Modified Method for the Determination of Grain Amylase Activity by the Falling Number

The standard method for determination of amylase activity by the falling number was modified by us to study the carbohydrate–amylase complex and time course of starch hydrolysis by amylases from rye grain. The method is based on the use of potato starch as a standard substrate and aqueous extract of grain amylases as an enzyme source.     

A modified method of determining amylase activity in grain by falling number

The standard method for determination of amylase activity by the falling number was modified by us to study the carbohydrate-amylase complex and time course of starch hydrolysis by amylases from rye grain. The method is based on the use of potato starch as a standard substrate and aqueous extract of grain amylases as an enzyme source.     

Modes of action of acarbose hydrolysis and transglycosylation catalyzed by a thermostable maltogenic amylase, the gene for which was cloned from a Thermus strain

A maltogenic amylase gene was cloned in Escherichia coli from a gram-negative thermophilic bacterium, Thermus strain IM6501. The gene encoded an enzyme (ThMA) with a molecular mass of 68 kDa which was expressed by the expression vector p6xHis119. The optimal temperature of ThMA was 60 degrees C, which was higher than those of other maltogenic amylases reported so far. Thermal inactivation kinetic analysis of ThMA indicated that it was stabilized in the presence of 10 mM EDTA. ThMA harbored both hydrolysis and transglycosylation activities. It hydrolyzed beta-cyclodextrin and starch mainly to maltose and pullulan to panose. ThMA not only hydrolyzed acarbose, an amylase inhibitor, to glucose and pseudotrisaccharide (PTS) but also transferred PTS to 17 sugar acceptors, including glucose, fructose, maltose, cellobiose, etc. Structural analysis of acarbose transfer products by using methylation, thin-layer chromatography, high-performance ion chromatography, and nuclear magnetic resonance indicated that PTS was transferred primarily to the C-6 of the acceptors and at lower degrees to the C-3 and/or C-4. The transglycosylation of sugar to methyl-alpha-D-glucopyranoside by forming an alpha-(1,3)-glycosidic linkage was demonstrated for the first time by using acarbose and ThMA. Kinetic analysis of the acarbose transfer products showed that the C-4 transfer product formed most rapidly but readily hydrolyzed, while the C-6 transfer product was stable and accumulated in the reaction mixture as the main product.     

<Emphasis Type="Italic">Rhizopus microsporus</Emphasis> var.<Emphasis Type="Italic"> rhizopodiformis</Emphasis>: a thermotolerant fungus with potential for production of thermostable amylases

The effect of several nutritional and environmental parameters on growth and amylase production from Rhizopus microsporus var. rhizopodiformis was analysed. This fungus was isolated from soil of the Brazilian "cerrado" and produced high levels of amylolytic activity at 45°C in liquid medium supplemented with starch, sugar cane bagasse, oat meal or cassava flour. Glucose in the culture medium drastically repressed the amylolytic activity. The products of hydrolysis were analysed by thin layer chromatography, and glucose was detected as the main component. The amylolytic activity hydrolysed several substrates, such as amylopectin, amylase, glycogen, pullulan, starch, and maltose. Glucose was always the main end product detected by high-pressure liquid chromatography analysis. These results indicated that the amylolytic activity studied is a glucoamylase, but there were also low levels of -amylase. As compared to other fungi, R. microsporus var. rhizopodiformis can be considered an efficient producer of thermostable amylases, using raw residues of low cost as substrates. This information is of technological value, considering the importance of amylases for industrial hydrolysis.     

Purification and characterisation of a malto-oligosaccharide-forming amylase active at high pH from Bacillus clausii BT-21

Bacillus clausii BT-21 produced an extracellular malto-oligosaccharide-forming amylase active at high pH when grown on starch substrates. The enzyme was purified to homogeneity by affinity and anion-exchange chromatography. The molecular weight of the enzyme estimated by sodium dodecyl sulfate polyacrylamide electrophoresis was 101 kDa. The enzyme showed an optimum of activity at pH 9.5 and 55 degrees C. Maltohexaose was detected as the main initially formed starch hydrolysis product. Maltotetraose and maltose were the main products obtained after hydrolysis of starch by the enzyme for an extended period of time and were not further degraded. The enzyme readily hydrolysed soluble starch, amylopectin and amylose, while cyclodextrins, pullulan or dextran were not degraded. The mode of action during hydrolysis of starch indicated an exo-acting type of amylolytic enzyme mainly producing maltohexaose and maltotetraose. Amino acid sequencing of the enzyme revealed high homology with the maltohexaose-forming amylase from Bacillus sp. H-167.     

