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PubMed1993

Cloning of the beta-amylase gene from Bacillus cereus and characteristics of the primary structure of the enzyme.

The gene encoding the beta-amylase of Bacillus cereus BQ10-S1 (SpoII) was cloned into Escherichia coli JM 109. A sequenced DNA fragment of 2,001 bp contains the beta-amylase gene. The N-terminal sequences (AVNGKG MNPDYKAYLMAPLKKI), the C-terminal sequences (SHTSSW), and the amino acid sequences of the five regions in the beta-amylase molecules were determined. The mature beta-amylase contains 514 amino acid residues with a molecular mass of 57,885 Da. The amino acid sequence homology with those of known beta-amylases was 52.7% for Bacillus polymyxa, 52.0% for Bacillus circulans, 43.4% for Clostridium thermosulfurogenes, 31.8% for Arabidopsis thaliana, 31.5% for barley, 29.9% for sweet potato, and 28.9% for soybean. Ten well-conserved regions were found between the N terminus and the area around residue 430, but the C-terminal region of 90 residues has no similarity with those of the plant beta-amylases. The homology search revealed that this C-terminal region has homology with C-terminal regions of the beta-amylase from C. thermosulfurogenes, some bacterial alpha-amylases, cyclodextrin glucanotransferase, and glucoamylase. Some of these sequences are known as the raw-starch-binding domain. These results suggest that B. cereus beta-amylase has an extra domain which has raw-starch-binding ability and that the domain has considerable sequence homology with those of other amylases or related enzymes from a wide variety of microorganisms.

Amino Acid Sequence↗

PubMed1974

Genetic control of the rate of alpha-amylase synthesis in Bacillus subtilis.

The level of extracellular alpha-amylase (EC 3.2.1.1) of Bacillus subtilis Marburg was increased about fivefold by introducing the amyR marker from B. natto 1212 through transformation. amyR2 of B. natto 1212 has been assumed to determine a high level of alpha-amylase of the organism. The gene acts specifically on alpha-amylase synthesis but not on the production of other extracellular enzymes. alpha-Amylase of an amyR2-carrying strain was found to be quite similar to that of an isogenic amyR1-carrying strain in the thermostability and electrophoretic behavior of whichever amylase the strain produces. Marburg-type alpha-amylase (amyEm) or B. natto-alpha-amylase (amyEn). Anti-amylase serum titration indicates that a high level of the enzyme activity in the amyR2-carrying strain is caused by the existence of more enzyme rather than the presence of an enzyme having higher efficiency. This is supported further by the fact that amyR controls the synthesis of the amyE gene product in mutant M9, which synthesizes a temperature-sensitive-alpha-amylase, and in mutant M07, which secretes cross-reacting material. The results indicate that amyR regulates the rate of alpha-amylase synthesis.

Amylases↗

PubMed1974

Genetic and biochemical studies on cell-bound alpha-amylase in Bacillus subtilis Marburg.

A small but significant amount of alpha-amylase activity was detected in the cells of Bacillus subtilis Marburg. The cell-associated activity was almost constant regardless of the level of extracellular alpha-amylase activity. The cell-bound amylase activity could be separated into three components, upon Sephadex G-75 chromatography, referred to as components A, B, and C. Component C showed the same properties as the extracellular alpha-amylases so far examined. Component A had a molecular weight greater than 70,000, as judged from the elution position on Sephadex G-75, and became smaller upon treatment with trypsin but was still larger than that of component C. An alpha-amylase mutant that lacked extracellular alpha-amylase completely because of a mutation within the structural gene of the enzyme was found to lose all three cell-bound amylase components simultaneously. These data suggest strongly that the cell-bound amylase components are precursors of the extracellular alpha-amylase and that the alpha-amylase of this organism is produced under the direction of the same gene whether the enzyme is within or outside the cell.

Amylases↗

PubMed1997

Properties and gene structure of the Thermotoga maritima alpha-amylase AmyA, a putative lipoprotein of a hyperthermophilic bacterium.

