Showing papers on "Amylase published in 1988"
TL;DR: The increased insulin response to finely ground flour may be relevant to the etiology of diseases associated with hyperinsulinemia and to the management of diabetes.
263 citations
TL;DR: The chemical forms and resistance to hydrolysis in vitro of raw and gelatinised starch from peas, maize, wheat and potatoes were measured as discussed by the authors, and the onset of resistance to starch-hydrolysis was confirmed using a simulated digestion technique.
255 citations
TL;DR: A variety of species of ruminal bacteria were screened for the ability to grow in starch-containing medium and produce amylase, and the highest levels of amyl enzyme were produced by Streptococcus bovis JB1 and Ruminobacter amylophilus H18.
Abstract: A variety of species of ruminal bacteria were screened for the ability to grow in starch-containing medium and produce amylase. Of those tested, the highest levels of amylase were produced by Streptococcus bovis JB1 and Ruminobacter amylophilus H18. Other strains that grew well on starch and produced amylase included Butyrivibrio fibrisolvens A38 and 49 and Bacteroides ruminicola 23 and B14. Varying the carbohydrate source provided for growth resulted in changes in the growth rate and level of amylase produced by these strains. All strains grew rapidly in starch-containing medium, and the rates of growth were generally more rapid than those observed for maltose-grown cultures. For S. bovis JB1, B. ruminicola 23 and B14, and B. fibrisolvens 49 and A38, amylase was produced when growth was on maltose or starch, but this activity was greatly reduced in glucose-grown cultures. The distribution of amylolytic activity between cellular and extracellular fractions was sometimes affected by the carbohydrate provided for growth. If S. bovis JB1 and B. fibrisolvens 49 were grown on starch, amylase was largely associated with cell pellets; however, if grown on maltose these strains produced activities that were almost entirely present in the extracellular fluid fractions. Although not as dramatic, a similar shift in the location of amylase activities was noted for the two B. ruminicola strains when grown on the same substrates. Growth on maltose or starch had little influence on either the predominantly cell-associated activity of B. fibrisolvens A38 or the activity of R. amylophilus H18, which was equally divided between cell pellet and extracellular fluid fractions.(ABSTRACT TRUNCATED AT 250 WORDS)
158 citations
TL;DR: The results suggest that starch degradation in tomato fruit is predominantly phosphorolytic, and starch biosynthetic capacity, as determined by levels of ADPglucose pyrophosphorylase rather than starch degradative capacity, regulate the transient accumulation of starch that occurs early in Tomato fruit development.
Abstract: In developing tomato (Lycopersicon esculentum Mill.) fruit, starch levels reach a peak early in development with soluble sugars (hexoses) gradually increasing in concert with starch degradation. To determine the enzymic basis of this transient partitioning of carbon to starch, the activities of key carbohydrate-metabolizing enzymes were investigated in extracts from developing fruits of three varieties (cv VF145-7879, cv LA1563, and cv UC82B), differing in final soluble sugar accumulation. Of the enzymes analyzed, ADPglucose pyrophosphorylase and sucrose synthase levels were temporally correlated with the transient accumulation of starch, having highest activities in cv LA1563, the high soluble sugar accumulator. Of the starch-degrading enzymes, phosphorylase levels were fivefold higher than those of amylase, and these activities did not increase during the period of starch degradation. Fiften percent of the amylase activity and 45 to 60% of the phosphorylase activity was localized in the chloroplast in cv VF145-7879. These results suggest that starch degradation in tomato fruit is predominantly phosphorolytic. The results suggest that starch biosynthetic capacity, as determined by levels of ADPglucose pyrophosphorylase rather than starch degradative capacity, regulate the transient accumulation of starch that occurs early in tomato fruit development. The results also suggest that ADPglucose pyrophosphorylase and sucrose synthase levels correlated positively with soluble sugar accumulation in the three varieties examined.
122 citations
TL;DR: A direct colorimetric assay for alpha-amylase, with 2-chloro-4-nitrophenyl-alpha-maltotrioside as substrate, which compares well with that of Pantrak Amylase (Behring Diagnostics) for both serum and urine samples.