Glycosylated salivary alpha-amylases are capable of maltotriose hydrolysis and glucose formation

The physiological and/or clinical significance of sugar chains in human salivary alpha-amylase was investigated in terms of substrate-specificity for synthesized malto-oligosaccharides. Glycosylated and non-glycosylated alpha-amylases were prepared on a Sephacryl S-200 column, in which the amylases were separated into four fractions from the different affinities for Sephacryl: fraction I, amylases bearing sugar chains with sialic acid; fraction II, amylases bearing sugar chains without sialic acid; fractions III and IV, non-glycosylated amylases. These were classified according to the differences in their affinities for lectins, molecular sizes and isoelectric points. The inhibitory effect of maltotriose (G3) on starch hydrolysis of the amylase fraction, suggests that starch and G3 can be the substrate for glycosylated amylase, and that the glycosylated amylases are capable of G3 hydrolysis for conversion into maltose and glucose. Using malto-oligosaccharides, G3, G4, G5 and G7, as substrates, the substrate-specificities and G3/G5 ratio of amylase activities in the four fractions were examined. Maltopentaose, G5, is routinely used as a substrate for alpha-amylase, and then we assumed that both glycosylated and non-glycosylated amylases react with G5. Moreover, the results indicate that the glycosylated amylases clearly had a higher capacity for G3 hydrolysis than the non-glycosylated amylases, although no substrate preference of either type of amylase was observed among G4, G5 and G7. Glycosylated amylases have the capacity for glucose formation from malto-oligosaccharides.     

Complete starch hydrolysis by the synergistic action of amylase and glucoamylase: impact of calcium ions

Starch hydrolysis was performed by the synergistic action of amylase and glucoamylase. For that purpose glucoamylase (Dextrozyme) and two amylases (Liquozyme and Termamyl) in different combinations were investigated. Experiments were carried out in the repetitive- and fed-batch modes at 65 °C and pH 5.5 with and without the addition of Ca²⁺ ions. 100 % conversion of starch to glucose was achieved in batch experiments. Calcium ions significantly enhanced stability of the amylase Termamyl. The intensity of synergism between amylase Termamyl and glucoamylase Dextrozyme was higher than in the experiments carried out with amylase Liquozyme and Dextrozyme. Mathematical model of the complete reaction system was developed. Using the model, a possible explanation of the synergism between the amylase and glucoamylase was provided.     

The Characterisation of an Alkali-Stable Maltogenic Amylase from Bacillus lehensis G1 and Improved Malto-Oligosaccharide Production by Hydrolysis Suppression

A maltogenic amylase (MAG1) from alkaliphilic Bacillus lehensis G1 was cloned, expressed in Escherichia coli, purified and characterised for its hydrolysis and transglycosylation properties. The enzyme exhibited high stability at pH values from 7.0 to 10.0. The hydrolysis of β-cyclodextrin (β-CD) produced malto-oligosaccharides of various lengths. In addition to hydrolysis, MAG1 also demonstrated transglycosylation activity for the synthesis of longer malto-oligosaccharides. The thermodynamic equilibrium of the multiple reactions was shifted towards synthesis when the reaction conditions were optimised and the water activity was suppressed, which resulted in a yield of 38% transglycosylation products consisting of malto-oligosaccharides of various lengths. Thin layer chromatography and high-performance liquid chromatography analyses revealed the presence of malto-oligosaccharides with a higher degree of polymerisation than maltoheptaose, which has never been reported for other maltogenic amylases. The addition of organic solvents into the reaction further suppressed the water activity. The increase in the transglycosylation-to-hydrolysis ratio from 1.29 to 2.15 and the increased specificity toward maltopentaose production demonstrated the enhanced synthetic property of the enzyme. The high transglycosylation activity of maltogenic amylase offers a great advantage for synthesising malto-oligosaccharides and rare carbohydrates.     

The mechanism of salivary amylase hydrolysis: role of residues at subsite S2'

Hydrolysis of starch or oligosaccharides by mammalian amylases, in general, results in maltose as the leaving group. The active site of these amylases harbors three aromatic residues Trp59, Tyr62, and Tyr151, which provide stacking interactions to the bound glucose moieties. We hypothesized that Tyr151, located at the S2' subsite, may influence the size of the leaving group. Therefore, using a baculovirus expression system, we generated a mutant Y151M in which the tyrosine at position 151 of human salivary amylase is replaced by a methionine. The specific activity, K(m), rate of hydrolysis, and the product distribution for Y151M were distinctly different from those of the wild-type enzyme using starch and oligosaccharides as substrates. The mutant enzyme Y151M consistently produced glucose as the minimal leaving group and exhibited a twofold increase in K(m). These results suggest that the stacking interaction at subsite S2' in the wild type plays a role in hydrolysis.     

