Identification of archaeon-producing hyperthermophilic α-amylase and characterization of the α-amylase

The extremely thermophilic anaerobic archaeon strain, HJ21, was isolated from a deep-sea hydrothermal vent, could produce hyperthermophilic alpha-amylase, and later was identified as Thermococcus from morphological, biochemical, and physiological characteristics and the 16S ribosomal RNA gene sequence. The extracellular thermostable alpha-amylase produced by strain HJ21 exhibited maximal activity at pH 5.0. The enzyme was stable in a broad pH range from pH 5.0 to 9.0. The optimal temperature of alpha-amylase was observed at 95 degrees C. The half-life of the enzyme was 5 h at 90 degrees C. Over 40% and 30% of the enzyme activity remained after incubation at 100 degrees C for 2 and 3 h, respectively. The enzyme did not require Ca(2+) for thermostability. This alpha-amylase gene was cloned, and its nucleotide sequence displayed an open reading frame of 1,374 bp, which encodes a protein of 457 amino acids. Analysis of the deduced amino acid sequence revealed that four homologous regions common in amylases were conserved in the HJ21 alpha-amylase. The molecular weight of the mature enzyme was calculated to be 51.4 kDa, which correlated well with the size of the purified enzyme as shown by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Influence of succinic acid on the extracellular α-amylase production by chemostat-crow Bacillus licheniformis

Summary An 8-fold increase in -amylase production by pulsing of succinic acid to a chemostat culture ofBacillus licheniformis has been shown. The -amylase concentration was found to be at the highest value two doubling times after the addition, indicating that the effect may be due to regulatory control.     

Extracellular α-amylase from Streptomyces rimosus

Summary A purification procedure for an extracellular -amylase from Streptomyces rimosus, oxytetracycline-producing strain, is described. The enzyme obtained was shown to be an acidic (pI 4.75) monomer with a relative molecular mass (M_r) of 43 000, containing three cysteines involved in the catalytic activity of the enzyme. Its amino-terminal part has 57–67% homology with amylases from other Streptomyces species. S. rimosus -amylase is sensitive to higher temperatures, and partially stabilized by Ca²⁺ ions. It hydrolyses starch (optimum at pH 5.0–6.0) in an endohydrolase manner giving rise to maltotriose, maltotetraose and higher oligosaccharides. Starch granules, except those from rice, were not significantly affected by the isolated -amylase.     

Production of α-amylase fromHalobacterium halobium

Maximum production of extracellular -amylase activity inHalobacterium halobium was at 40°C in a medium containing 25% (w/v) NaCl, 1% (w/v) soluble starch and 1% (w/v) peptone, in presence of 0.1mm ZnSO₄ after 5 days in shaking cultures. The amylase had optimal activity at pH 6.5 in the presence of 1 to 3% (w/v) NaCl at 53°C.S. Patel, N. Jain and D. Madamwar are with the Post Graduate Department of Biosciences, Sadar Patel University, Vallabh Vidyanagar-388120, India.     

Wheat protein inhibitors of α-amylase

The location in the seed, molecular properties and biological role of protein α-amylase inhibitors from wheat are discussed. Inhibition specificity of albumin inhibitors and structural features essential for interaction with inhibited amylases are also examined. The possible significance of these naturally occurring inhibitors in relation to their presence in foods in active form is described. Finally, genetic aspects of the albumin inhibitor production and the possibility of improving nutritional value and insect re     

Thermostable liquefying α-amylase fromBacillus amyloliquefaciens

Summary An extracellular -amylase is purified to homogeneity with 62 % recovery of the enzyme activity using heat treatment, ion-exchange and gel filtration chromatographies. The purified enzyme has a molecular weight of 68, 000, isoelectric point 6.25, optimal activity at pH 6 and temperature 65 °C, k_est 8.8×10⁸ s¹ liquefying amylase units, and K_m for starch 2.9 mg ml^–1. The enzyme is further characterized for its endo action on the starch and related polymers. Calcium stabilizes the active conformation of the enzyme during prolonged exposure to the extremes of pH and temperature. The enzyme retains 100 % activity for 24 h at 65°C and exhibits a half-life of 9, 3, and 0.5 h at 80°, 85° and 90°C, respectively.     