Thermotoga maritima MSB8 has a chromosomal alpha-amylase gene, designated amyA, that is predicted to code for a 553-amino-acid preprotein with significant amino acid sequence similarity to the 4-alpha-glucanotransferase of the same strain and to alpha-amylase primary structures of other organisms. Upstream of the amylase gene, a divergently oriented open reading frame which can be translated into a polypeptide with similarity to the maltose-binding protein MalE of Escherichia coli was found. The T. maritima alpha-amylase appears to be the first known example of a lipoprotein alpha-amylase. This is in agreement with observations pointing to the membrane localization of this enzyme in T. maritima. Following the signal peptide, a 25-residue putative linker sequence rich in serine and threonine was found. The amylase gene was expressed in E. coli, and the recombinant enzyme was purified and characterized. The molecular mass of the recombinant enzyme was estimated at 61 kDa by denaturing gel electrophoresis (63 kDa by gel permeation chromatography). In a 10-min assay at the optimum pH of 7.0, the optimum temperature of amylase activity was 85 to 90 degrees C. Like the alpha-amylases of many other organisms, the activity of the T. maritima alpha-amylase was dependent on Ca2+. The final products of hydrolysis of soluble starch and amylose were mainly glucose and maltose. The extraordinarily high specific activity of the T. maritima alpha-amylase (about 5.6 x 10(3) U/mg of protein at 80 degrees C, pH 7, with amylose as the substrate) together with its extreme thermal stability makes this enzyme an interesting candidate for biotechnological applications in the starch processing industry.

Amino Acid Sequence↗

PubMed1988

Concerted evolution of human amylase genes.

Cosmid clones containing 250 kilobases of genomic DNA from the human amylase gene cluster have been isolated. These clones contain seven distinct amylase genes which appear to comprise the complete multigene family. By sequence comparison with the cDNAs, we have identified two pancreatic amylase genes and three salivary amylase genes. Two truncated pseudogenes were also recovered. Intergenic distances of 17 to 22 kilobases separate the amylase gene copies. Within the past 10 million years, duplications, gene conversions, and unequal crossover events have resulted in a very high level of sequence similarity among human amylase gene copies. To identify sequence elements involved in tissue-specific expression and hormonal regulation, the promoter regions of the human amylase genes were sequenced and compared with those of the corresponding mouse genes. The promoters of the human and mouse pancreatic amylase genes are highly homologous between nucleotide -160 and the cap site. Two sequence elements thought to influence pancreas-specific expression of the rodent genes are present in the human genes. In contrast, similarity in the 5' flanking sequences of the salivary amylase genes is limited to several short sequence elements whose positions and orientations differ in the two species. Some of these sequence elements are also associated with other parotid-specific genes and may be involved in their tissue-specific expression. A glucocorticoid response element and a general enhancer element are closely associated in several of the amylase promoters.

Amylases↗

PubMed1976

Differential expression of salivary (Amy1) and pancreatic (Amy2) human amylase loci in prenatal and postnatal development.

The age-dependent development of alpha-amylase expression in utero and during the first two years of life is reported. Separation of salivary and pancreatic amylase isozymes in a discontinuous buffered sheet polyacrylamide electrophoretic system, with subsequent densitometry, provides a reliable semiquantitative method of estimating the proportions of salivary and pancreatic amylases in urine and amniotic fluid samples. In the newborn the predominant amylase isozymes seen in the urine are of salivary origin. As the child ages the level of amylase in the urine rises and an increase in the proportion of pancreatic amylase isozymes occurs. Amniotic fluids of late first and early second trimester pregnancies contain salivary isozymes. None of the amniotic fluid samples examined has pancreatic amylase isozymes. These data reflect a differential development of the expression of the two amylase approaches adult levels by 16 months of age. Conversely, the salivary (Amy1) locus is expressed as early as 18 weeks of gestation and remains relatively constant with but a small increase in salivary amylase (units/ml) activity during early development, as the total amylase activity approaches adult values.

