucose, fructose and sucrose mixtures, both Y(amy/x) and the specific rate of alpha-amylase production decreased markedly. In fructose- or sucrose-containing mixtures, mannitol was formed stoichiometrically indicating that fructose served as electron acceptor, and acetate was produced at constant yield from biomass (Y(ac/x)) (1 g acetate g biomass(-1)). Acetate production was expected to confer to the strain a competitive advantage during natural fermentation by improving biomass formation and growth through an increase in the ATP gain. Y(ATP) varied depending on the carbohydrate mixture, indicating different effects of substrate mixtures on the efficiency in ATP coupling to biomass formation. Compared to starch fermentation, the highest value of Y(ATP) (29 g biomass mol ATP(-1)) was estimated for the starch/fructose mixture but no increase in mu(max) was observed. The lowest value (16 g biomass mol ATP(-1)) was obtained for the starch, glucose and fructose mixture, whereas for the mixture of all carbohydrates, Y(ATP) was similar to that obtained with starch alone (20 g biomass mol ATP(-1)) and it was intermediary for the starch and sucrose mixture (17 g biomass mol ATP(-1)). It is concluded that competitiveness of the strain cannot be based on expected energy gain in mixed substrate fermentation involving fructose and sucrose with glucose and starch, but rather on its ability to simultaneously use carbohydrates while producing alpha-amylase and to produce acetic acid. Acetic acid production could enhance the strain capacity to inhibit nonacid-tolerant, competitive microflora at the earlier stage of natural fermentation.

Acetic Acid↗
PubMed1985

Serum amylase isoenzyme alterations in acute abdominal conditions.

To determine the accuracy of the serum amylase in identifying a pancreatic source, amylase isoenzymes were determined prospectively in 65 patients initially evaluated with a complaint of abdominal pain and associated hyperamylasemia. Isoenzyme patterns were demonstrated by an electrophoretic technique, and the results were correlated with clinical diagnoses. Patients were divided into two diagnostic groups. Group I consisted of 42 patients with clinical findings suggesting pancreatitis. P-type isoenzymes were normal or elevated in 31 of these patients (74%), and s-type isoenzymes were normal or elevated in 11 (26%). Group 2 consisted of 23 patients with abdominal pain attributed to causes other than pancreatitis. Four patients (17%) had elevation of p-type isoenzymes, and 19 patients (83%) had predominantly s-type patterns. We conclude that amylase isoenzymes cannot determine unequivocally the cause of hyperamylasemia, but they can enhance the diagnostic specificity of the serum amylase. Elevated serum amylase with a predominant p-type pattern suggests pancreatic disease; elevation of s-type isoenzymes suggests but is not conclusive for, diagnoses other than pancreatitis. Hyperamylasemia with a normal isoenzyme pattern occurred in a few patients in both groups, and it was nondiagnostic.

Abdomen↗
PubMed2002

The effect of guar galactomannan and water availability during hydrothermal processing on the hydrolysis of starch catalysed by pancreatic alpha-amylase.

The effects of water-soluble nonstarch polysaccharides (sNSP) on human metabolism are considered to be beneficial because they decrease postprandial glycaemia and insulinaemia following ingestion of starch-rich foods. The mechanisms by which sNSP attenuate the postprandial rise in blood glucose are not well understood but their presence increases the viscosity of gastrointestinal contents, which affects physiological functions, e.g. gastric emptying and peristalsis. Increased viscosity and decreased water activity during hydrothermal treatment of starch could influence alpha-amylase action. Using guar galactomannan as a representative of sNSP, we found that galactomannan has a direct noncompetitive inhibitory effect on alpha-amylase with a K(i) value of approximately 0.5% (3.3 microM). The inhibition is not time dependent and studies suggest direct binding of the enzyme to galactomannan; the resulting galactomannan-amylase complex being inactive. Processing of starch at low water levels greatly affects the catalytic efficiency of alpha-amylase. The Km value for starch heat treated in limited water is raised and kcat is lowered relative to starch gelatinised in excess water. Since galactomannan has no effect on the Km of alpha-amylase, we conclude that the inhibitory action of the polymer is not secondary to a decrease in available water. Neither does it seem to be a consequence of impaired diffusion of enzyme, substrate and products because of an increase in viscosity of the medium.Thus, the effects of sNSP in lowering postprandial glycaemia not only involve modifications of gut physiology, but also include direct inhibition of the first stage in the biochemical degradation of starch.

Amylose↗
PubMed1997

Histamine does not potentiate cyclic AMP-mediated amylase secretion in the guinea-pig pancreatic acinar cells.