Thermostable extracellular α-amylase and α-glucosidase ofLipomyces starkeyi

Summary Thermostable, extracellular -amylase and -glucosidase were produced byLipomyces starkeyi CBS 1809 in a medium containing maize starch and soya bean meal. Contrary to published findings which suggested a single cell-bound amylolytic system for another strain ofL. starkeyi, this study revealed the presence of two enzymes — an -amylase and an -glucosidase inL. starkeyi CBS 1809. The enzymes were separated by solvent and salt precipitation and ion-exchange chromatography on DEAE-Biogel-A. The -amylase and -glucosidase had pH optima at 4.0 and 4.5 and temperature optima at 70°C and 60°C, respectively. While the low pH optima are not unique the enzymes are very distinctive in yeasts in having very high temperature optima. The -glucosidase had highest activities on maltose and isomaltose (100) with relative rates of activity on maltotriose, isomaltotriose and p-nitrophenyl--d-glucoside of 59, 48 and 22, respectively. It was inactive towards sucrose. Both the -amylase and -glucosidase ofL. starkeyi were located extracellularly and had molecular weights of 76,000 and 35,000, respectively.     

On the secretion of α-amylase by barley aleurone layers after incubation in gibberellic acid

Summary Gel filtration and centrifugation studies were used to study the distribution of -amylase activity in homogenates of barley (Hordeum vulgare L.) aleurone layers. The results obtained were consistent with the hypothesis that -amylase is secreted via membrane-bound vesicles. The -amylase activity in an homogenate of barley aleurone layers was derived not only from the enzyme retained in the aleurone cells but also from enzyme previously secreted from the cells but apparently retained by the cell walls. The amount of -amylase retained by the cell wall was influenced by factors such as the buffer in which the layers were incubated or the presence of Actinomycin D in the incubation medium.Abbreviations GA₃ gibberellic acid - RER rough endoplasmic reticulum - Act. D Actinomycin D     

Effects of gibberellic acid and abscisic acid on α-amylase activity and ultrastructure of aleurone cells ofAvena sativa L.

The effects of GA₃ and/or ABA on the α-amylase activity and the ultrastructure of aleurone cells in halves of seeds without embryos (embryo-less half seeds) of oats (Avena sativa L.) were studied. α-Amylase activity was detected by the starch-agar gel method in the aleurone layers of embryo-less half seeds soaked in 1 μM GA₃ solution or 100 μM GA₃+10 μM ABA solution but not in those of seeds soaked in distilled water, 10 μM ABA solution, or 1 μM GA₃+10 μM ABA solution. Ultrastructural examinations of aleurone cells with α-amylase activity showed a decrease in the number of sphaerosomes, the appearance of flattened saccules pressed to the surface of aleurone grains, and the development and transformations of the rER from a slender form to the one with wide inner spaces. In the aleurone cells in which the enzyme activity was not detected, components of the rER showed only slender profiles. The number of sphaerosomes did not decrease, and no flattened saccules appeared in the aleurone cells treated with 10 μM ABA or 1 μM GA₃+10 μM ABA.     

Multiple forms of porcine pancreatic α-amylase

Porcine pancreatic α-amylase can be fractionated into two components by DEAE-cellulose chromatography and by disc electrophoresis. The basis for fractionation is tentatively ascribed to a charge difference. The two components displayed the same specific activity and their thermal and pH stability, as well as the variation of V_max and K_m with pH, were identical within experimental error. It is concluded that the multiple forms of the amylase are physically distinct, but structurally related, with a common active site.     