Age Factors↗

PubMed1990

Differences in the renal handling of pancreatic and salivary amylase in the rat.

Plasma activity and excretion of pancreatic (P) amylase in the rat was found to be negligible. In contrast, the excretion rate of salivary (S) amylase was substantial and variable, depending on diuresis. P-amylase had a higher isoelectric point, a greater sieving coefficient, and a shorter half-life than S-amylase. A bolus injection of 125I-labelled enzymes was followed by the appearance of 125I-labelled enzyme- as well as protein-free 125I activity in the urine. The enzyme loss was smaller and the fraction of protein-free 125I activity higher following injection of P-amylase. The affinity of P-amylase to paraffin oil exceeded that of S-amylase in partition experiments with water and paraffin oil in vitro. It is concluded that both renal filtration and reabsorption of P-amylase exceed those of S-amylase. This might be due to the higher lipophility of P-amylase in comparison to the salivary type.

Absorption↗

PubMed1996

Exposure-sensitization relationship for alpha-amylase allergens in the baking industry.

Fungal alpha-amylase is an important occupational allergen in the bakery industry. Epidemiologic studies focusing on the relationship between alpha-amylase allergen exposure and work-related respiratory allergy, however, have not been reported yet. In this cross-sectional study, sensitization to occupational allergens and work-related symptoms were studied in 178 bakery workers and related to allergen exposure. Alpha-amylase allergen concentrations were measured in personal dust samples, using a sandwich enzyme immunoassay. All workers were categorized into groups on the basis of their job histories and the alpha-amylase exposure levels of their job titles. Of all workers 25% had one or more work-related symptoms. As much as 9% of the bakery workers showed a positive skin prick test reaction to fungal amylase, and in 8% amylase-specific IgE was demonstrated. Alpha-amylase exposure and atopy appeared to be the most important determinants of skin sensitization, with prevalence ratios for atopy of 20.8 (95% CI, 2.74 to 158) and for medium and high alpha-amylase exposure groups of 8.6 (95% CI, 1.01 to 74) and 15.9 (95% CI, 1.95 to 129), respectively. Furthermore, a positive association was found between positive skin prick tests to alpha-amylase and work-related respiratory symptoms. In conclusion, this study has shown that there is a strong and positive relationship between alpha-amylase allergen exposure levels in bakeries and specific sensitization in bakery workers.

Adult↗

PubMed1989

Comparison of some recent methods for the differentiation of elevated serum amylase and the detection of macroamylasaemia.

A pancreatic isoamylase method (Pancreatic Alpha-Amylase EPS, Boehringer) that uses monoclonal antibodies showed almost complete immunoinhibition of salivary (S) amylase activity with only a minor decrease of pancreatic (P) amylase activity. The method displayed good sensitivity and linearity. The correlations of P-amylase activities determined by this technique with a wheat-germ inhibition method and with agarose electrophoresis followed by densitometric scanning were excellent. However, both the wheat-germ and monoclonal inhibition methods failed to detect macroamylasaemia. To recognise macroamylases we used the PEG precipitation method and confirmed the results with agarose electrophoresis. Of 161 serum samples with elevated amylase activities, only four out of five with macroamylasaemia were detected by the PEG precipitation method. No false positives were demonstrated. After PEG precipitation of 28 samples, P-amylase determinations were performed on the supernatants. Again, four out of five with macroamylasaemia were recognised. We consider P-amylase measurement and, when macroamylasaemia is suspected, the combined use of the PEG precipitation method and P-amylase or total amylase determination to be the most practical way to differentiate between elevated serum amylase levels.

Amylases↗

PubMed1995

Salivary amylase promotes adhesion of oral streptococci to hydroxyapatite.

