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PubMed1991

Effects of replacement of promoters and modification of the leader peptide region of the amy gene of Streptomyces griseus on synthesis and secretion of alpha-amylase by Streptomyces lividans.

Five different mutations were introduced into the leader peptide region of the alpha-amylase gene of Streptomyces griseus IMRU 3570. A mutation which increased the positive charge of the N-terminal region of the leader peptide enhanced the secretion of alpha-amylase by two- to threefold. Replacement of the native promoter of the amylase gene by the promoter of the Tn5 neo gene or by the promoter of the saf gene resulted in a 16-fold increase in alpha-amylase secretion. The enhanced secretion of alpha-amylase obtained by using the most efficient promoters was due to a correlated increase in the amount of transcript formed. The translation and secretion processes in S. lividans are not a bottleneck for enzyme secretion even at very high transcription rates, since stimulation of transcription of the alpha-amylase gene results in a proportionate increase in secretion of the enzyme.

Amino Acid Sequence↗
PubMed1984

Cloning and expression of a thermophilic alpha-amylase gene from Bacillus stearothermophilus in Escherichia coli.

A 6.4 Kb HindIII fragment of Bacillus stearothermophilus DY-5 DNA cloned in Escherichia coli using pBR322 as a vector was shown to direct the synthesis of a thermophilic alpha-amylase. In attempts to reduce the size of the insert, the alpha-amylase gene was shown to be contained in a 3.1 Kb HindIII - BamHI fragment of the donor strain DNA. The alpha-amylase gene was stably maintained and expressed efficiently in E. coli. The enzymic properties of alpha-amylase produced in E. coli closely resembled those of the donor strain alpha-amylase and the temperature range for the maximal activity was from 65 degrees C to 80 degrees C. Nearly 100% of the activity remained after heating at 80 degrees C for 15 min. The alpha-amylase was shown to be accumulated in the periplasmic space. It was purified to a nearly homogenous protein with a molecular weight of 61,000, which was very similar in size to that produced by B. stearothermophilus DY-5.

Cloning, Molecular↗
PubMed1979

Amylase isoenzymes in mumps.

The amylase isoenzymes activities in serum and urine from 75 children treated for complications of mumps were evaluated. The clinical observations, especially in cases with suspected pancreatitis, were compared with the P- and S-type amylase activities. Increased total activity of serum amylase in 79% of patients was due to increased S-type amylase. However, in 39% of patients there was also a markedly increased P-type serum amylase. Amylase isoenzymes may offer a new insights into the relationship between the pancreas and the parotid glands in mumps.

Amylases↗
PubMed1986

Amylase in human lungs and the female genital tract. Histochemical and immunohistochemical localization.

We investigated the localization of amylase in normal human lungs and the female genital tract using immunohistochemical and histochemical methods. Immunohistochemical procedures were applied to formaldehyde-fixed, paraffin-embedded specimens as well as to cryostat sections of periodate/lysine/paraformaldehyde (PLP)-fixed tissues. The starch-substrate-film method was used for the histochemical investigation of unfixed frozen sections. Amylase immunoreactivity was observed in ciliated epithelial cells of the bronchus and in serous cells of the bronchial glands but not in the alveolar epithelium. Immunoreactive amylase was also found in the cytoplasm of the ciliated epithelium of the fallopian tubes, especially in the apical part of the cytoplasm and in ciliary vesicles. Immunoreactive amylase was also found to be present in the surface epithelial cells and glands of the uterine cervix, as well as in the superficial part of the endometrial glands. The distribution of amylase activity revealed using histochemistry was similar to that observed in cryostat sections of PLP-fixed tissues after immunohistochemical staining. Amylase antigenicity was better preserved in cryostat sections of PLP-fixed materials than in formaldehyde-fixed, paraffin-embedded specimens. The results are discussed in relation to pulmonary and female-genital-tract diseases.

Adolescent↗
PubMed1977

The relationship between stimulation-induced potassium release and amylase secretion in the mouse parotid.

1. The output of amylase from superfused mouse parotid segments in response to stimulation with acetylcholine (ACh), phenylephrine and isoprenaline during exposure to solutions with varying potassium concentrations was monitored by an on line automated fluorometric method. 2. During stimulation with ACh or phenylephrine a 10-fold increase in superfusion fluid potassium concentration caused an immediate very marked reduction in amylase output which was fully reversible. A 10-fold reduction in potassium concentration resulted in a prominent rise in amylase output. During stimulation with isoprenaline there was no effect on the amylase output of varying the extracellular potassium concentration. Acetylcholine and phenylephrine caused potassium release from the mouse parotid whereas isoprenaline had no such effect. 3. It appears that under conditions where stimulation-induced potassium release is enhanced there is also an enhanced amylase secretion and vice cersa. There may therefore be a link between passive potassium transport and amylase secretion.

