alpha-Amylase adsorption on starch crystallites

The goal of this work was to characterize the adsorption of Bacillus subtills alpha-amylase onto crystalline starchy materials of the B-type polymorph. Monodisperse spherulitic particles (R z6; 5.0 mum), essentially resistant to alpha-amylolysis at 25 degrees C were prepared from short amylose chains (DP(n) approximately 15). The alpha-amylase adsorbed specifically onto the spherulites, and adsorption was found to be a prerequisite step for hydrolysis. Adsorption was inhibited by the presence of maltose and maltotriose in the reaction mixture. Adsorption isotherm of the enzyme on the particles showed a well developed plateau of 1.62 mug/cm(2) at 25 degrees C corresponding to a monolayer adsorption process. The binding free energy calculated from the initial slope of the isotherm was DeltaG approximately -20.7 kJ/mol. This is smaller than published values for the binding of alpha-amylase to soluble amylosic chains (DeltaG < -30 kJ/mol).     

New chromogenic substrates for the assay of alpha-amylase and (1 leads to 4)-beta-D-glucanase

New chromogenic substrates have been developed for the quantitative assay of alpha-amylase and (1 leads to 4)-beta-D-glucanase. These were prepared by chemically modifying amylose or cellulose before dyeing, to increase solubility. After dyeing, the substrates were either soluble or could be readily dispersed to form fine, gelatinous suspensions. Assays based on the use of these substrates are sensitive and highly specific for either alpha-amylase or (1 leads to 4)-beta-D-glucanase. The method of preparation can also be applied to obtain substrates for other endo-hydrolases.     

An ESR assay for alpha-amylase activity toward succinylated starch, amylose and amylopectin

The esterification of the three polysaccharides, starch, amylose and amylopectin was carried out in pyridine-DMSO by succinic anhydride. The carboxylic groups in the succinylated polysaccharides were measured by FT-IR spectroscopy. The succinic derivatives were tested as alpha-amylase (1,4-alpha-D-glucan glucano hydrolase, E.C. 3.2.1.1) substrates. A colorimetric assay of the alpha-amylase activity indicated that this enzyme is active on succinic esters of starch and amylose and that the activity shows a linear decrease with the number of succinic units introduced into the polysaccharide. Since the colorimetric test was not suitable for the detection of the alpha-amylase activity when succinylated amylopectin was the substrate, we set-up an assay based on the labeling by a paramagnetic probe of the free carboxylic groups of succinylated polysaccharides. The kinetics of the alpha-amylase reaction were monitored by ESR spectroscopy through the increase of the mobility of the paramagnetic probe. The spin label used was the commercially available 4-amino-tempo. By this method we demonstrated that alpha-amylase is active on succinylated amylopectin. The utility of the assay for monitoring alpha-amylase activity when other methods (i.e. colorimetric tests) fail, is discussed.     

Solubilization and partial purification of an enzyme involved in rat liver microsomal fatty acid chain elongation: beta-hydroxyacyl-CoA dehydrase.

The solubilization and partial purification of beta-hydroxyacyl-CoA dehydrase from rat liver microsomes has been accomplished through deoxycholate solubilization, ammonium sulfate fractionation, and ion exchange chromatography. A purification of about 90-fold based on total soluble activity was achieved, with an overall yield of 40%. However, the initial solubilization is accompanied by the loss of the secondary portion of the v/s curve observed with intact microsomes. The enzyme requires detergent during the purification procedure to remain "soluble," and is strongly activated by the inclusion of Triton-X-100 at concentrations above its critical micelle concentration in the assay mixture. In addition a preference for micelles has been inferred based on discontinuities in the v/s curves relative to the measured critical micelle concentration of the substrates in the absence of Triton X-100. Kinetic parameters calculated on the basis of micelle-specific activity indicated that beta-hydroxyacyl-CoA substrates possessing even-numbered alkyl chains from 14 to 20 carbon atoms differed little in Vm', but had progressively larger Km' as the chain length increased. The partially purified preparation was also active with beta-hydroxy-8,11-eicosadienoyl-CoA; and with 2-trans-enoyl-CoA substrates in a reverse (hydration) reaction.     

