Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “AMYLASE”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 523 records · Page 29Linked to original sources
PubMed1989

Some thermodynamic parameters of pancreatic and salivary alpha-amylase in serum.

Using monoclonal antibodies and 4,6-ethylidene-protected 4-nitrophenylmaltoheptaoside as substrate we determined the Arrhenius slope, the apparent energy of activation and the apparent enthalpy changes of total amylase, pancreatic amylase and salivary amylase in serum. The Arrhenius slope and hence the other thermodynamic parameters of pancreatic amylase are significantly different from those of total amylase or salivary amylase, but identical to those obtained for purified human pancreatic amylase. Similarly, the thermodynamic parameters of serum salivary amylase activity are the same as those of the purified enzyme. Temperature conversion factors are given for amylase and its isoenzymes. The results are discussed briefly.

Humans↗
PubMed1984

Amylase from human serous ovarian tumors: purification and characterization.

Human serous-type ovarian tumors contain an acidic isoenzyme of amylase. Previous attempts at purification of tumor amylases have yielded preparations contaminated with other proteins. The purification scheme presented here incorporates an affinity-chromatography procedure, with use of cycloheptaamylose linked to epoxy-activated Sepharose, that is specific for alpha-amylase (EC 3.2.1.1). Purified amylase isoenzyme from a human serous ovarian tumor was characterized and compared with the purified salivary and pancreatic isoenzymes. All three were similar in amino acid composition, pH optimum, substrate specificity, calcium requirement, heat inactivation, and Km for maltotetraose substrate. The ovarian tumor amylase was similar to the salivary and distinct from the pancreatic enzyme by apparent molecular mass and doublet formation on sodium dodecyl sulfate--polyacrylamide electrophoresis, specific activity of pure enzyme, and sensitivity to specific alpha-amylase inhibitors. All three isoenzymes differed in net electrical charge as evidenced by diethylaminoethyl-Sephadex ion-exchange chromatography and isoelectric focusing. The tumor amylase is clearly distinct from the pancreatic and differs from the salivary enzyme in net electrical charge. Evidence is presented that this charge difference may reflect, at least in part, deamidation of an amylase that is similar to or identical with salivary amylase.

Amylases↗
PubMed1984

Amylase and isoamylase activities in serum of pregnant women.

The data previously reported for serum amylase activity in the blood of pregnant women are conflicting with respect to normal values and the relationship of activity to duration of pregnancy. By use of a DuPont Automatic Clinical Analyzer, amylase activity in serum from the blood of 413 asymptomatic healthy women who were six to 40 weeks pregnant was determined. Samples with amylase activity greater than 100 IU/L were tested for pancreatic and salivary isoenzymes. In every case except one, the isoenzyme levels were normal. In the blood of all women, except this one, the serum amylase activity was less than 150 IU/L and mean serum amylase activity did not correlate with gestational duration. Amylase activities at all gestational ages did not differ from the activity in serum obtained from the blood of women six weeks post partum. The authors conclude that 1) serum amylase levels may be as high as 150 IU/L in the blood of normal pregnant women; 2) serum amylase activities vary widely among pregnant women, but not in a manner related to the stage of gestation; and 3) pregnancy does not cause elevation of serum amylase isoenzyme activity.

Amylases↗
PubMedDate not supplied

Effect of decreased feed intake on serum and pancreatic alpha-amylase of broiler chickens.

Removal of feed from 20-day-old broiler chicks for 24 hours caused serum and pancreatic alpha-amylase activities to increase almost twofold. A 24-hour feeding period following feed removal caused a reduction in serum alpha-amylase to basal levels and a sixfold reduction in pancreatic alpha-amylase activity. Serum alpha-amylase levels remained elevated after long-term feed restriction in adult broilers compared with levels in full-fed controls. Reduction in feed intake in chicks caused by coccidial infections also resulted in increased serum alpha-amylase. In all cases, the degree of change in serum alpha-amylase corresponded inversely to feed intake. It is proposed that the pancreas synthesizes a specific quantity of alpha-amylase, which does not change even under conditions of extended underconsumption of feed. The rate of alpha-amylase secretion is determined, at least indirectly, by the rate of carbohydrate metabolism, and the remainder of the enzyme is stored in the pancreatic cells. A small percentage of the stored enzyme diffuses into the blood and thus directly reflects increased secretion or accumulation of alpha-amylase in the pancreas in response to conditions of carbohydrate utilization.