Abstract: We describe a direct colorimetric assay for alpha-amylase, with 2-chloro-4-nitrophenyl-alpha-maltotrioside as substrate. Both human pancreatic and salivary amylase split this substrate without the use of helper enzymes, yielding free 2-chloro-4-nitrophenol, which is monitored at 405 nm. The performance of this reagent compares well with that of Pantrak Amylase (Behring Diagnostics) for both serum and urine samples. The reagent is very stable in dry powder form and is stable for one month at 2 to 8 degrees C after reconstitution. Because of the rapid color development and linear kinetics (less than 30 s), the assay is easily automated. Results can be obtained in less than 5 min.
120 citations
TL;DR: 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.
Abstract: 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.
114 citations
TL;DR: Results confirm the existence of feedback control of pancreatic enzyme secretion in humans and reveal a twofold to threefold increase in the output and concentration of all four enzymes in period 3 compared with period 2.
114 citations
TL;DR: A newly isolated bacterium, identified as Bacillus subtilis 65, was found to produce raw-starch-digesting alpha-amylase, which could digest raw potato starch almost as fast as it could corn starch, but it showed no adsorbability onto any kind of raw starch at any pH.
Abstract: A newly isolated bacterium, identified as Bacillus subtilis 65, was found to produce raw-starch-digesting α-amylase The electrophoretically homogeneous preparation of enzyme (molecular weight, 68,000) digested and solubilized raw corn starch to glucose and maltose with small amounts of maltooligosaccharides ranging from maltotriose to maltoheptaose This enzyme was different from other amylases and could digest raw potato starch almost as fast as it could corn starch, but it showed no adsorbability onto any kind of raw starch at any pH The mixed preparation with Endomycopsis glucoamylase synergistically digested raw potato starch to glucose at 30°C The raw-potato-starch-digesting α-amylase showed strong digestibility to small substrates, which hydrolyzed maltotriose to maltose and glucose, and hydrolyzed p-nitrophenyl maltoside to p-nitrophenol and maltose, which is different from the capability of bacterial liquefying α-amylase
99 citations
TL;DR: In this article, a simple mathematical treatment is proposed to explain the results of the kinetics of protein degradation by alpha-amylase, and the results support evidence for an active disentanglement of chains, carried out by the different subsites of alpha-AMylase molecules.
Abstract: Native starch granules from wheat have been subjected to enzymatic depolymerization with an alpha-amylase from Bacillus subtilis. Crystallites made from short-chain amylose and residues from mild acid hydrolysis have been also tested. Electron microscopy, particle size analysis, DSC, and x-ray diffractometry reveal that enzymatic degradation occurs granule by granule. Gel permeation chromatography shows off the macromolecular nature of the remaining material. In contrast, acid erodes simultaneously all the granules, leading to a splitting into small particles. Crystalline fractions are completely degraded by alpha-amylase. These results support evidence for an active disentanglement of chains, carried out by the different subsites of alpha-amylase molecules. A simple mathematical treatment is proposed to explain the results of the kinetics.
97 citations
TL;DR: The results of substrate specificity studies, action pattern and viscometric analyses indicate that the extrachloroplastic amylases are exolytic.
Abstract: Amylolytic enzymes of Arabidopsis leaf tissue were partially purified and characterized. Endoamylase, starch phosphorylase, d-enzyme (transglycosylase), and possibly exoamylase were found in the chloroplasts. Endoamylase, fraction A2, found only in the chloroplast, was resolved from the exoamylases by chromatography on a Mono Q column and migrated with an RF of 0.44 on 7% polyacrylamide gel electrophoresis. Exoamylase fraction, A1, has an RF of 0.23 on the polyacrylamide gel. Viscometric analysis showed that A1 has a slope of 0.013, which is same as that of A3, the extrachloroplastic amylase. A1, however, can be distinguished from A3 by having much higher amylolytic activity in succinate buffer than acetate buffer, and having much less reactivity with amylose. A1 probably is also localized in the chloroplast, and contributes to the 30 to 40% higher amylolytic activity of the chloroplast preparation in succinate than acetate buffer at pH 6.0. The high activity of d-enzyme compared to the amylolytic activity in the chloroplast suggests that transglycosylation probably has an important role during starch degradation in Arabidopsis leaf. Extrachloroplastic amylase, A3, has an RF of 0.55 on 7% electrophoretic gel and constitutes 80% of the total leaf amylolytic activity. The results of substrate specificity studies, action pattern and viscometric analyses indicate that the extrachloroplastic amylases are exolytic.