Acetylcholine↗
PubMed1989

Effects of butyric acid and analogues on amylase release from pancreatic segments of sheep and goats.

The specificity of the structural elements of a short-chain fatty acid for stimulating amylase release was investigated in superfused pancreatic segments of sheep and goats in vitro using butyric acid and analogues. Monocarboxylic (cyclohexanecarboxylic and benzoic) acids were as effective as butyric acid, whilst 4-phenyl-n-butyric and dicarboxylic (succinic and phthalic) acids were weak stimulants. The amylase release evoked by butyric acid was markedly reduced in the presence of these compounds. Replacement of hydrogens by a hydroxyl group or amino group or reduction of the carboxyl group to alcohol diminished, while replacement of hydrogen by chloride at the 3 carbon position did not change, the ability to stimulate amylase release. The dose-response curve for butyric acid was shifted to the right in parallel in the solution containing succinic acid at 8 x 10(-4) mol/l. The maximal increment (but not ED50) of amylase release evoked by ACh was severely reduced in the solution simultaneously containing butyric acid at 10(-3) mol/l. These results suggest that short-chain fatty acids are required to possess both carboxyl (hydrophilic) group and hydrophobic tails in order to have the ability to stimulate amylase release, and that amylase release evoked by butyrate is caused through specific recognizing sites for short-chain fatty acids which might be different from ACh receptors, in the pancreatic segments of sheep and goats.

Acetylcholine↗
PubMed1980

[Macroamylasemia. Immunoglobulin a-amylase-complexes as rare cause of hyperamylasemia (author's transl)].

A 56 year old diabetic woman with persistent hyperamylasemia, with an extremely low renal clearance of amylase and without signs of pancreatic disease was found to have an abnormally large serum amylase. This macroamylase had a molecular size of 450,000 Daltons in gel filtration and could be dissociated at low pH values. By the use of chromatographic, electrophoretic and immuno-chemical tests, it was possible to identify the macroamylase as a complex formed by immunoglobin A (IgA kappa) along with normal pancreatic and parotic amylase. The immunoglobulins had a high affinity for human pancreatic and salivary amylase as well as for hog pancreatic amylase. It remains unclear if the amylase binding immunoglobulins are autoantibodies. The finding of macroamylasemia should prevent patients from being subjected to superflous investigation and meaningless treatment.

Amylases↗
PubMed1982

[Dissociation of serum kinetics of amylase and trypsin following stimulation with secretin and pancreozymin].

The diagnostic value of basal serum trypsin and amylase values in the assessment of pancreatic exocrine function is limited. Secretin injection evokes a significantly different response in the serum kinetics of trypsin and amylase, due probably to their difference in molecular weight (21,000 vs 52,000 Daltons). Serum trypsin increases in 70% of people with normal secretin-pancreozymin test after stimulation with secretin, whereas amylase remains unchanged. The post-stimulatory rise in trypsin is lower in mild exocrine insufficiency and almost completely abolished in severe exocrine insufficiency. The diagnosis of severe exocrine insufficiency is confirmed in 93% and that of mild insufficiency in 54% by low basal and post-stimulatory levels of serum trypsin. In diabetics with low basal values the post-stimulatory rise in serum trypsin confirmed normal pancreatic function. The poststimulatory kinetics of serum amylase shows no clear correlation to pancreatic function. From the divergent serum kinetics of trypsin and amylase it may be concluded that trypsin is primarily of ductular and amylase primarily of acinar origin.

Adult↗
PubMed1987

Enzyme-coding genes as molecular clocks: the molecular evolution of animal alpha-amylases.

We constructed a cDNA library for the beetle, Tribolium castaneum. This library was screened using a cloned amylase gene from Drosophila melanogaster as a molecular probe. Beetle amylase cDNA clones were isolated from this bank, and the nucleotide sequence was obtained for a cDNA clone with a coding capacity for 228 amino acids. Both the nucleotide sequence and predicted amino acid sequence were compared to our recent results for D. melanogaster alpha-amylases, along with published sequences for other alpha-amylases. The results show that animal alpha-amylases are highly conserved over their entire length. A broader comparison, which includes plant and microbial alpha-amylase sequences, indicates that parts of the gene are conserved between prokaryotes, plants, and animals. We discuss the potential importance of this and other enzyme-coding genes for the construction of molecular phylogenies and for the study of the general question of molecular clocks in evolution.

Amino Acid Sequence↗
PubMed1988

DNA rearrangement causes multiple changes in gene expression at the amylase locus in Drosophila melanogaster.