Animals↗
PubMed1995

[Usefulness of amylase isoenzyme determination for the diagnosis of pancreatic diseases].

Serum amylase shows the greatest increase among the various pancreatic enzymes that increase at the onset of acute pancreatitis. However, the diagnostic value of the total serum amylase activity has been questioned due to its lack of specificity. To differentiate hyperamylasemia due to pancreatic disease from that due to other causes, the activity of pancreatic amylase should be determined by using a monoclonal antibody that specifically binds to pancreatic or salivary amylase, or by electrophoresis. The most useful and accurate method for distinguishing pancreatic from salivary-type hyperamylasemia is isoamylase analysis by electrophoresis. In patients with acute pancreatitis, increase of Amylase-1 and -2 is accompanied by the appearance of Amylase-4, a minor component of the pancreatic-type isoamylases, and by disappearance of the salivary-type isoenzymes, thereby leaving a pattern of the pancreatic isoenzymes alone. This pancreatitis pattern persists for about 10 days after the onset of illness. Therefore, if such a pattern is found in a patient with clinical findings suggesting acute pancreatitis despite a normal serum amylase level, the patient can be diagnosed as having acute pancreatitis or a recent attack of the disease. However, the existence of an inherited trait of the pancreatitis pattern in some healthy individuals must be borne in mind. Patients with recurrent chronic pancreatitis also show pancreatic-type hyperamylasemia, whereas the pancreatic amylase activity decreases when pancreatic exocrine insufficiency progresses. Hyperamylasemia due to elevated salivary amylase activity is also common in patients with diabetic ketosis or malignancies such as lung cancer (adenocarcinoma). Hyperamylasemia is also found following various types of operation. In most cases, it is salivary-type hyperamylasemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Amylases↗
PubMed1977

Isolation and in vitro translation of the messenger RNA coding for pancreatic amylase.

RNA prepared from dog pancreas polysomes or microsomes directs the synthesis of pancreas-specific proteins in heterologous cell-free translation systems. A translation product, approximately 1500 daltons larger than authentic amylase, corresponding to pancreatic amylase was identified by immunoprecipitation with anti-amylase gamma-globulin and tryptic peptide analysis. We suggest that this larger form of amylase is an amylase precursor. Using amylase immunoprecipitation of reticulocyte translation reactions as an assay, we have shown that greater than 99% of the mRNA for amylase is associated with polysomes bound to the endoplasmic reticulum. Electrophoresis of pancreatic mRNA preparations in formamide-containing polyacrylamide gels and subsequent translation of the fractions have shown that amylase mRNA is of a discrete size with a mobility equivalent to that of 18 S ribosomal RNA, and therefore significantly larger than required to code solely for the amino acid sequence of the amylase precursor.

Amylases↗
PubMed1996

Serum amylase and lipase in the evaluation of acute abdominal pain.

The purpose of this study was to determine 1) the incidence and magnitude of elevation in admission serum amylase and lipase levels in extrapancreatic etiologies of acute abdominal pain, and 2) the test most closely associated with the diagnosis of acute pancreatitis. Serum amylase and lipase levels were obtained in 306 patients admitted for evaluation of acute abdominal pain. Patients were categorized by anatomic location of identified pathology. Logistic regression analysis was used to compare the enzyme levels between patient groups and to determine the correlation between elevation in serum amylase and lipase. Twenty-seven (13%) of 208 patients with an extrapancreatic etiology of acute abdominal pain demonstrated an elevated admission serum amylase level with a maximum value of 385 units (U)/L (normal range 30-110 U/L). Twenty-six (12.5%) of these 208 patients had an elevated admission serum lipase value with a maximum of 3685 U/L (normal range 5-208 U/L). Of 48 patients with abdominal pain resulting from acute pancreatitis, admission serum amylase ranged from 30 to 7680 U/L and lipase ranged from 5 to 90,654 U/L. Both serum amylase and lipase elevations were positively associated with a correct diagnosis of acute pancreatitis (P < 0.001) with diagnostic efficiencies of 91 and 94 per cent, respectively. A close correlation between elevation of admission serum amylase and lipase was observed (r = 0.87) in both extrapancreatic and pancreatic disease processes. Serum amylase and lipase levels may be elevated in nonpancreatic disease processes of the abdomen. Significant elevations (greater than three times upper limit of normal) in either enzyme are uncommon in these disorders. The strong correlation between elevations in the two serum enzymes in both pancreatic and extrapancreatic etiologies of abdominal pain makes them redundant measures. Serum lipase is a better test than serum amylase either to exclude or to support a diagnosis of acute pancreatitis.