91 citations
TL;DR: A highly heat-stable amylase gene from an obligately anaerobic and extremely thermophilic bacterium was cloned and expressed in Escherichia coli and was found to be a highly liquefying-type.
Abstract: A highly heat-stable amylase gene from an obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, was cloned and expressed in Escherichia coli. The nucleotide sequence of the amylase gene predicts a 686-amino-acid protein of relative molecular mass 81200, which is consistent with that determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified enzyme. The NH2-terminal sequence determined using the enzyme purified from E. coli cells corresponds precisely to that predicted from the nucleotide sequence, except for the absence of the NH2-terminal methionine in the mature protein. When the amylase gene was expressed in E. coli cells, the enzyme was localized in the cytoplasmic fraction; this is probably explained by the absence of the signal sequence for secretion. By using the amylase purified from the E. coli transformant, some enzymatic properties, such as optimum pH, optimum temperature, pH-stability and heat-stability, were examined. The amylase was found to be a highly liquefying-type.
TL;DR: X-ray structural results for lysozyme, glycogen phosphorylase, and amylase are described in an attempt to draw together some general principles that appear to be important in generating a protein-oligosaccharide recognition site and compare these features with protein monosACcharide sites.
Abstract: Polysaccharides have many diverse biological roles. They comprise the insoluble structural and supportive elements of bacterial and plant cell walls. They serve as storage macromolecules (e.g., glycogen and starch) to provide fuel for cells, and they form important components in the heteromacromolecules, proteoglycans and glycoproteins, which are involved in the structural elements of connective tissue, in the lubrication of skeletal joints, in cell-cell adhesion and in cell-surface recognition. The enzymes that catalyze the biosynthesis and degradation of these polysaccharides are highly specific, resulting, in the case of biosynthesis, in polymers of defined constitution and sequence and, in the case of degradation in the cleavage of specific linkages. In this article we describe the X-ray structural results for three of these enzymes: lysozyme, glycogen phosphorylase, and amylase. Each catalyzes a specific step in the degradation of different polysaccharides. (To date there are no structural data on any of the biosynthetic enzymes.) Lysozyme catalyzes the hydrolysis of a β-(1–4) linkage of bacterial cell wall polysaccharides; glycogen phosphorylase catalyzes the reversible phosphorylation of α-(1–4) linkages of glycogen and amylase catalyzes the hydrolysis of α-(1–4) linkages in starch. We use these results in an attempt to draw together some general principles that appear to be important in generating a protein-oligosaccharide recognition site and compare these features with protein monosaccharide sites.
TL;DR: The deduced amino acid sequence of the extracellular mature enzyme was more than 60% homologous to those of the liquefying type alpha-amylases but not to those
TL;DR: The surface of starch granules was smooth and the unique modification obsenred during ripening was the reduction of the granule dimensions; at advanced ripening stages, some striations were detected on the surface of both small and large granules as mentioned in this paper.
Abstract: Banana starch disappearance during ripening originated at the central portion of the fruit radiating, afterwards, to the surface; the amylose/amylopectin ratio remained constant during the process. The amyloplast was investigated by light and scanning electron microscopy. The surface of starch granules was smooth, and the unique modification obsenred during ripening was the reduction of the granule dimensions; at advanced ripening stages, some striations were detected on the surface of both small and large granules. Several amylolytic enzymes were followed during banana ripening. Seven amylases were detected at all stages, presumably three a- and four p-amylases and, like glucosidases, their activity increased at the climacteric phase; only p-amylase activity increased before the onset of the respiratory peak. For the first time α-l, 6-glucosidase activity was detected in banana.