Glycosylated salivary α-amylases are capable of maltotriose hydrolysis and glucose formation

The physiological and/or clinical significance of sugar chains in human salivary αamylase was investigated in terms of substrate-specificity for synthesized malto-oligosaccharides. Glycosylated and non-glycosylated α-amylases were prepared on a Sephacryl S-200 column, in which the amylases were separated into four fractions from the different affinities for Sephacryl: fraction I, amylases bearing sugar chains with sialic acid; fraction II, amylases bearing sugar chains without sialic acid; fractions III and IV, non-glycosylated amylases. These were classified according to the differences in their affinities for lectins, molecular sizes and isoelectric points. The inhibitory effect of maltotriose (G₃) on starch hydrolysis of the amylase fraction, suggests that starch and G₃ can be the substrate for glycosylated amylase, and that the glycosylated amylases are capable of G₃ hydrolysis for conversion into maltose and glucose. Using malto-oligosaccharides, G₃, G₄, G₅ and G₇, as substrates, the substrate-specificities and G₃/G₅ ratio of amylase activities in the four fractions were examined. Maltopentaose, G₅, is routinely used as a substrate for α-amylase, and then we assumed that both glycosylated and non-glycosylated amylases react with G₅. Moreover, the results indicate that the glycosylated amylases clearly had a higher capacity for G₃ hydrolysis than the non-glycosylated amylases, although no substrate preference of either type of amylase was observed among G₄, G₅ and G₇. Glycosylated amylases have the capacity for glucose formation from malto-oligosaccharides.     

An enzymatic signal amplification system for calorimetric studies of cellobiohydrolases

The study of cellulolytic enzymes has traditionally been carried out using endpoint measurements by quantitation of reaction products using high-performance liquid chromatography (HPLC) or overall determination of produced reducing ends. To measure catalytic activity, model substrates such as solubilized cellulose derivates, soluble chromogenic, and flourogenic oligomeric substrates are often employed even though they do not reflect the natural insoluble substrate hydrolysis. Thermochemical methods using, for example, isothermal titration calorimetry (ITC) yield data where the primary observable is heat production. This can be converted to the rate of reaction and allows direct and continuous monitoring of the hydrolysis of complex substrates. To overcome the low molar enthalpy of the hydrolysis of the glycosidic bond, which is typically on the order of −2.5 kJ mol⁻¹, an enzymatic signal amplification method has been developed to measure even slow hydrolytically active enzymes such as cellobiohydrolases. This method is explained in detail for the amplification of the heat signal by more than 130 times by using glucose oxidase and catalase. The kinetics of this complex coupled reaction system is thoroughly investigated, and the potential use to generate kinetic models of enzymatic hydrolysis of unmodified cellulosic substrates is demonstrated.     

Starch Degradation in Spinach Leaves: ISOLATION AND CHARACTERIZATION OF THE AMYLASES AND R-ENZYME OF SPINACH LEAVES

The properties of two amylase activities which differ in their substrate specificity and subcellular location as well as a chloroplast-associated R-enzyme (debranching activity) are reported. An extrachloroplastic amylase is resolved by gel filtration chromatography into two activities of 80,000 and 40,000 daltons. Both extrachloroplastic activities hydrolyze amylopectin and shellfish glycogen and only slowly hydrolyze rabbit liver glycogen, β-limit amylopectin, and amylose. In contrast, the major chloroplastic amylase attacks all of these glucans at comparable rates. Glucan hydrolysis by both the extrachloroplastic and chloroplastic amylase generates not only maltose but appreciable amounts of other oligosaccharides, whereas maltotetraose hydrolysis produces glucose, maltose, and maltotriose. The action patterns displayed by the amylase activities indicate that both are endoamylases, although they lack the typical Ca²⁺ requirement or heat stability of seed endosperm α-amylases. Dithiothreitol, glutathione (oxidized or reduced), ascorbate, dehydroascorbate, and dithiothreitol plus thioredoxin have no effect on either the chloroplastic or extrachloroplastic amylase activities.     