Abdominal Pain↗
PubMed1998

[Quantification of inhaled exposure to alpha-amylase in 2 bakeries].

BACKGROUND: Baker's asthma and baker's rhinitis are among the most frequent occupational diseases. A major cause is the high exposure to flour dust in the workplace and to allergenic enzymes like alpha-amylase from Aspergillus oryzae (allergen name: Asp o 2). METHODS: To quantify allergen exposure in the workplace, 31 personal dust samples in a conventional small bakery (six workers) and in a biobakery (seven workers) were collected. Using a recently developed two-site enzyme-linked immunosorbent assay based on monoclonal antibodies to alpha-amylase from Aspergillus oryzae, the allergen content of these dust samples was determined. RESULTS: Dust exposure in the biobakery was in the range between 3.5 and 12 mg/m3 (median: 5.2 mg/m3) and in the conventional bakery between 0.9 and 118 mg/m3 (median 8.5 mg/m3). 23 out of 31 exposure measurements showed values higher than 4 mg/m3 (threshold limit value for inhalable dust). In the biobakery, no fungal alpha-amylase could be detected. 15 out of 17 samples taken in the conventional bakery contained fungal alpha-amylase in the range between 0.2 and 88 ng per mg dust. The geometric mean of alpha-amylase exposure in this bakery was 13 ng Asp o 2/m3, and the maximum exposure was 4.8 micrograms/m3. In four cases, fungal alpha-amylase was detected although exposure to dust was below the threshold limit of 4 mg/m3. CONCLUSIONS: This study in two German bakeries shows that preventive measures to reduce contact to allergens have not been sufficiently realised. Relevant alpha-amylase exposure occurred at low dust levels illustrating that dust measurements are not adequate to control alpha-amylase exposure. For fungal alpha-amylase an additional threshold limit should be established.

Aspergillus oryzae↗
PubMed1963

INDUCED BIOSYNTHESIS OF ALPHA-AMYLASE BY GROWING CULTURES OF BACILLUS STEAROTHERMOPHILUS.

Welker, N. E. (Western Reserve University, Cleveland, Ohio), and L. Leon Campbell. Induced biosynthesis of alpha-amylase by growing cultures of Bacillus stearothermophilus. J. Bacteriol. 86:1196-1201. 1963.-The maximal differential rate (K) of alpha-amylase synthesis was usually two to three times that of the sucrose control culture, over an inducer concentration range of 5 x 10(-4) to 1 x 10(-3)m. With maltotetraose, higher concentrations decreased the K value, whereas higher concentrations of maltose were needed to obtain maximal K values. Glucose, in concentrations from 10(-5) to 10(-2)m, had no effect on the differential rate of enzyme synthesis. Cultures growing on maltotriose, maltotetraose, maltopentaose, and maltohexaose exhibited the same growth rate (k) and differential rate of alpha-amylase synthesis over a concentration range of 2.92 x 10(-4) to 1.46 x 10(-2)m. Growth of cultures in various concentrations of pure maltose revealed that with concentrations of maltose ranging from 2.92 x 10(-3) to 1.46 x 10(-2)m the K value for alpha-amylase production increased 18-fold. The amount of maltose utilized, during the growth period, at each concentration of maltose, was constant. Diauxic type growth was observed when maltose was used in addition to another carbon source (i.e., glucose, glycerol, fructose, or sucrose). Maltose was not utilized until the other carbon source had been metabolized. Phenyl-, methyl-, and ethyl-alpha-d-glucoside and methyl-beta-d-maltoside were good inducers of alpha-amylase and would not serve as a carbon source in a chemically defined medium supplemented with 0.1% casein hydrolysate. These compounds were therefore gratuitous inducers of alpha-amylase. Isomaltose, panose, butyl-alpha-d-glucoside, and methyl-alpha-d-maltotetraoside were not effective as inducers of alpha-amylase. Fructose had an inhibitory effect on constitutive (41%) and inducible (55%) alpha-amylase formation; glucose had no effect.

Amylases↗
PubMedDate not supplied

[Immunochemical characterization of alpha-amylases in wheat seeds at different ontogenical steps (author's transl)].