C-type cytochromes in the photosynthetic electron transfer pathways in green sulfur bacteria and heliobacteria

Green sulfur bacteria and heliobacteria are strictly anaerobic phototrophs that have homodimeric type 1 reaction center complexes. Within these complexes, highly reducing substances are produced through an initial charge separation followed by electron transfer reactions driven by light energy absorption. In order to attain efficient energy conversion, it is important for the photooxidized reaction center to be rapidly rereduced. Green sulfur bacteria utilize reduced inorganic sulfur compounds (sulfide, thiosulfate, and/or sulfur) as electron sources for their anoxygenic photosynthetic growth. Membrane-bound and soluble cytochromes c play essential roles in the supply of electrons from sulfur oxidation pathways to the P840 reaction center. In the case of gram-positive heliobacteria, the photooxidized P800 reaction center is rereduced by cytochrome c-553 (PetJ) whose N-terminal cysteine residue is modified with fatty acid chains anchored to the cytoplasmic membrane.     

Comparative characterization of complete and truncated forms of Lactobacillus amylovorus alpha-amylase and role of the C-terminal direct repeats in raw-starch binding

Two constructs derived from the alpha-amylase gene (amyA) of Lactobacillus amylovorus were expressed in Lactobacillus plantarum, and their expression products were purified, characterized, and compared. These products correspond to the complete (AmyA) and truncated (AmyADelta) forms of alpha-amylase; AmyADelta lacks the 66-kDa carboxyl-terminal direct-repeating-unit region. AmyA and AmyADelta exhibit similar amylase activities towards a range of soluble substrates (amylose, amylopectin and alpha-cyclodextrin, and soluble starch). The specific activities of the enzymes towards soluble starch are similar, but the K(M) and V(max) values of AmyADelta were slightly higher than those of AmyA, whereas the thermal stability of AmyADelta was lower than that of AmyA. In contrast to AmyA, AmyADelta is unable to bind to beta-cyclodextrin and is only weakly active towards glycogen. More striking is the fact that AmyADelta cannot bind or hydrolyze raw starch, demonstrating that the carboxyl-terminal repeating-unit domain of AmyA is required for raw-starch binding activity.     

Preparation, stability and antimicrobial activity of cationic cross-linked starch-iodine complexes

Cationic cross-linked starch (CCS)-iodine complexes containing different amounts of quaternary ammonium groups (different degrees of substitution (DS)) and iodine have been obtained by iodine adsorption on CCS from aqueous iodine potassium iodide solution. Equilibrium adsorption studies showed that with an increase of DS the amount of iodine adsorbed on CCS and the affinity of iodine to CCS increased linearly. The influences of the DS of CCS and the amount of adsorbed iodine on the stability of CCS-iodine complexes in a solution of 0.02M sodium acetate and reactivity toward l-tyrosine have been investigated. At the same DS, the stability of CCS-iodine complexes decreased with an increase of the amount of adsorbed iodine. With increasing the DS, the stability of CCS-iodine complexes increased. The iodine consumption in the reaction with l-tyrosine increased significantly with an increase of the amount of adsorbed iodine. The influence of DS on iodine consumption was lower and depended on the amount of adsorbed iodine. The antibacterial activity of CCS-iodine complexes against Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli was determined by the broth-dilution and spread-plate methods. The obtained results have demonstrated that an appropriate selection of the CCS-iodine complex composition (the DS of CCS and the amount of adsorbed iodine) could ensure good antimicrobial properties by keeping a low concentration of free iodine in the system. The main advantage of using CCS-iodine complexes as antimicrobial agents is the biodegradability of the polymeric matrix.     

Purification and characterization of the extracellular alpha-amylase from Clostridium acetobutylicum ATCC 824.

The extracellular alpha-amylase (1,4-alpha-D-glucanglucanohydrolase; EC 3.2.1.1) from Clostridium acetobutylicum ATCC 824 was purified to homogeneity by anion-exchange chromatography (mono Q) and gel filtration (Superose 12). The enzyme had an isoelectric point of 4.7 and a molecular weight of 84,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was a monomeric protein, the 19-amino-acid N terminus of which displayed 42% homology with the Bacillus subtilis saccharifying alpha-amylase. The amino acid composition of the enzyme showed a high number of acidic and hydrophobic residues and only one cysteine residue per mole. The activity of the alpha-amylase was not stimulated by calcium ions (or other metal ions) or inhibited by EDTA, although the enzyme contained seven calcium atoms per molecule. alpha-Amylase activity on soluble starch was optimal at pH 5.6 and 45 degrees C. The alpha-amylase was stable at an acidic pH but very sensitive to thermal inactivation. It hydrolyzed soluble starch, with a Km of 3.6 g . liter-1 and a Kcat of 122 mol of reducing sugars . s-1 . mol-1. The alpha-amylase showed greater activity with high-molecular-weight substrates than with low-molecular-weight maltooligosaccharides, hydrolyzed glycogen and pullulan slowly, but did not hydrolyze dextran or cyclodextrins. The major end products of maltohexaose degradation were glucose, maltose, and maltotriose; maltotetraose and maltopentaose were formed as intermediate products. Twenty seven percent of the glucoamylase activity generally detected in the culture supernatant of C. acetobutylicum can be attributed to the alpha-amylase.     