Complete amino acid sequence and location of the five disulfide bridges in porcine pancreatic α-amylase

Porcine pancreatic α-amylase (1,4-α-d-glucan glucanohydrolase EC 3.2.1.1), a single polypeptide chain, contains nine residues of methionine. Eight different fragments resulting from cleavage of this molecule by cyanogen bromide were characterized. The sequences of six of them have previously been reported. Two missing fragments, CN2 (82 residues) and CN3b1 (76 residues) were purified after breaking of the interpeptidic disulfide bridge and their complete sequence as well as that of the previously purified CN1 peptide (102 residues) are reported here. The location of the three disulfide bridges present in these peptides was determined. Ordering of the carboxymethylated cyanogen bromide fragments was carried out by pulse labeling the amylase chain in vivo. The complete sequence of the porcine pancreatic amylase chain (496 residues) and the location of its five disulfide bridges is presented. Comparison with human and mouse pancreatic and salivary α-amylases and with rat pancreatic amylase obtained from the corresponding cDNA nucleotidic sequences shows a high degree of homology between mammalian α-amylases.     

Action pattern of human pancreatic α-amylase on maltoheptaose,a substrate for determining α-amylase in serum

An enzymatic assay for the determination of α-amylase in serum was developed which employed a soluble substrate, maltoheptaose, and a coupled enzymatic indicator reaction consisting of α-glucosidase and the hexokinase—glucose-6-phosphate dehydrogenase system. We used high-performance liquid chromatography (HPLC) to establish the action pattern of maltoheptaose under the test conditions: (A) the action pattern of α-amylase alone, (B) that of the combined action of α-amylase and α-glucosidase. Conducive to this effort was: the availability of pure maltoheptaose and human pancreatic α-amylase; the development of an adequate procedure for sample pretreatment (partition chromatography on a mixed-bed ion exchanger) and of an HPLC system for separation of substrate and reaction products without interference from by-products of the assay (partition chromatography on a cation-exchange column with acetonitrile—water); and the use of a new, very sensitive refractometric detector revealing sugar amounts as low as 40 ng.We derived the following stoichiometric equations:

The standard deviation of the rate coefficients is about 5%.     

The effect of abscisic acid on the differential expression of α-amylase isozymes in barley aleurone layers

Summary The treatment of barley aleurone layers with gibberellic acid (GA₃) results in the synthesis of two groups of -amylase isozymes. Addition of abscisic acid (ABA) at the same time as GA₃ inhibited the synthesis of both groups of isozymes. However, midcourse ABA addition (12 h or later after GA₃) had a more inhibitory effect on the high pI -amylase group than on the low pI -amylase group. This midcourse inhibition was detectable within 2 h of ABA addition. Northern analysis results using cDNA probes for the high pI and low pI -amylase groups paralleled the protein synthesis results for both isozyme groups. High pI -amylase mRNA levels began to decrease within 2 h of midcourse ABA treatment and were less than 10% of the original level by 4 h. The levels of low pI -amylase mRNA were decreased less by midcourse ABA addition than were high pI mRNA levels. Cordycepin and cycloheximide blocked the effects of midcourse ABA addition on -amylase mRNA. These observations indicate that ABA inhibits -amylase expression at the pretranslational level and that protein and RNA synthesis are required for midcourse ABA action to occur. Our results also show that -amylase mRNA, which has been thought to be very stable, is degraded after midcourse ABA treatment.     

Purification of α-amylase from Bacillus lentus cultures

Acetone fractionation of Bacillus lentus culture filtrate yielded the highest -amylase activity and the 66.6% fraction reached 13-fold that of the crude enzyme preparation. Gel filtration and ion exchange chromatography afforded a pure -amylase (relative molecular mass, 42 000). The pure enzyme was highly active on starch and dextrin. It produced a mixture of oligosaccharides as major products of starch hydrolysis. Maximal activity was reached at 70° C and pH 6.1. Ca²⁺, Na⁺, K⁺ and Sr²⁺ ions stabilized or slightly stimulated the enzyme whereas Ag⁺, Co²⁺, Hg²⁺, Zn²⁺, Cd²⁺ and Fe³⁺ ions strongly inhibited the activity. The enzyme contained 16 amino acids, of which aspartic and glutamic acids were present in the highest proportions. Correspondence to: S. H. Omar     