Recent studies have demonstrated that several species of oral streptococci, such as Streptococcus gordonii, bind soluble salivary alpha-amylase. The goal of the present study was to determine if amylase immobilized onto a surface such as hydroxyapatite can serve as an adhesion receptor for S. gordonii. Initially, human parotid saliva was fractionated on Bio-Gel P60, and fractions were screened for their ability to promote adhesion of S. gordonii to hydroxyapatite. Fractions containing alpha-amylase and proline-rich proteins promoted the adhesion of [3H]-labeled S. gordonii to hydroxyapatite. Similar findings were obtained with purified amylase and acidic proline-rich protein 1 (PRP1). Incubation of S. gordonii G9B in the presence of starch and maltotriose increased the binding of this strain to amylase-coated hydroxyapatite, while the adhesion of S. sanguis 10556 to amylase-coated hydroxyapatite was not affected by these saccharides. These results suggest that amylase may serve as a hydroxyapatite pellicle receptor for amylase-binding streptococci. Furthermore, starch and starch metabolites may enhance the adhesion of amylase-binding streptococci to amylase in dental pellicles to augment the formation of dental plaque.

Adhesins, Bacterial↗

PubMed1981

Immunocytochemical localization of amylase in the parotid gland of developing and adult rats.

An antiserum against purified rat parotid amylase was used to localize the protein in parotid glands of developing and adult rats. The unlabeled antibody peroxidase-antiperoxidase method and the protein A-gold colloid technique were used at the light and electron microscope levels, respectively. Immunoreactive amylase was detected in a few scattered cells in the glands of 2-day-old rats. During the following days the number of cells stained immunocytochemically for amylase increased rapidly; at 15 days of age all acinar cells revealed amylase, but the intensity of immunostaining varied from cell to cell. Electron microscopically, amylase was localized in the secretory granules, and by using a more concentrated antiserum, in the rough endoplasmic reticulum and Golgi complex. At early stages of development the acinar cells contained fewer and smaller secretory granules than in adult animals; the gold particles indicative of amylase were randomly distributed over the secretory granules. In the glands of adult rats, amylase was distributed inhomogeneously within the secretory granules. In the majority of secretory granules gold colloid particles were located over the electron-dense portions of the granules. However, secretory granules in which an amylase-rich shell surrounded an amylase-poor or amylase-negative "core" were not infrequent.

Aging↗

PubMed1986

Parotid gland recovery from an obstruction--changes of amylase release from the tissue.

The recovery of the parotid gland in the rat was studied by isoproterenol-induced amylase release from the parotid tissue in vitro after the removal of duct ligation following 2 and 7 days of obstruction. The percentage of amylase release did not change, but the activity of amylase in the medium (the released amylase activity) increased gradually after the removal of ligation. When the duct was ligated for 2 days, a complete recovery of the released amylase activity was seen 21 days after the removal of ligature. However, in the case of 7-day-ligation, the recovery was about 70 percent after the same period. An obstruction of a shorter duration produced a more rapid recovery of amylase release. Amylase activity in the parotid tissue increased gradually with time after the removal of ligature, and the recovery rates were very similar to that of the released amylase activity. The present results suggest that the recovery of released amylase activity after the removal of ligature is due to the increase of amylase content in the parotid tissue.

Amylases↗

PubMed1986

Effect of duct ligation on amylase release from rat parotid slices.

The effect of parotid duct ligation on amylase release from the rat parotid gland by isoproterenol was investigated in vitro. Unilateral ligation of the excretory duct progressively reduced amylase activity in the medium (the released amylase activity), but did not change the percentage of amylase release. Amylase activity in the parotid tissue decreased progressively after duct ligation, and the decrease rates of the amylase activity were very similar to that of the released amylase activity. Accordingly, the decrease of the released amylase activity after the ligation may be not due to an alteration in the amylase release mechanism, but due to the decrease of amylase content in the parotid tissue.

Amylases↗

PubMed1986

Effects of cholinergic and adrenergic nerve stimulations on amylase release from superfused small segment of rat parotid gland.