Protein extracts of wheat seeds taken at an early stage of development, at maturation and after seven days of germination were investigated by using immunochemical techniques with immune sera prepared against alpha-amylases purified from developing seeds and alpha-amylases purified from germinated seeds. After immunochemical analyses carried out in agarose gel, alpha-amylase characterization was performed by using beta-limit dextrin followed by iodine staining. Detection of three antigenic alpha-amylases separated by agarose immunoelectrophoretic analysis at pH 8.6 and called I, II, III from the anode to the cathode, as well as an antigenical relationship between the anodic enzymes I and II were confirmed. Three constituents in I and four in II were further distinguished by using long duration electrophoresis in agarose gel. The immune sera reacted with all of these constituents. Thus with these immune sera a quantitative determination of all anodic alpha-amylase proteins can be attempted as quantitation of two antigens. During this identification consitutents with a apparent activity on beta-limit dextrin but delivering incomplete unstained substrate were detected. These constituents found in developing seeds have electrophoretic mobility close to that of constituents I but differ antigenically from alpha-amylases I and II. Combination of rocket-line-immunoelectrophoresis and alpha-amylase characterization reaction on precipitin bands was developed for comparing amounts of each of three alpha-amylase antigens in different seed extracts. The use of this technique for quantitating at the same time two antigen groups having a certain cross reactivity is discussed. Preliminary results of quantitative study on each of these antigen in developing, mature and germinating seeds are reported.

Amylases↗
PubMed1987

Passage of salivary amylase through the stomach in humans.

With an inhibitor assay technique rates of passage of salivary and pancreatic isoamylase through the jejunum were measured in six healthy volunteers after different liquid, intragastric meals. In all subjects and in 13/17 experiments, more than 2500 units of salivary amylase were passed over 200 postcibal minutes. Salivary amylase comprised 13.8 +/- 3.9% (mean +/- SEM) of the total amylase and appeared predominantly as single, distinct peak. The inhibitor method was validated by isoelectric focusing (r = 0.988; P less than 0.001; N = 7). The frequency of detection of salivary amylase in gastric or jejunal samples fell as gastric pH fell below 3.0. In vitro, amylase was inactivated in gastric juice as pH fell between 3.8 and 3.3. Salivary amylase accounted for 11% of total amylase output in a normal and 27% in an achlorhydric subject after a hamburger meal. We conclude that amylase should not be measured in postprandial studies of pancreatic secretion in humans without correction for salivary amylase.

Adult↗
PubMed1995

Light-Induced Chloroplast [alpha]-Amylase in Pearl Millet (Pennisetum americanum).

In pearl millet (Pennisetum americanum) seedlings light induces the appearance of a leaf [alpha]-amylase isozyme. The leaf [alpha]-amylase isozyme was present in enriched amounts in isolated chloroplast but it could not be detected in isolated etioplasts. The chloroplast [alpha]-amylase was present in both mesophyll and bundle-sheath chloroplasts. Preliminary characterization indicated that molecular properties of chloroplast [alpha]-amylase were like those of a typical [alpha]-amylase. The plastidic [alpha]-amylase had a molecular mass of 46 kD, pH optimum of 6.2, required Ca2+ for activity and thermostability, but lost activity in the presence of ethylenediaminetetracetate. Plastidic [alpha]-amylase activity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis could be renatured in situ by Triton X-100. Western blot analysis demonstrated that this protein was antigenically similar to a maize seed [alpha]-amylase. In vivo [35S]methionine labeling of bundle-sheath strands isolated from light-grown leaves followed by immunoprecipitation revealed that bundlesheath strands synthesized plastidic [alpha]-amylase de novo.

Journal Article↗
PubMed1980

Effect of temperature on the synthesis and secretion of alpha-amylase in barley aleurone layers.

The effect of temperature on alpha-amylase synthesis and secretion from barley (c.v. Himalaya) half-seeds and aleurone layers is reported. Barley half-seeds incubated at 15 C in gibberellic acid (GA) concentrations of 0.5 and 5 micromolar for 16 hours do not release alpha-amylase. Similarly, isolated aleurone layers of barley do not release alpha-amylase when incubated for 2 or 4 hours at temperatures of 15 C or below following 12 hours incubation at 25 C at GA concentrations from 50 nanomolar to 50 micromolar. There is an interaction between temperature and GA concentration for the process of alpha-amylase release from aleurone layers; thus, with increasing GA concentration, there is an increase in the Q(10) of this process. A thermal gradient bar was used to resolve the temperature at which the rate of alpha-amylase release changes; thermal discontinuity was observed between 19 and 21 C. The time course of the response of aleurone tissue to temperature was determined using a continuous monitoring apparatus. Results show that the effect of low temperature is detectable within minutes, whereas recovery from exposure to low temperature is also rapid. Although temperature has a marked effect on the amount of alpha-amylase released from isolated aleurone layers, it does not significantly affect the accumulation of alpha-amylase within the tissue. At all GA concentrations above 0.5 nanomolar, the level of extractable alpha-amylase is unaffected by temperatures between 10 and 28 C. It is concluded that the effect of temperature on alpha-amylase production from barley aleurone layers is primarily on the process of enzyme secretion.