Effect of pH on complex formation between debranched waxy rice starch and fatty acids

Complex formations between debranched waxy rice starch (DBS) and fatty acids (FA) of different hydrocarbon chain lengths (8:0, 10:0, 12:0, 14:0, 16:0, and 18:0) were studied in an aqueous solution by measuring the blue colour stained with iodine. The objective of this study was to understand the effects of the solubility and hydrophobicity of guest molecules (FA) on the complex formation with DBS. Lauric acid (12:0) displayed the greatest complex forming ability with DBS by showing the least blue colour developed with iodine. The effect of pH (3-7) on the DBS/FA complex formation was evaluated by measuring the iodine-scanning spectra of the mixture. Short-chain FA (8:0) displayed less complex formation at pH>or=5, above the pK(a) of fatty acid (approximately 4.8), which suggested that the charge formation of the short-chain FA caused a lower partitioning of the FA into the hydrophobic cavity of the DBS single helix. On the contrary, FA of 10:0-18:0 displayed an increased complex formation at pH>5, which could be attributed to increased solubility of these longer-chain FA at a dissociated and ionized form. The hydrocarbon chain length of the FA had an important impact on the extent of the complex formation. A FA that had a shorter hydrocarbon chain was more soluble in an aqueous solution and more readily formed a complex with DBS. At pH 6 and 7 (above the pK(a)), 10:0 formed less inclusion complexes with DBS than did 12:0. Iodine-scanning spectra showed that the absorbances of all iodine-stained DBS/FA solutions at higher wavelength were substantially lower than that of the iodine-stained DBS alone, suggesting that FA preferentially formed inclusion complexes with DBS of longer chains.     

Purification, biochemical characterization, and gene cloning of a new extracellular thermotolerant and glucose tolerant maltooligosaccharide-forming alpha-amylase from an endophytic ascomycete Fusicoccum sp. BCC4124

An endophytic fungus, Fusicoccum sp. BCC4124, showed strong amylolytic activity when cultivated on multi-enzyme induction enriched medium and agro-industry substrates. alpha-Amylase and alpha-glucosidase activities were highly induced in the presence of maltose and starch. The purified target alpha-amylase, Amy-FC1, showed strong hydrolytic activity on soluble starch (kcat/Km=6.47 x 10(3) min(-1)(ml/mg)) and selective activity on gamma- and beta-cyclodextrins, but not on alpha-cyclodextrin. The enzyme worked optimally at 70 degrees C in a neutral pH range with t(1/2) of 240 min in the presence of Ca(2+) and starch. Maltose, matotriose, and maltotetraose were the major products from starch hydrolysis but prolonged reaction led to the production of glucose, maltose, and maltotriose from starch, cyclodextrins, and maltooligosaccharides (G3-G7). The amylase showed remarkable glucose tolerance up to 1 M, but was more sensitive to inhibition by maltose. The deduced protein primary structure from the putative gene revealed that the enzyme shared moderate homology between alpha-amylases from Aspergilli and Lipomyces sp. This thermotolerant, glucose tolerant maltooligosaccharide-forming alpha-amylase is potent for biotechnological application.     

Molecular cloning, nucleotide sequencing, and expression of the Bacillus subtilis (natto) IAM1212 alpha-amylase gene, which encodes an alpha-amylase structurally similar to but enzymatically distinct from that of B. subtilis 2633.