1. This study investigates the interaction between histamine and the adenylate cyclase systems involved in the secretion of amylase in isolated guinea-pig pancreatic acinar cells. 2. Histamine caused does-related enhancement of amylase release. Similarly, incubation of acini with increasing concentrations of vasoactive intestinal peptide (VIP) resulted in a typical dose-dependent increase in amylase output. 3. When pancreatic acinar cells were incubated with histamine in combination with VIP, amylase secretion did not differ statistically from secretion induced by histamine or VIP alone and was significantly lower than theoretical additivity. Additionally, amylase secretion in the presence of histamine plus forskolin was significantly less than additive. The action of histamine was equally effective as VIP in causing cyclic adenosine monophosphate (cAMP) increase. 4. These results indicate that histamine may exert its secretory effects via the cyclic AMP pathway in the exocrine guinea-pig pancreas.

Amylases↗
PubMedDate not supplied

The effect of L-arginine/nitric oxide pathway on salivary amylase secretion in conscious rats.

In a recent study we have demonstrated the presence of nitric oxide synthase immunoreactive neurons and also perivascular, periacinar and periductal nerve fibres in feline submandibular salivary gland. The role of nitric oxide (NO) in salivary vasoregulation has been suggested by other data too, but the effect of NO on salivary amylase secretion has not been investigated yet. Under ether anaesthesia a catheter was introduced into the oesophagus for salivary juice collections, and a cannula was inserted into the jugular vein for infusions. After postanaesthesia recovery, submaximal carbachol infusion was given as a background to obtain steady secretion because of the low basal secretory rate. Then different groups of animals received NO synthase inhibitor NOLA (NG-nitro-L-arginine), L-arginine, D-arginine or NO donor SIN-1 (3-morpholinosydnonimine). Volume and amylase activity were determined in mixed saliva samples collected for 30 min. Carbachol background infusion alone induced an elevated, sustained salivary secretion. NOLA (30 mg/kg) increased both volume and amylase output (P < 0.001). This effect was prevented by L-arginine but not by D-arginine. SIN-1 did not change either volume or amylase secretion. The present results suggest that the L-arginine/NO pathway has a modulatory effect on salivary fluid and amylase secretion, which is probably not related to its effect on salivary blood flow. NO may block certain presently unidentified secretagogue mechanisms and/or may relax myoepithelial cells.

Amylases↗
PubMed1998

Activation of Bacillus licheniformis alpha-amylase through a disorder-->order transition of the substrate-binding site mediated by a calcium-sodium-calcium metal triad.

BACKGROUND: The structural basis as to how metals regulate the functional state of a protein by altering or stabilizing its conformation has been characterized in relatively few cases because the metal-free form of the protein is often partially disordered and unsuitable for crystallographic analysis. This is not the case, however, for Bacillus licheniformis alpha-amylase (BLA) for which the structure of the metal-free form is available. BLA is a hyperthermostable enzyme which is widely used in biotechnology, for example in the breakdown of starch or as a component of detergents. The determination of the structure of BLA in the metal-containing form, together with comparisons to the apo enzyme, will help us to understand the way in which metal ions can regulate enzyme activity. RESULTS: We report here the crystal structure of native, metal-containing BLA. The structure shows that the calcium-binding site which is conserved in all alpha-amylases forms part of an unprecedented linear triadic metal array, with two calcium ions flanking a central sodium ion. A region around the metal triad comprising 21 residues exhibits a conformational change involving a helix unwinding and a disorder-->order transition compared to the structure of metal-free BLA. Another calcium ion, not previously observed in alpha-amylases, is located at the interface between domains A and C. CONCLUSIONS: We present a structural description of a major conformational rearrangement mediated by metal ions. The metal induced disorder-->order transition observed in BLA leads to the formation of the extended substrate-binding site and explains on a structural level the calcium dependency of alpha-amylases. Sequence comparisons indicate that the unique Ca-Na-Ca metal triad and the additional calcium ion located between domains A and C might be found exclusively in bacterial alpha-amylases which show increased thermostability. The information presented here may help in the rational design of mutants with enhanced performance in biotechnological applications.

Bacillus↗
PubMed1989

Restriction map and alpha-amylase activity variation among Drosophila mutation accumulation lines.

The specific activities of alpha-amylase were measured for two sets of mutation accumulation lines, each set having originated from a different lethal-carrying second chromosome and SM1(Cy) chromosome and having been maintained by a balanced lethal system for about 300 generations. Significant variation was found to have accumulated among lines of both sets. Because of dysgenic crosses in the early generations of mutation accumulation, insertions or deletions of transposable elements in the Amy gene region were suspected of being the cause of this variation. In order to test this possibility, the structural changes in the 14 kb region of these chromosomes that includes the structural genes for alpha-amylase were investigated by restriction map analysis. We found that most part of the activity variation is due to replacements of a chromosomal region of SM1(Cy), including the structural genes for alpha-amylase, by the corresponding regions of the lethal chromosomes. One line also contained an insertion in this region but this line has an intermediate activity value. Thus, insertions of transposable elements into the Amy gene region were not found to be responsible for the new variation observed in alpha-amylase activity. If we remove those lines with structural changes from the analysis, the genetic variance of alpha-amylase specific activity among lines becomes non-significant in both sets of chromosomes.

Animals↗