Journal Article↗
PubMed1988

Hormonal regulation of alpha-amylase expression in barley aleurone layers : the effects of gibberellic Acid removal and abscisic Acid and phaseic Acid treatments.

The expression of alpha-amylase isozymes in barley (Hordeum vulgare L.) aleurone layers is known to be maximally induced between 12 and 20 hours after addition of the phytohormone, gibberellic acid (GA(3)). Addition of another hormone, abscisic acid (ABA), or its metabolite, phaseic acid, during this time period resulted in reduced alpha-amylase expression. Expression of the high isoelectric point alpha-amylase isozyme group was affected much more by both of these treatments than was expression of the low isoelectric point alpha-amylase isozyme group. Addition of either the translation inhibitor cycloheximide or the transcription inhibitor cordycepin prevented the decrease in alpha-amylase mRNA levels after ABA treatment. Cordycepin also prevented the decreases in alpha-amylase expression that result from phaseic acid treatment. Midcourse GA(3) removal experiments were performed to determine whether ABA treatment and the removal of GA(3) have analogous effects on alpha-amylase expression. It was found that cordycepin treatment also prevented decreases in alpha-amylase mRNA levels resulting from GA(3) removal. We conclude that the suppression of alpha-amylase expression caused by ABA or midcourse GA(3) removal is dependent on continuous RNA and protein synthesis.

Journal Article↗
PubMed1991

Isolation and Partial Characterization of a Factor from Barley Aleurone that Modifies alpha-Amylase in Vitro.

Posttranslational modifications that give rise to multiple forms of alpha-amylase (EC 3.2.1.1) in barley (Hordeum vulgare L. cv Himalaya) were studied. When analyzed by denaturing polyacrylamide gel electrophoresis, barley alpha-amylase has a molecular mass of 43 to 44 kilodaltons, but isoelectric focusing resolves the enzyme into a large number of isoforms. To precisely identify these isoforms, we propose a system of classification based on their isoelectric points (pl). alpha-Amylases with pls of approximately 5, previously referred to as low pl or Amy1 isoforms, have been designated HAMY1, and alpha-amylases with pls of approximately 6, referred to as high pl or Amy2, are designated HAMY2. Individual isoforms of HAMY1 and HAMY2 are identified by their pls. For example, the most acidic alpha-amylase synthesized and secreted by barley aleurone layers is designated HAMY1(4.56). Some of the diversity in the pls of barley alpha-amylases arises from posttranslational modifications of the enzyme. We report the isolation of a factor from barley aleurone layers and incubation media that can modify HAMY1 isoforms in vitro. This factor has a molecular mass between 30 and 50 kilodaltons, and it can catalyze the conversion of HAMY1(4.90) and HAMY1(4.64) to isoforms 4.72 and 4.56, respectively. The in vitro conversion of HAMY1 isoforms by the factor is favored by pH values of approximately 5 and is inhibited at approximately pH 7. The level of this factor in aleurone layers and incubation media is not affected by treatment of the tissue with gibberellic acid. The amylase-modifying activity from barley will also modify alpha-amylases isolated from human saliva and porcine pancreas. An activity that can modify HAMY1 isoforms in vitro has also been isolated from Onozuka R10 cellulase. Because the activity isolated from barley lowers the pl of alpha-amylase from barley, human saliva, and porcine pancreas, we speculate that it is a deamidase.