The high maltose-producing α-amylase of Penicillium expansum

Summary An -amylase capable of producing exceptionally high levels of maltose (74%) from starch has been identified from a strain of Penicillium expansum. The enzyme is produced extracellularly and was purified to homogeneity by starch adsorption and Sephadex gel filtration chromatography. P. expansum -amylase has a pH optimum of 4.5 and is stable in the pH range of 3.6–6.0. Other properties include a temperature optimum of 60° C, a molecular weight of 69 000 and an isoelectric point of 3.9. The most outstanding feature of the P. expansum enzyme is its ability to yield 14% more maltose and 17.1% less maltotriose than a currently used commercial enzyme. This may be partly explained by the greater affinity of this new enzyme for maltotriose (K _m=0.76 mM) relative to the commerical enzyme, Fungamyl (K _m=2.9 mM). The enzyme reported here is unique among fungal -amylases in being able to produce such high levels of maltose and its physicochemical properties suggest that it has potential for commercial development.     

Stability of fungal α-amylase in sodium dodecylsulfate

Unfolding of a fungal -amylase in aqueous sodium dodecylsulfate (SDS) solution was examined by SDS-polyacrylamide gel electrophoresis (PAGE). When the -amylase was incubated with 1% SDS at room temperature and subjected to SDS-PAGE, it showed a much higher mobility than expected from the molecular weight. Circular dichroic and gel filtration analyses indicated that the protein is apparently in the native conformation upon incubation with 1% SDS. When the protein was heated in the presence of 1% SDS at 90°C for 10 min, it had a lower mobility in SDS-PAGE and showed characteristics of an unfolded protein by circular dichroism and gel filtration. The melting temperatures of the protein were determined in the absence and presence of SDS by incubating it for 10 min at various temperatures. The melting temperatures were 70, 55, and 49°C in the presence of 0, 1, and 2% SDS, respectively. The observed small shift of the melting temperatures by SDS suggests that the destabilizing action of SDS on the -amylase is weak. However, the unfolding in SDS is not reversible process, since prolonged incubation of the protein with 1% SDS at 50°C gradually increased the amount of unfolded protein. This indicates that the SDS-induced unfolding of the -amylase is a slow process.     

Effects of bean and wheat α-amylase inhibitors on α-amylase activity and growth of stored product insect pests

Insect α-amylase inhibiting and/or growth inhibiting activities of proteinaceous inhibitors from red kidney bean (Phaseolus vulgaris) and hard red winter wheat (Triticum aestivum) were examined. The bean inhibitor was most effectivein vitro against α-amylases from the red flour beetle (Tribolium castaneum) and the confused flour beetle (T. confusum), followed by those from the rice weevil (Sitophilus oryzae) and yellow mealworm (Tenebrio molitor). The insect enzymes were from two- to 50-fold more susceptible than human salivary α-amylase. When the inhibitors were added at a 1% level to a wheat flour plus germ diet, the growth of red flour beetle larvae was slowed relative to that of the control group of larvae, with the bean inhibitor being more effective than the wheat inhibitor. Development of both the red flour beetle and flat grain beetle (Cryptolestes pusillus) was delayed by 1% bean inhibitor, but development of the sawtoothed grain beetle (Oryzaephilus surinamensis) and lesser grain borer (Rhyzopertha dominica) was not affected by either the bean or wheat inhibitor at the 1% level. Rice weevil adults fed a diet containing 1% bean or wheat inhibitor exhibited more mortality than weevils fed the control diet. When the wheat amylase inhibitor was combined with a cysteine protease inhibitor, E-64, and fed to red flour beetle larvae, a reduction in the growth rate and an increase in the time required for adult eclosion occurred relative to larvae fed either of the inhibitors separately. The bean inhibitor was just as effective alone as when it was combined with the protease inhibitor. These results demonstrate that plant inhibitors of insect digestive enzymes act as growth inhibitors of insects and possibly as plant defense proteins, and open the way to the use of the genes of these inhibitors for genetically improving the resistance of cereals to storage pests. Cooperative investigation between the Agricultural Research Service, the University of California, San Diego, and the Kansas Agricultural Experiment Station (Contribution no. 94-416-J). Supported in part by a grant from the Ministry of Education and Science, Spain-Fulbright Program to J.J.P. Mention of a proprietary product does not constitute a recommendation or endorsement by the USDA. The USDA is an equal opportunity/affirmative action employer and all agency services are available without discrimination.