Thermus aquaticus ATCC 33923 amylomaltase gene cloning and expression and enzyme characterization: production of cycloamylose. (1/213)

The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of the Escherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75 degrees C in a 10-min reaction with maltotriose and was stable at temperatures up to 85 degrees C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic alpha-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coli amylomaltase and the plant D-enzyme.  (+info)

Granule-bound starch synthase I in isolated starch granules elongates malto-oligosaccharides processively. (2/213)

Isoforms of starch synthase belonging to the granule-bound starch synthase I (GBSSI) class synthesize the amylose component of starch in plants. Other granule-bound isoforms of starch synthase, such as starch synthase II (SSII), are unable to synthesize amylose. The kinetic properties of GBSSI and SSII that are responsible for these functional differences have been investigated using starch granules from embryos of wild-type peas and rug5 and lam mutant peas, which contain, respectively, both GBSSI and SSII, GBSSI but not SSII and SSII but not GBSSI. We show that GBSSI in isolated granules elongates malto-oligosaccharides processively, adding more than one glucose molecule for each enzyme-glucan encounter. Granule-bound SSII can elongate malto-oligosaccharides, but has a lower affinity for these than GBSSI and does not elongate processively. As a result of these properties GBSSI synthesizes longer malto-oligosaccharides than SSII. The significance of these results with respect to the roles of GBSSI and SSII in vivo is discussed.  (+info)

Stable, inducible thermoacidophilic alpha-amylase from Bacillus acidocaldarius. (3/213)

Bacillus acidocaldarius Agnano 101 produces an inducible thermoacidophilic alpha-amylase. The enzyme production occurs during the stationary phase of growth in the presence of compounds with alpha-1,4-glucosidic linkages. The enzymatic activity is both present in the culture medium and associated with the cells; the enzymes purified from both sources show identical molecular and catalytic properties. The purified amylase has a single polypeptide chain of molecular weight 68,000 and behaves like an alpha-amylase with affinity constants for starch and related substances of 0.8 to 0.9 mg/ml. The pH and temperature optima for activity are 3.5 and 75degreesC, respectively. The amylase is stable at acidic pH (below 4.5). Its thermal stability is strictly dependent upon protein concentration; the half-life at 60degreesC of the amylase in a 70-mug/ml solution is about 5 days.  (+info)

Mechanism of porcine pancreatic alpha-amylase inhibition of amylose and maltopentaose hydrolysis by kidney bean (Phaseolus vulgaris) inhibitor and comparison with that by acarbose. (4/213)

The effects of Phaseolus vulgaris inhibitor (alpha-AI) on the amylose and maltopentaose hydrolysis catalysed by porcine pancreatic alpha-amylase (PPA) were investigated. Based on a statistical analysis of the kinetic data and using the general velocity equation, which is valid at equilibrium for all types of inhibition in a single-substrate reaction, it was concluded that the inhibitory mode is of the mixed noncompetitive type involving two molecules of inhibitor. In line with this conclusion, the Lineweaver-Burk primary plots intersect in the second quadrant and the secondary plots of the slopes and the intercepts versus the inhibitor concentrations are parabolic curves, whether the substrate used was amylose or maltopentaose. A specific inhibition model of the mixed noncompetitive type applies here. This model differs from those previously proposed for acarbose [Al Kazaz, M., Desseaux, V., Marchis-Mouren, G., Payan, F., Forest, E. & Santimone, M. (1996) Eur. J. Biochem. 241, 787-796 and Al Kazaz, M., Desseaux, V., Marchis-Mouren, G., Prodanov, E. & Santimone, M. (1998) Eur. J. Biochem. 252, 100-107]. In particular, with alpha-AI, the inhibition takes place only when PPA and alpha-AI are preincubated together before the substrate is added. This shows that the inhibitory PPA-alphaAI complex is formed during the preincubation period. Secondly, other inhibitory complexes are formed, in which two molecules of inhibitor are bound to either the free enzyme or the enzyme-substrate complex. The catalytic efficiency was determined both with and without inhibitor. Using the same molar concentration of inhibitor, alpha-AI was found to be a much stronger inhibitor than acarbose. However, when the inhibitor amount is expressed on a weight basis (mg x L-1), the opposite conclusion is drawn. In addition, limited proteolysis was performed on PPA alone and on the alpha-AI-PPA complex. The results show that, in the complex, PPA is more sensitive to subtilisin attack, and shorter fragments are obtained. These data reflect the conformational changes undergone by PPA as the result of the protein inhibitor binding, which differ from those previously observed with acarbose.  (+info)

Psyllium shifts the fermentation site of high-amylose cornstarch toward the distal colon and increases fecal butyrate concentration in rats. (5/213)