An alpha-amylase gene of Bacillus subtilis (natto) IAM1212 was cloned in a lambda EMBL3 bacteriophage vector, and the nucleotide sequence was determined. An open reading frame encoding the alpha-amylase (AMY1212) consists of 1,431 base pairs and contains 477 amino acid residues, which is the same in size as the alpha-amylase (AMY2633) of B. subtilis 2633, an alpha-amylase-hyperproducing strain, and smaller than that of B. subtilis 168, Marburg strain. The amino acid sequence of AMY1212 is different from that of AMY2633 at five residues. Enzymatic properties of these two alpha-amylases were examined by introducing the cloned genes into an alpha-amylase-deficient strain, B. subtilis M15. It was revealed that products of soluble starch hydrolyzed by AMY1212 are maltose and maltotriose, while those of AMY2633 are glucose and maltose. From the detailed analyses with oligosaccharides as substrates, it was concluded that the difference in hydrolysis products of the two similar alpha-amylases should be ascribed to the different activity hydrolyzing low-molecular-weight substrates, especially maltotriose; AMY1212 slowly hydrolyzes maltotetraose and cannot hydrolyze maltotriose, while AMY2633 efficiently hydrolyzes maltotetraose and maltotriose. Further analyses with chimeric alpha-amylase molecules constructed from the cloned genes revealed that only one amino acid substitution is responsible for the differences in hydrolysis products.     

Kinetics and energetics of ligand binding determined by microcalorimetry: insights into active site mobility in a psychrophilic alpha-amylase

A new microcalorimetric method for recording the kinetic parameters k(cat), K(m) and K(i) of alpha-amylases using polysaccharides and oligosaccharides as substrates is described. This method is based on the heat released by glycosidic bond hydrolysis. The method has been developed to study the active site properties of the cold-active alpha-amylase produced by an Antarctic psychrophilic bacterium in comparison with its closest structural homolog from pig pancreas. It is shown that the psychrophilic alpha-amylase is more active on large macromolecular substrates and that the higher rate constants k(cat) are gained at the expense of a lower affinity for the substrate. The active site is able to accommodate larger inhibitory complexes, resulting in a mixed-type inhibition of starch hydrolysis by maltose. A method for recording the binding enthalpies by isothermal titration calorimetry in a low-affinity system has been developed, allowing analysis of the energetics of weak ligand binding using the allosteric activator chloride. It is shown that the low affinity of the psychrophilic alpha-amylase for chloride is entropically driven. The high enthalpic and entropic contributions of activator binding suggest large structural fluctuations between the free and the bound states of the cold-active enzyme. The kinetic and thermodynamic data for the psychrophilic alpha-amylase indicate that the strictly conserved side-chains involved in substrate binding and catalysis possess an improved mobility, responsible for activity in the cold, and resulting from the disappearance of stabilizing interactions far from the active site.     

Improved methods for the structural analysis of the amylose-rich fraction from rice flour

Cooking and sensory properties of rice are largely determined by the amylose content and structure. For relationships between functional and structural properties, a more accurate method to determine the structure of amylose is required. Here we calibrate size exclusion chromatography (SEC) columns, using Mark-Houwink parameters for linear starch and pullulan standards, to obtain the true molecular weight distribution of linear starch. When the molecular weight distribution is reported relative to pullulan, rather than the actual molecular weight which is readily obtained from universal calibration, it is seen that the molecular weights of longer amylose chains are greatly underestimated. We validate the SEC method to enable the measurement of the hydrodynamic volume distribution of the starch by examining reproducibility and recovery. Analysis of the starch in the sample pre- and post-SEC shows that 20% of the carbohydrate is not recovered. Comparison of the weight-average degree of polymerization, X(w), of (undebranched) starch of pre- and post-SEC is made using iodine binding as well as Berry plots of data from multi-angle laser light scattering (MALLS). These both show that current SEC techniques for starch analysis lead to significant loss of high molecular weight material. Indeed, for the systems studied here, the values for X(w) after SEC are about three times lower than those before SEC. Iodine-starch complexes of pre- and post-SEC samples reveals that the SEC techniques give reliable data for the amylose fraction but not for amylopectin. We address reports in the literature suggesting that the conventional isoamylase method for debranching starch would lead to incomplete debranching and thus incorrect molecular weight distributions. However, it is shown using (1)H NMR that isoamylase can completely debranch the amylose (to within the detection limit of 0.5%), and by SEC that successive incubation with isoamylase, alpha-amylase, and beta-amylase can degrade the amylose-rich fraction completely to maltose. We develop a method to obtain a hot water soluble fraction (HWSF), rich in undamaged amylose molecules, directly from rice flour, avoiding the structural degradation of previous techniques. With appropriate sample handling, the formation of associations between starch chains is minimized. With the combination of calibrated and validated SEC methods, and an improved extraction of amylose from rice, the X(w) for both HWSF and debranched HWSF are found to be much larger than has previously been reported.     