TL;DR: The crude enzyme preparation well degraded not only cereal starches but also tuber and root starches, and the initial velocity for potato starch was 72% of that for corn starch.
Abstract: Aspergillus sp. K-27, isolated from soil, produced extracellular glucoamylase and α-amylase using wheat starch as a carbon source, and its productivity was doubled by the addition of α-methyl-d-glucoside to the medium. The crude enzyme preparation, which was found to be a mixture of 70% glucoamylase and 30% α-amylase, well degraded not only cereal starches but also tuber and root starches, and the initial velocity for potato starch was 72% of that for corn starch.
TL;DR: The N-terminal of the mature amylase has been determined and shown to be in accordance with signal peptidase processing after a typical Gram-positive signal sequence of 33 amino acids.
Abstract: We have cloned and expressed a novel maltogenic alpha-amylase from B. stearothermophilus on plasmid in B. subtilis. Originally the plasmid was very unstable in the absence of selection, but was stabilized due to a spontaneous, copy number reducing mutation. The promoter region and the extension of the gene have been analysed, and a provisional DNA sequence has been determined. The N-terminal of the mature amylase has been determined and shown to be in accordance with signal peptidase processing after a typical Gram-positive signal sequence of 33 amino acids.
TL;DR: As the amylase inhibitor improves carbohydrate homeostasis and is not associated with continuing diarrhea, it may be a useful adjuvant in the treatment of patients with non-insulin-dependent diabetes mellitus.
Journal Article•
TL;DR: An extracellular beta-amylase from Clostridium thermosulphurogenes was purified 811-fold to homogeneity, and its general molecular, physico-chemical and catalytic properties were determined.
Abstract: An extracellular beta-amylase from Clostridium thermosulphurogenes was purified 811-fold to homogeneity, and its general molecular, physico-chemical and catalytic properties were determined The native enzyme was a tetramer of 210 kDa composed of a single type subunit; its 20 amino acid N-terminus displayed 45% homology with Bacillus polymyxa beta-amylase The beta-amylase was enriched in both acidic and hydrophobic amino acids The pure enzyme displayed an isoelectric point of 51 and a pH activity optimum of 55 The optimum temperature for beta-amylase activity was 75 degrees C, and enzyme thermostability at 80 degrees C was enhanced by substrate and Ca2+ addition The beta-amylase hydrolysed amylose to maltose and amylopectin and glycogen to maltose and limit dextrins, and it was inhibited by alpha- and beta-cyclodextrins The enzyme displayed kcat and Km values for boiled soluble starch of 400,000 min-1 per mol and 168 mg/ml, respectively The enzyme was antigenically distinct from plant beta-amylases
TL;DR: The increased concentration of the components studied is most probably caused by an increased adrenergic and VIP'ergic tonus of the individuals after the run.
Abstract: The concentration of epidermal growth factor (EGF), amylase, haptocorrin, total protein, sodium and potassium was studied in mixed saliva collected from 25 individuals before and after a 2 h-long cross-country race. The concentration was higher following the run for all components studied. The concentrations obtained before and after the run, given as extreme values and median, are 0.2-1.3 (0.4), 0.3-1.5 (0.8) nmol/l for EGF, 15-72 (25), 23-162 58) nmol/l for haptocorrin and 24-502 (120), 155-5,030 (1,200) kU/l for amylase. The increased concentration of the components studied is most probably caused by an increased adrenergic and VIP'ergic tonus of the individuals after the run.
TL;DR: Results indicate that dietary factors that reduce Zn availability may also reduceZn toxicity, and reduce exportable pancreatic enzyme activities or tissue alpha-tocopherol concentrations.