We examined the combination effects of psyllium (PS) and resistant starch on large bowel short-chain fatty acids (SCFA). Rats were fed one of the following four diets: low amylose (LAS) or high amylose cornstarch diets (HAS, 50 g/kg diet) with or without 15 g PS/kg diet (LAS/PS and HAS/PS diets). HAS and/or PS were substituted for the same amounts of LAS in diets. Cecal butyrate concentrations were significantly higher in rats fed the HAS and HAS/PS diets than in those fed the LAS and LAS/PS diets. However, butyrate and total SCFA concentrations in rats fed the HAS diet decreased along the length of the colon and fecal butyrate concentration was reduced to one-third of that in the cecum. In contrast, the HAS/PS diet maintained higher butyrate concentrations throughout the large bowel. Fecal butyrate concentration in the HAS/PS diet-fed group significantly exceeded the sum of the concentrations in rats fed the LAS/PS and HAS diets. PS supplementation to the HAS diet significantly increased fecal starch excretion by 10 fold compared with that of rats fed the HAS diet. There was a positive correlation between fecal butyrate concentration and fecal starch excretion (r = 0.709, P < 0.0001). In a further experiment, ileorectostomized rats were fed the HAS and HAS/PS diets. From the difference in fecal starch excretion between normal and ileorectostomized rats, starch degradation by large bowel microflora in rats fed the HAS and HAS/PS diets was deduced to be 96% and 63%, respectively. These findings support the hypothesis that PS may delay the fermentation rate of HAS in the cecum and shift the fermentation site of HAS toward the distal colon, leading to the higher butyrate concentration in the distal colon and feces.  (+info)

Characterization and crystallization of an active N-terminally truncated form of the Escherichia coli glycogen branching enzyme. (6/213)

The prokaryotic glycogen branching enzymes (GBE) can be divided into two groups on the basis of their primary structures: the first group of enzymes, which includes GBE from Escherichia coli, is characterized by a long N-terminal extension that is absent in the enzymes of the second group. The extension consists of approximately 100 amino-acid residues with unknown function. In order to characterize the function of this region, the 728 amino-acid residue, full-length E. coli GBE, and a truncated form (nGBE) missing the first 107 amino-acid residues were overexpressed in E. coli. Both enzymes were purified to homogeneity by a simple purification procedure involving ammonium sulphate precipitation, ion-exchange chromatography, and a second ammonium sulphate precipitation. Purified full-length enzyme was poorly soluble and formed aggregates, which were inactive, at concentrations above 1 mg.mL-1. In contrast, the truncated form could be concentrated to 6 mg.mL-1 without any visible signs of aggregation or loss of activity on concentration. The ability to overexpress nGBE in a highly soluble form has allowed us to produce diffracting crystals of a branching enzyme for the first time. A comparison of the specific activities of purified GBE and nGBE in assays where amylose was used as substrate demonstrated that nGBE retained approximately half of the branching activity of full-length GBE and is therefore a suitable model for the study of the enzymes' catalytic mechanism.  (+info)

Mapping of genes for cooking and eating qualities in Thai jasmine rice (KDML105). (7/213)

Thai jasmine rice, KDML 105, is known as the best quality rice. It is known not only for its aroma but also for its good cooking and eating qualities. Amylose content (AC), gel consistency (GC) and gelatinization temperature (GT) are important traits determining rice quality. A population of recombinant inbred lines (RIL) derived from KDML105 x CT9993 cross was used to study the genetic control of AC, GC and GT traits. A total of 191 markers were used in the linkage map construction. The 1605.3 cM linkage map covering nearly the whole rice genome was used for QTL (define QTL) analysis. Four QTLs for AC were detected on chromosomes 3, 4, 6 and 7. These QTLs accounted for 80% of phenotypic variation explained (PVE) in AC. The presence of one major gene as well as several modifiers was responsible for the expression of the trait. Two QTLs on chromosome 6 and one on chromosome 7 were detected for GC, which accounts for 57% of PVE. A single gene of major effect along with modifier genes controls GC from this cross. The QTLs in the vicinity of waxy locus were major contributors in the expression of AC and GC. The finding that the position of QTLs for AC and GC were near each other may reflect tight linkage or pleiotropy. Three QTLs were detected, one on chromosome 2 and two on chromosome 6, which accounted for 67% of PVE in GT. Just like AC and GC, one major gene and modifier genes governed the variation in GT resulting from the KDML105 x CT9993 cross. Breeding for cooking and eating qualities will largely rely on the preferences of the end users.  (+info)

Suppression of extablished Friend virus leukemia by statolon: potentiation of statolon's leukemosuppressive activity by chlorite-oxidized oxyamylose. (8/213)

Treatment of Friend virus (FV)-infected mice, 3 days after FV inoculation, with statolon, an extract of the mold Penicillium stoloniferum, induces interferon and restores immunocompetence to viral and nonviral antigens such as sheep erythrocytes. Clinical remissions are established in 20 to 70% of the infected mice. Cholorite-oxidized oxyamylose administered intraperitoneally 24 h before, 3 h before, or 3 h after statolon enhanced interferon production, but the increased number of mice protected against FV disease was more closely related to the associated enhanced synthesis of FV cytotoxic antibody. The prolonged selective immunodepression to intraperitoneal sheep erythrocytes after intraperitoneal administration of chlorite-oxidized oxyamylose-statolon appeared to be related to a stimulation in number and erythrocyte-phagocytic capacity of peritoneal macrophages. The marked activation of macrophages in FV leukemic mice after such treatment may also have contributed to the enhanced FV leukemosuppressive effects of chlorite-oxidized oxyamylose-statolon.  (+info)

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