Separation and characterization of four different amylases of Entamoeba histolytica. II. Characterization of amylases

Purified E. histolytica amylases III to VI were characterized by their hydrolytic behaviour towards 4-nitrophenyl alpha-malto-oligosaccharides, malto-oligosaccharides, amylose, amylopectin, glycogen and Y-cyclodextrin. The influence of specific inhibitors on the amylase activity of E. histolytica was examined and compared with typical alpha- and beta-amylases. Amylases III and IV showed alpha-glucosidase and glucosyltransferase activity by cleaving terminal non-reducing glucose from pNPG1 (III, IV) and pNPG2 to pNPG7 (III). Both enzymes were able to cleave malto-oligosaccharides and glucopolysaccharides to a large number of malto-oligosaccharides. Also transglucosidation reactions were observed, but maltose was not hydrolysed. Amylase V showed exoamylase-like properties by preferentially cleaving maltose units from the non-reducing end of synthetic and biogenic malto-oligosaccharides by a multiple-attack mechanism. Amylase VI was characterized as an alpha-amylase, showing great similarities with porcine pancreatic alpha-amylase in the hydrolysis pattern of 4-nitrophenyl alpha-malto-oligosaccharides and glucopolysaccharides. With biogenic malto-oligosaccharides amylase VI showed a transglucosidation reaction.     

Molecular subtyping within a single Salmonella typhimurium phage type, DT204c, with a PCR-generated probe for IS200

Abstract Pullulan is an industrial biopolymer produced by the yeast-like fungus Aureobasidium , usually by direct fermentation of starch. Despite evidence that autogenous amylases produced during these fermentations are detrimental to the final molecular mass of the product, fundamental studies of these enzymes have not been reported. Total extracellular amylases were studied from the promising production strain NRRL Y-12,974. Growth rates and yields were equivalent in cultures grown on glucose, maltose, soluble starch, or cornstarch. Total amylase levels were low and varied only three-fold, from 0.01 IU ml⁻¹ in glucose-grown cultures to 0.03 IU ml⁻¹ in soluble-starch-grown cultures. All cultures showed both α-amylase activity and activity against pullulan. Synthetic oligosaccharide substrates were apparently attacked by an α-glucosidase, produced in highest levels by maltose-grown cultures.     

Expression and characterization of amylase encoded by a gene cloned from Cellulomonas sp. NCIM 2353

A Cellulomonas genomic fragment encoding extracellular amylase activity was isolated as a clone (ACs2) in Escherichia coli DH10B. The amylase was expressed in the absence of IPTG and in the presence of starch or maltose. This enzyme corresponded to the low mobility activity of Cellulomonas amylases as demonstrated on gel electrophoresis. Maltose, as well as lactose, xylose and xylan cross-induced the amylase of clone ACs2. Maltose-induced amylase was purified to homogeneity. ACs2-coded amylase is a 70kDa acidic protein, with a pH optimum of 7.0 at 45°C. This enzyme exhibited an endo mode of action, similar to the corresponding Cellulomonas enzyme.     

Chromatographic methods for amylases

This review surveys recent developments in chromatographic methods for the separation of amylases from complex extracts, including the separation of isozymes. It contains two tables with the properties and molecular characteristics of α- and β-amylases from different sources as well as an updated review of methods for the determination of amylase activity. The main subject of this review is a detailed evaluation of the application of newly developed chromatographic methods for the purification of amylases.     

Purification and Properties of Extracellular Amylase from the Hyperthermophilic Archaeon Thermococcus profundus DT5432

A hyperthermophilic archaeon, Thermococcus profundus DT5432, produced extracellular thermostable amylases. One of the amylases (amylase S) was purified to homogeneity by ammonium sulfate precipitation, DEAE-Toyopearl chromatography, and gel filtration on Superdex 200HR. The molecular weight of the enzyme was estimated to be 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amylase exhibited maximal activity at pH 5.5 to 6.0 and was stable in the range of pH 5.9 to 9.8. The optimum temperature for the activity was 80(deg)C. Half-life of the enzyme was 3 h at 80(deg)C and 15 min at 90(deg)C. Thermostability of the enzyme was enhanced in the presence of 5 mM Ca(sup2+) or 0.5% soluble starch at temperatures above 80(deg)C. The enzyme activity was inhibited in the presence of 5 mM iodoacetic acid or 1 mM N-bromosuccinimide, suggesting that cysteine and tryptophan residues play an important role in the catalytic action. The amylase hydrolyzed soluble starch, amylose, amylopectin, and glycogen to produce maltose and maltotriose of (alpha)-configuration as the main products. Smaller amounts of larger maltooligosaccharides were also produced with a trace amount of glucose. Pullulan; (alpha)-, (beta)-, and (gamma)-cyclodextrins; maltose; and maltotriose were not hydrolyzed.     