Abstract: The effects of excess dietary zinc (Zn) on exocrine pancreatic function were studied in chicks. A purified diet based on crystalline amino acids and sucrose was employed in several experiments, and a practical type of diet based on corn and soybean meal was used in one experiment for the purpose of comparison. Additions of as little as 100 mg Zn/kg as ZnO to the purified diet markedly elevated pancreatic Zn concentration, whereas liver and plasma Zn were moderately increased. Histological examination of the pancreas showed alterations in acinar structure due to Zn intoxication. The activities of the pancreatic exportable enzymes amylase, lipase, trypsinogen and chymotrypsinogen were each decreased by such levels of Zn feeding. Reductions of pancreatic enzyme activities were associated with reductions in the digestibility of dietary starch and in tissue alpha-tocopherol concentrations. Chicks fed the nonpurified (i.e., soy-containing) diet accumulated much less Zn in the pancreas (about one-tenth as compared with the purified diet); the addition of as much as 2000 mg Zn/kg as ZnO to this diet produced only a small increase in pancreas Zn concentration, and did not affect exportable pancreatic enzyme activities or tissue alpha-tocopherol concentrations. These results indicate that dietary factors that reduce Zn availability may also reduce Zn toxicity. Chicks fed the purified diet with excess Zn had slightly higher liver selenium (Se) concentrations, but this effect was not associated with changes in the activity of Se-dependent glutathione peroxidase in that organ. Nutritional deprivation of Se did not affect the extent of Zn-induced acinar damage.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: Unexpected fragments protected by liver RNA led to the discovery that the 5' regions of the five human amylase genes contain a processed gamma-actin pseudogene.
Abstract: The human genes encoding salivary amylase (AMY1) and pancreatic amylase (AMY2) are nearly identical in structure and sequence. We have used ribonuclease protection studies to identify the functional gene copies in this multigene family. Riboprobes derived from each gene were hybridized to RNA from human pancreas, parotid and liver. The sizes of the protected fragments demonstrated that both pancreatic genes are expressed in pancreas. One of the pancreatic genes, AMY2B, is also transcribed at a low level in liver, but not from the promoter used in pancreas. AMY1 transcripts were detected in parotid, but not in pancreas or liver. Unexpected fragments protected by liver RNA led to the discovery that the 5' regions of the five human amylase genes contain a processed gamma-actin pseudogene. The promoter and start site for transcription of AMY1 are recently derived from the 3' untranslated region of gamma-actin. In addition, insertion of an endogenous retrovirus has interrupted the gamma-actin pseudogene in four of the five amylase genes.
TL;DR: Starch protected amylase activity even in the presence of pepsin, and maltotriose conferred striking protection below pH 3, indicating that salivary-type isoamylases are protected in a simulated gastric environment by substrates of amyl enzyme as well as its end products of hydrolysis.
Abstract: Salivary-type amylase may significantly contribute to duodenal starch hydrolysis in exocrine pancreatic insufficiency, provided that gastric inactivation does not occur. We investigated the effect of starch and its hydrolytic products, therefore, on salivary amylase activity in vitro at low pH. When incubated at pH 3 in the presence of 1% starch, 56% of the initial activity of amylase purified from saliva remained after 60 min at 37 degrees C compared with only 6% without starch. Similar protection of amylolytic activity was observed using human milk, which also contains a salivary-type amylase. In addition, partially hydrolyzed starch protected salivary amylase activity at pH 3, and purified glucose oligomers ranging in length from two to seven glucose molecules protected amylase in a concentration-dependent manner. Lactose, sucrose, and glucose, however, were ineffective in sparing amylase. Starch protected amylase activity even in the presence of pepsin, and maltotriose conferred striking protection below pH 3. These studies indicate that salivary-type isoamylases are protected in a simulated gastric environment by substrates of amylase as well as its end products of hydrolysis.
TL;DR: The rgr1 mutation was not allelic with ssn6 and cyc9, and failed to suppress snf1, and showed pleiotropic effects on the following cellular functions: resistance to glucose repression, temperature-sensitive lethality, sporulation deficieny in homozygous diploid cells, and (4) abnormal cell morphology.