The effects of the cholinergic and adrenergic nerve stimulations on amylase release from the segment isolated from the rat parotid gland were investigated, employing the combined techniques of electrical field stimulation (FS) and tyramine application with automated fluorescence method for measuring amylase. The maximum amylase release in response to FS for a short period (1 min) was attained at 16 Hz frequency, 2 ms pulse width and 8 V strength stimulation in the absence of any autonomic antagonist. Periodic short-lasting FS using these parameters at intervals of about 15 min could reproduce similar sizes of amylase release for about 2 h. Continuous long-lasting FS (3 V, 2 ms, 16 Hz) caused a transient sharp increase in amylase release followed by a sustained one. The FS-evoked amylase release was completely abolished by tetrodotoxin (TTX) (10(-7) g/ml) and markedly reduced by atropine (7 X 10(-6) M) or by propranolol (10(-5) M), while it was scarcely affected by hexamethonium (3 X 10(-4) M) and phentolamine (10(-5) M). The maximum stimulus frequency of short-lasting FS for amylase release in the presence of propranolol was similar to that (16 Hz) in the control, but it was higher (32 Hz) in the presence of atropine. Reduced response in amylase release to FS by propranolol was completely restored by the superimposed addition of dibutyryl cyclic AMP (10(-4) M). Application of tyramine (5 X 10(-4) M) evoked amylase release in the presence of atropine, which was blocked mostly by propranolol and partly by phentolamine, while tyramine was ineffective in the tissue segment from rats pretreated with 6-hydroxydopamine. From these results the following were suggested; that the intrinsic cholinergic neurotransmitter activates a muscarinic receptor of the acinar cell, while the adrenergic neurotransmitter stimulates mainly a beta-adrenergic and partly an alpha-adrenergic receptor, resulting in amylase release.

Amylases↗

PubMed1989

Use of 2-ethoxyethanol and alpha-amylase in the neutral detergent fiber method of feed analysis.

Use of 2-ethoxyethanol in the NDF procedure for feed analysis was reevaluated, because justification for its use in the NDF procedure has diminished and 2-ethoxyethanol poses potential health hazards. Initial rationale for addition of 2-ethoxyethanol was to minimize inhibitory effects of decalin on filtration and to facilitate starch solubilization. Decalin is no longer recommended in the procedure; alpha-amylase is commonly used to facilitate starch solubilization. Objectives of this study were to evaluate effects on NDF values of: 1) eliminating 2-ethoxyethanol from the procedure and 2) addition of and timing of addition of alpha-amylase. Deletion of 2-ethoxyethanol resulted in lower NDF values than neutral detergent solution with 2-ethoxyethanol (61.95 and 62.06%, respectively). Neutral detergent fiber values were lower for samples treated with alpha-amylase just prior to filtration (61.86%) than for samples not treated with alpha-amylase (62.13%). Addition of alpha-amylase 30 min after onset of boiling resulted in lower values (62.07%) than with no alpha-amylase addition (62.29%) in solutions containing 2-ethoxyethanol but not in solutions without 2-ethoxyethanol. Observed treatment differences were small, however, and probably not of practical significance. No differences in NDF values were noted due to timing of alpha-amylase addition. Inclusion of samples requiring alpha-amylase to filter increased overall mean CV and resulted in no statistical differences between neutral detergent solutions or alpha-amylase treatments. Suggested modifications to the NDF procedure include elimination of 2-ethoxyethanol and addition of alpha-amylase just prior to filtering.

Animal Feed↗

PubMed2001

The ram1 mutant of Arabidopsis exhibits severely decreased beta-amylase activity.