Purification and properties of a maltoheptaose- and maltohexaose-forming amylase produced by Bacillus subtilis US116

A new bacterial strain, identified as Bacillus subtilis US116, was isolated from Tunisian soil and selected for its potential production of an atypical amylase with an industrial interest. The identification was founded on physiological tests and molecular techniques related to the 16S rRNA, 23S rRNA genes and intergenic sequences showing the highest similarity of 98% with regions in the complete genome of Bacillus subtilis 168 (accession no. Z99104). This strain produces an atypical amylase that was purified to homogeneity by a combination of acetone precipitation, size exclusion and ion exchange chromatography. The molecular mass of the enzyme is about 60 kDa as determined by SDS–PAGE. Optimal conditions for the activity of the purified enzyme are pH 6 and 65 °C. The half-life duration is about 3 h at 70 °C and 5 h at 65 °C. This enzyme belongs to the endo-type amylases according to the hydrolytic mode study using Ceralpha and Betamyl methods. It is classified as a maltoheptaose- and maltohexaose-forming amylase since it generates about 30% maltohexaose (DP6) and 20% maltoheptaose (DP7) from starch. Moreover, the minimum length of maltosaccharide cleaved by this enzyme was maltoheptaose.     

Inhibition of amylases from different origins by albumins from the wheat kernel.

The amylase activity of water extracts from 18 insect species, from 23 marine species and from 17 different species of birds and mammals was determined quantitatively. The inhibition of amylase in these extracts by three albumin fractions from the mature wheat kernel, which had been separated according to their molecular weights (60 000, 24 000 and 12 500 D), was determined as well. The inhibition activity of the three albumin fractions toward amylases extracted from a number of cereal species or from immature and germinating wheat kernel was also tested. The extracts from insects that are destructive of wheat grain and stored wheat products showed much higher amylase activities as compared to the other insect species that do not attack wheat and wheat products. On the basis of the effectiveness with which the three albumin fractions inhibit their activities, the amylase preparations tested were divided into susceptible, partially susceptible and resistent. Susceptible amylases, inhibited by any of the three albumin fractions, were found mainly in insects that attack wheat and in marine species. Partially susceptible amylases, inhibited by only one or two of the three albumin fractions, were present in a few avain and mammalian species including man. Resistent amylases were largely distributed in cereal, avian and mammalian species as well as in insect species that do not usually attack wheat grain or wheat flour products. At no stage of development, wheat alpha-amylase was inhibited by the albumin fractions from the mature kernel. The 12 500 dalton albumin fraction was the most effective in inhibiting insect amylases, but it was inactive toward avian and mammalian amylases. The 24 000 dalton albumin fraction was the most effective in inhibiting amylases from marine avian and mammalian species and inhibited as much as 33 amylases over 66 different amylases tested. It is suggested that protein inhibitors of amylase contributed to natural selection of polyploid wheats by giving some insect resistence to such wheats, even though some insect species were able to overcome this biochemical defense toa large degree by producing higher amylase activities.     

The cloning and characterization of a second alpha-amylase of A. hydrophila JMP636

AIMS: The aim of this study was to identify, clone and characterize the second amylase of Aeromonas hydrophila JMP636, AmyB, and to compare it to AmyA.METHODS AND RESULTS: The amylase activity of A. hydrophila JMP636 is encoded by multiple genes. A second genetically distinct amylase gene, amyB, has been cloned and expressed from its own promoter in Escherichia coli. AmyB is a large alpha-amylase of 668 amino acids. Outside the conserved domains of alpha-amylases there is limited sequence relationship between the two alpha-amylases of A. hydrophila JMP636 AmyA and AmyB. Significant (80%) similarity exists between amyB and an alpha-amylase of A. hydrophila strain MCC-1. Differences in either the functional properties or activity under different environmental conditions as possible explanations for multiple copies of amylases in JMP636 is less likely after an examination of several physical properties, with each of the properties being very similar for both enzymes (optimal pH and temperature, heat instability). However the reaction end products and substrate specificity did vary enough to give a possible reason for the two enzymes being present. Both enzymes were confirmed to be alpha-type amylases.CONCLUSIONS: AmyB has been isolated, characterized and then compared to AmyA.SIGNIFICANCE AND IMPACT OF THE STUDY: The amylase phenotype is rarely encoded by more than one enzyme within one strain, this study therefore allows the better understanding of the unusual amylase production by A. hydrophila.