Abstract: We have isolated mutants responsible for an oversecretion phenotype in Saccharomyces cerevisiae, using a promoter of SUC2 and the gene coding for alpha-amylase from mouse as a marker of secretion. These mutations defined two complementation groups, designated as ose1 (over secretion) and rgr1 (resistant to glucose repression). The ose1 mutant produced an oversecretion of amylase by 12- to 15-fold under derepressing conditions; however, the amylase mRNA was present at nearly the same amount as it was in the parent cells. No expression of the amylase gene was detected under repressing conditions. The rgr1 mutant oversecreted amylase by 11- to 13-fold under repressing conditions by 15- to 18-fold under derepressing conditions. The rgr1 mutant showed pleiotropic effects on the following cellular functions: (1) resistance to glucose repression, (2) temperature-sensitive lethality, (3) sporulation deficieny in homozygous diploid cells, and (4) abnormal cell morphology. The rgr1 mutation was not allelic with ssn6 and cyc9, and failed to suppress snf1.
TL;DR: This study confirms the existence of a transglycosylase that disproportionates maltotriose and higher maltodextrins by transferring maltosyl or Maltodextrinyl groups between maltodesxtrinins resulting in the production of glucose and different maltodeXTrins, but not maltose, in leaf tissue with enzymic properties very similar to the previously reported d-enzyme in potato.
Abstract: Two major forms of d-enzyme (4-α-glucanotransferase, EC 2.4.1.25) were successfully separated from most of the amylase activity using FPLC-Mono Q column chromatography. Transfer of a maltosyl group was observed upon the incubation of d-enzyme with maltotriose and d-[U-14 C]glucose. About 4.5% of the radioactivity was transferred to maltotriose in 2 hours. End product analysis showed the accumulation of glucose and maltopentaose from maltotriose within the first 10 minutes of the reaction. Several other maltodextrins were also observed with longer incubation times, although maltose was never produced. A quantitative measurement of maltodextrin production from the reaction of [14 C]maltotriose with d-enzyme showed that the quantity of maltotriose decreased from 100% to 31% after 3 hours incubation, while glucose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose, and higher maltodextrins increased in amount. Glucose is the major product throughout the course of the reaction of d-enzyme with maltotriose. Maltotriose, in addition to glucose, are the major products in the reaction of d-enzyme with maltodextrins with a chain length greater than maltotriose. This study confirms the existence of a transglycosylase that disproportionates maltotriose and higher maltodextrins by transferring maltosyl or maltodextrinyl groups between maltodextrins resulting in the production of glucose and different maltodextrins, but not maltose, in leaf tissue with enzymic properties very similar to the previously reported d-enzyme in potato.
TL;DR: The structural gene for excreted amylase from Aeromonas hydrophila JMP636 has been cloned within a 2.1-kilobase SmaI fragment of DNA.
Abstract: The structural gene for excreted amylase from Aeromonas hydrophila JMP636 has been cloned within a 2.1-kilobase SmaI fragment of DNA. The amylase gene is transcribed from its own promoter in Escherichia coli, producing a gene product of Mr 49,000. The amylase gene product is secreted to the periplasm of E. coli; however, it is not excreted. Nucleotide sequencing revealed an open reading frame of 1,392 base pairs corresponding to a protein of 464 amino acid residues. A potential signal peptide of 21 amino acid residues is present at the NH2 terminal of the predicted protein. Three regions of homology with other procaryotic and eucaryotic alpha-amylases were detected within the predicted amino acid sequence.
TL;DR: Wood tissues, by depositing starch, may play a role in keeping the balance of demand and supply in carbohydrates, as well as providing a supply of energy and substrates for the early growth of shoots in spring.
TL;DR: Investigation of the role of eyestalk factors in the neuroendocrine control of the crustacean midgut gland concerning the release of amylase found the presence of forskolin in the incubation medium had an amyl enzyme-releasing effect, which points to a role of cAMP in the mode of action.