Journal Article↗
PubMed1988

Production of alpha-Amylase by the Ruminal Anaerobic Fungus Neocallimastix frontalis.

alpha-Amylase production was examined in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released mainly into the culture fluid and had temperature and pH optima of 55 degrees C and 5.5, respectively, and the apparent K(m) for starch was 0.8 mg ml. The products of alpha-amylase action were mainly maltotriose, maltotetraose, and longer-chain oligosaccharides. No activity of the enzyme was observed towards these compounds or pullulan, but activity on amylose was similar to starch. Evidence for the endo action of alpha-amylase was also obtained from experiments which showed that the reduction in iodine-staining capacity and release in reducing power by action on amylose was similar to that for commercial alpha-amylase. Activities of alpha-amylase up to 4.4 U ml (1 U represents 1 mumol of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of starch ml in shaken cultures. No growth occurred in unshaken cultures. With elevated concentrations of starch (>2.5 mg ml), alpha-amylase production declined and glucose accumulated in the cultures. Addition of glucose to cultures grown on low levels of starch, in which little glucose accumulated, suppressed alpha-amylase production, and in bisubstrate growth studies, active production of the enzyme only occurred during growth on starch after glucose had been preferentially utilized. When cellulose, cellobiose, glucose, xylan, and xylose were tested as growth substrates for the production of alpha-amylase (initial concentration, 2.5 mg ml), they were found to be less effective than starch, but maltose was almost as effective. The fungal alpha-amylase was found to be stable at 60 degrees C in the presence of low concentrations of starch (</=5%), suggesting that it may be suitable for industrial application.

Journal Article↗
PubMed1995

Effect of a wheat amylase inhibitor on canine carbohydrate digestion, gastrointestinal function, and pancreatic growth.

BACKGROUND/AIMS: Chronic amylase inhibition might be useful to treat diabetes mellitus and obesity. Duodenal and ileal cannulas were placed in 8 dogs to determine if long-term ingestion of a wheat amylase inhibitor maintained amylase inhibition or affected gastrointestinal or metabolic function or pancreatic growth. METHODS: Five dogs were fed and 3 were not fed 1.5 g of the inhibitor with meals for 9 weeks. Postprandial and cholecystokinin octapeptide stimulated pancreatic secretion, and fecal balance studies were performed at intervals. After the experiment, the pancreas was analyzed. RESULTS: Weight loss was similar in both groups. Amylase inhibition persisted throughout the 9 weeks; it declined from 91% to 37% from the first to the sixth postprandial hour. Amylase inhibition decreased plasma glucose levels during the first hour (P < 0.05), increased carbohydrate delivery to the ileum (315 vs. 555 mg/h; P = 0.002), and increased cholecystokinin octapeptide-stimulated amylase secretion. However, amylase inhibition did not significantly change plasma concentrations of insulin, peptide YY or neurotensin, postprandial pancreatic secretion, gastrointestinal transit or pancreatic weight, and protein or DNA content. CONCLUSIONS: Prandial ingestion of 1.5 g of the inhibitor for 9 weeks reduces postprandial amylase levels enough to delay carbohydrate digestion and absorption and lower plasma glucose levels without altering pancreatic growth. This dose may be effective to treat diabetes mellitus but not obesity.

Amylases↗
PubMed1983

Bacillus stearothermophilus contains a plasmid-borne gene for alpha-amylase.

The gene for thermostable alpha-amylase from the thermophilic bacterium Bacillus stearothermophilus has been cloned and expressed in Escherichia coli. Each alpha-amylase-producing colony contained at least a 9.7-kilobase-pair (kb) chimeric plasmid composed of the vector pBR322 and a common 5.4-kb HindIII fragment of DNA. B. stearothermophilus contains four plasmids with sizes from 12 kb to over 108 kb. Restriction endonuclease analysis of these naturally occurring plasmids showed they also contain a 5.4-kb HindIII fragment of DNA. Cloning experiments with the four plasmids yielded alpha-amylase-producing E. coli that contained the same 9.7-kb chimeric plasmid. Restriction endonuclease analysis and further recombinant DNA experiments identified a 26-kb plasmid that contains the gene for alpha-amylase. A spontaneous mutant of B. stearothermophilus unable to produce alpha-amylase was missing the 26-kb plasmid but contained a 20-kb plasmid. A 6-kb deletion within the region of the 5.4-kb HindIII fragment yielded the 20-kb plasmid unable to code for alpha-amylase. A nick-translated probe for the alpha-amylase coding region did not hybridize to either plasmid or total cellular DNA from this mutant strain of B. stearothermophilus. These results demonstrate the gene for alpha-amylase is located exclusively on a 26-kb plasmid in B. stearothermophilus with no genetic counterpart present on the chromosome.

Amylases↗