A spontaneous null mutation at the alpha-amylase locus in Drosophila melanogaster was recovered from a laboratory population. The mutant strain was found to lack amylase enzyme production and to produce low, but detectable, levels of amylase mRNA. Moreover, the null strain is also lacking the glucose repression of amylase mRNA production which is seen in wild-type strains. The mutant phenotype correlates with a rearrangement in genomic DNA which, in turn, corresponds to a simple inversion in the arrangement observed most frequently in North American populations of D. melanogaster, including the common laboratory strain, Oregon-R. These results have implications for our understanding of both the evolution of the duplicated amylase gene structure and the regulation of amylase gene expression.

Amylases↗
PubMed1980

The contribution of pancreas and kidney in regulating serum amylase levels in dogs.

Amylase activity in canine serum before and after total pancreatectomy with or without additional removal of duodenum and other small intestines were determined and resulted that pancreas would provide approximately fifty per cent of amylase detected in the canine serum. Serum amylase levels were also measured before and after intravenous injection of canine pancreatic juice as a source of pancreatic amylase into normal or nephrectomized dogs in order to investigate the mode of disappearance of amylase from circulating blood and following result was obtained that intravenously injected amylase were removed from the blood of extraurinary mechanism in dogs.

Amylases↗
PubMed1985

Effect of obstructive jaundice on amylase secretion in rat pancreatic acini.

The effect of obstructive jaundice on pancreatic amylase secretion was studied in isolated pancreatic acini prepared from bile duct ligated rats (7 days postoperatively), sham operated rats being used as control. Obstructive jaundice caused increase in pancreatic wet weight, pancreatic protein content and pancreatic amylase content by 27.9%, 40.1% and 33.2%, respectively. In acini prepared from obstructive jaundice group, compared with acini from sham operation group, responsiveness to cholecystokinin (CCK) and carbachol was decreased when amylase release was expressed as the percentage of total amylase activity initially present in acini. However, sensitivity to both secretagogues was unchanged when expressed as the percentage of maximally stimulated amylase release. The dose-response curves to Ca2+ ionophore for amylase release were similarly shaped in both groups. These results suggested that a pancreatico-trophic effect, compared with altered responsiveness of pancreatic acini, should play a major role in hypersecretion in obstructive jaundice.

Amylases↗
PubMed1976

Serum amylase isozyme changes in chronic pancreatitis and their clinical significance.

The diagnostic usefulness of serum amylase isozyme measurement was investigated in 17 cases of definite chronic pancreatitis and 13 cases of suspected chronic pancreatitis, a total of 30 cases. Six types of serum isoamylase patterns were distinguished according to the character of the two main amylase activities at the fraction of fast-gamma globulin and pre-gamma globulin. The amylase activity of the fast-gamma fraction rises in acute exacerbation, and falls in cases of chronic calcifying pancreatitis and in cases with a marked decrease in pancreatic exocrine function. Especially, a fall in fast-gamma amylase activity excellently reflects a decrease in pancreatic enzyme production. The measurement of amylase activity at the fast-gamma fraction therefore appears to be a useful method for assessing pancreatic damage. The significance of a frequent rise in pre-gamma amylase activity in chronic pancreatitis remains unknown.

Amylases↗
PubMed1984

Amylase inhibitor from wheat: its action and clinical application.

The electrophoretic and column chromatographic characteristics of an amylase inhibitor of wheat origin were investigated. Further, the clinical usefulness of this inhibitor for determining the ratio of pancreatic to salivary isoamylase activity in serum was evaluated. Amylase inhibitor inhibits the action of salivary alpha-amylase by making an amylase-inhibitor complex, which is easily separated into its individual component during electrophoresis with full recovery of amylase activity. Using the specific inhibitory effect of this inhibitor on salivary alpha-amylase activity, the ratio of pancreatic to salivary isoamylase activity (P/S) in serum was determined. There was a good correlation in P/S ratio in serum between the results obtained with the inhibitor method and those with electrophoretic method. The P/S ratio in sera from patients with acute pancreatitis was over 8.0, whereas that in sera from patients with salivary-type hyperamylasemia such as mumps, pulmonary diseases and following surgery was less than 0.1. However, hyperamylasemia due to macroamylase or renal failure could not be identified by the inhibitor method.

Amylases↗
PubMed2002

Deletion analysis of the C-terminal region of the alpha-amylase of Bacillus sp. strain TS-23.

The alpha-amylase from Bacillus sp. strain TS-23 is a secreted starch hydrolase with a domain organization similar to that of other microbial alpha-amylases and an additional functionally unknown domain (amino acids 517-613) in the C-terminal region. By sequence comparison, we found that this latter domain contained a sequence motif typical for raw-starch binding. To investigate the functional role of the C-terminal region of the alpha-amylase of Bacillus sp. strain TS-23, four His(6)-tagged mutants with extensive deletions in this region were constructed and expressed in Escherichia coli. SDS-PAGE and activity staining analyses showed that the N- and C-terminally truncated alpha-amylases had molecular masses of approximately 65, 58, 54, and 49 kDa. Progressive loss of raw-starch-binding activity occurred upon removal of C-terminal amino acid residues, indicating the requirement for the entire region in formation of a functional starch-binding domain. Up to 98 amino acids from the C-terminal end of the alpha-amylase could be deleted without significant effect on the raw-starch hydrolytic activity or thermal stability. Furthermore, the active mutants hydrolyzed raw corn starch to produce maltopentaose as the main product, suggesting that the raw-starch hydrolytic activity of the Bacillus sp. strain TS-23 alpha-amylase is functional and independent from the starch-binding domain.