Despite extensive biochemical analyses, the biological function(s) of plant beta-amylases remains unclear. The fact that beta-amylases degrade starch in vitro suggests that they may play a role in starch metabolism in vivo. beta-Amylases have also been suggested to prevent the accumulation of highly polymerized polysaccharides that might otherwise impede flux through phloem sieve pores. The identification and characterization of a mutant of Arabidopsis var. Columbia with greatly reduced levels of beta-amylase activity is reported here. The reduced beta-amylase 1 (ram1) mutation lies in the gene encoding the major form of beta-amylase in Arabidopsis. Although the Arabidopsis genome contains nine known or putative beta-amylase genes, the fact that the ram1 mutation results in almost complete loss of beta-amylase activity in rosette leaves and inflorescences (stems) indicates that the gene affected by the ram1 mutation is responsible for most of the beta-amylase activity present in these tissues. The leaves of ram1 plants accumulate wild-type levels of starch, soluble sugars, anthocyanin, and chlorophyll. Plants carrying the ram1 mutation also exhibit wild-type rates of phloem exudation and of overall growth. These results suggest that little to no beta-amylase activity is required to maintain normal starch levels, rates of phloem exudation, and overall plant growth.

Anthocyanins↗

PubMed1976

Electrophoretic identification of an isoenzyme of amylase which increases in serum in liver diseases.

Isoenzymes of amylase were studied in serum from 72 persons by means of polyacrylamide gel electrophoresis and a direct saccharogenic assay for amylase activity. In 37 normal individuals, there were two major peaks of amylase actvity with mobilities similar to pancreatic and salivary amylases. In 11 patiets with acute pancreatitis, the area of activity corresponding with pancreatic amylases increased disproportionately. Electrophoretic patterns of amylase activity in normal and pancreatitis urine were almost identical to the respective serum patterns from the same persons. In contrast, a prominent slower-moving peak of amylase activity occurred in the serum of 8 of 12 patients who had hyperamylasemia associated with various liver diseases. Traces of this third peak were identifiable in one-third of normal serum specimens, but no increases in its activity were observed in any specimen from 11 patients with pancreatitis or from 12 other patients with hyperamylasemia unassociated with liver disease. The slower-moving peak was absent from the urine of patients whose serum contained it. The origin of the slower-moving serum amylase appearing in patients with liver disease is not established by these studies. It is possible either that a hepatic amylase is liberated from damaged liver cells or that the metabolism of an amylase not originating in the liver is altered as a result of liver dysfunction.

Amylases↗

PubMedDate not supplied

Determination of alpha-amylase activity in serum and dialysate from patients using icodextrin-based peritoneal dialysis fluid.

OBJECTIVE: Low serum activity of alpha-amylase has been reported in peritoneal dialysis (PD) patients following treatment with icodextrin-based peritoneal dialysis fluid (IPDF). However, these results have been questioned because icodextrin interferes with the polysaccharide reagent included in the assay as a substrate for alpha-amylase in the sample. DESIGN: We adapted a routine method using p-nitrophenol maltoheptaoside as substrate for the analysis of total alpha-amylase in serum and dialysate from 27 patients using IPDF. Serum from 12 healthy volunteers and serum and dialysate from 19 PD patients using glucose-based peritoneal dialysis fluid (GPDF) were used as controls. For the PD patients, time on dialysis ranged from 1 to 24 months (mean 5.7 months) and time of exposure to IPDF ranged from 1 to 52 weeks. RESULTS: To test for interference and recovery, and thus to validate the alpha-amylase assay, samples were spiked with IPDF and synthetic alpha-amylase. This revealed that addition of up to 75% IPDF did not interfere with the assay. Furthermore, alpha-amylase was fully recovered when spiked in serum from patients treated with IPDF. We show that total alpha-amylase activity is considerably lower in the serum of IPDF patients (20.3 +/- 16.5 U/L, p < 0.001) than GPDF patients (85.5 +/- 51.7 U/L) and healthy persons (55.1 +/- 13.6 U/L). CONCLUSIONS: We have shown that the IL method (ILTest; Instrumentation Laboratory, Lexington, MA, USA) measures alpha-amylase activity in samples containing icodextrin metabolites. The clinical significance of reduced plasma alpha-amylase activity, as well as the relative importance of pancreatic versus salivary and tissue-bound alpha-amylase, in PD patients using IPDF is not known.

Aged↗