Adsorption↗
PubMed2006

Purification and characterization of a maltooligosaccharide-forming alpha-amylase from a new Bacillus subtilis KCC103.

A maltooligosaccharide-forming alpha-amylase was produced by a new soil isolate Bacillus subtilis KCC103. In contrast to other Bacillus species, the synthesis of alpha-amylase in KCC103 was not catabolite-repressed. The alpha-amylase was purified in one step using anion exchange chromatography after concentration of crude enzyme by acetone precipitation. The purified alpha-amylase had a molecular mass of 53 kDa. It was highly active over a broad pH range from 5 to 7 and stable in a wide pH range between 4 and 9. Though optimum temperature was 65-70 degrees C, it was rapidly deactivated at 70 degrees C with a half-life of 7 min and at 50 degrees C, the half-life was 94 min. The K (m) and V (max) for starch hydrolysis were 2.6 mg ml(-1) and 909 U mg(-1), respectively. Ca(2+) did not enhance the activity and stability of the enzyme; however, EDTA (50 mM) abolished 50% of the activity. Hg(2+), Ag(2+), and p-hydroxymercurybenzoate severely inhibited the activity indicating the role of sulfydryl group in catalysis. The alpha-amylase displayed endolytic activity and formed maltooligosaccharides on hydrolysis of soluble starch at pH 4 and 7. Small maltooligosaccharides (D2-D4) were formed more predominantly than larger maltooligosaccharides (D5-D7). This maltooligosaccharide forming endo-alpha-amylase is useful in bread making as an antistaling agent and it can be produced economically using low-cost sugarcane bagasse.

Bacillus subtilis↗
PubMed1998

Effect of N-linked glycosylation on secretion, activity, and stability of alpha-amylase from Aspergillus oryzae.

The effect of N-linked glycosylation on secretion, activity, and stability of alpha-amylase from Aspergillus oryzae grown as dispersed filaments was studied. In the presence of tunicamycin the fungus grew either as dispersed filaments or as one large pellet, whereas growth was as dispersed filaments in all control cultures. The presence of tunicamycin affected neither biomass, level of secreted alpha-amylase, nor total amount of secreted protein in cultures growing as dispersed filaments. In these cultures both glycosylated and nonglycosylated alpha-amylase appeared in the culture medium as well as in the cells, whereas in control cultures only the glycosylated form of alpha-amylase was found in the medium and in the cells. The presence of nonglycosylated alpha-amylase in the medium seemed to result from active secretion rather than from autolysis of the mycelium or extracellular deglycosylation. Deglycosylation with Endo H of crude alpha-amylase in culture filtrate did not affect its stability towards heat, acid pH, or proteolytic degradation.

Aspergillus oryzae↗
PubMed2006

Cloning and functional expression of the gene encoding an inhibitor against Aspergillus flavus alpha-amylase, a novel seed lectin from Lablab purpureus (Dolichos lablab).

Maize is one of the more important agricultural crops in the world and, under certain conditions, prone to attack from pathogenic fungi. One of these, Aspergillus flavus, produces toxic and carcinogenic metabolites, called aflatoxins, as byproducts of its infection of maize kernels. The alpha-amylase of A. flavus is known to promote aflatoxin production in the endosperm of these infected kernels, and a 36-kDa protein from the Lablab purpureus, denoted AILP, has been shown to inhibit alpha-amylase production and the growth of A. flavus. Here, we report the isolation of six full-length labAI genes encoding AILP and a detailed analysis of the activities of the encoded proteins. Each of the six labAI genes encoded sequences of 274 amino acids, with the deduced amino acid sequences showing approximately 95-99% identity. The sequences are similar to those of lectin members of a legume lectin-arcelin-alpha-amylase inhibitor family reported to function in plant resistance to insect pests. The labAI genes did not show any of the structures characteristic of conserved structures identified in alpha-amylase inhibitors to date. The recombinant proteins of labAI-1 and labAI-2 agglutinated human red blood cells and inhibited A. flavus alpha-amylase in a manner similar to that shown by AILP. These data indicate that labAI genes are a new class of lectin members in legume seeds and that their proteins have both lectin and alpha-amylase inhibitor activity. These results are a valuable contribution to our knowledge of plant-pathogen interactions and will be applicable for developing protocols aimed at controlling A. flavus infection.

Amino Acid Sequence↗