Microcrystals of tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium are catalytically active

An improved and efficient method has been developed for the purification of the tryptophan synthase alpha 2 beta 2 complex (EC 4.2.1.20) from Salmonella typhimurium containing a multicopy plasmid. Microcrystals prepared in 12% poly(ethylene glycol) 8000 containing 2.5 mM spermine are shown by scanning electron microscopy to have the same crystal habit as the larger crystals that are being used for structural analysis by X-ray crystallography. The average dimensions of the crystals are 33 microns (length) X 9 microns (width) X 3 microns (maximum thickness). Our finding that suspensions of microcrystals are active in several reactions catalyzed by the active sites of the alpha and beta 2 subunits demonstrates that both active sites are functional in the crystal and accessible to substrates. Thus the larger crystals being used for X-ray crystallographic studies should form complexes with substrates and analogues at both active sites and should yield functionally relevant structural information. A comparison of the reaction rates of suspensions of microcrystals with those of the soluble enzyme shows that the maximum rate of the crystalline enzyme is 0.8 that of the soluble enzyme in the cleavage of indole-3-glycerol phosphate (alpha reaction), 0.3 that of the soluble enzyme in the synthesis of L-tryptophan by the beta reaction or the coupled alpha beta reaction, and 2.7 that of the soluble enzyme in the serine deaminase reaction. These small differences in rates probably reflect functional differences between the crystalline and soluble enzymes since the reaction rates of the microcrystals are calculated to be virtually free of diffusional limitation under these reaction conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of binding energy in catalysis analyzed by mutagenesis of the tyrosyl-tRNA synthetase

The utilization of enzyme-substrate binding energy in catalysis has been investigated by experiments on mutant tyrosyl-tRNA synthetases that have been generated by site-directed mutagenesis. The mutants are poorer enzymes because they lack side chains that form hydrogen bonds with ATP and tyrosine during stages of the reaction. The hydrogen bonds are not directly involved in the chemical processes but are at some distance from the seat of reaction. The free energy profiles for the formation of enzyme-bound tyrosyl adenylate and the equilibria between the substrates and products were determined from a combination of pre-steady-state kinetics and equilibrium binding methods. By comparison of the profile of each mutant with wild-type enzyme, a picture is built up of how the course of reaction is affected by the influence of each side chain on the energies of the complexes of the enzyme with substrates, transition states, and intermediates (tyrosyl adenylate). As the activation reaction proceeds, the apparent binding energies of certain side chains with the tyrosine and nucleotide moieties increase, being weakest in the enzyme-substrate complex, stronger in the transition state, and strongest in the enzyme-intermediate complex. Most marked is the interaction of Cys-35 with the 3'-hydroxyl of the ribose. Removal of the side chain of Cys-35 leads to no change in the dissociation constant of ATP but causes a 10-fold lowering of the catalytic rate constant. It contributes no net apparent binding energy in the E X Tyr X ATP complex and stabilizes the transition state by 1.2 kcal/mol and the E X Tyr-AMP complex by 1.6 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformation of amylose in aqueous solution: Optical rotatory dispersion and circular dichroism of amylose–iodine complexes and dependence on chain length of retrogradation of amylose

The dependence on chain length of two characteristic properties of amylose, i.e., retrogradation and complex formation with iodine, have been studied by using enzymatically synthesized, homodisperse amyloses. The association rates of amyloses in water containing 5% dimethyl sulfoxide have a sharp maximum at a degree of polymerization P?_n of 80; shorter and longer molecules are much more soluble. The iodine complexes of amylose exhibit a strong Cotton effect in the range of the long-wave absorption maximum (position depending on chain length) and two weaker Cotton effects at 480 and 350 nm. The long-wave Cotton effect is most intense at about P?_n 50 and decreases rapidly for shorter and longer chains. This behavior is unexpected and is not in accordance with the further increase of λ_max and λ_max. The experiments can best be interpreted by assuming well ordered, stiff chains in the low molecular weight range (P?_n 50–80). For longer chains, the findings are discussed in the light of current concepts of amylose conformation in aqueous solution, namely the model of the broken helical chain (alternating stiff helical segments and unordered regions) and the model of a flexible coil without a significant helical content. However, according to the results given in this paper, a wormlike helical chain seems to be the most adequate model for amylose conformation in neutral solution.     

Dye Complexes Produced in Gram Staining

Complexes of crystal violet and iodine containing (a) one molecule and four atoms, and (b) one molecule and two atoms respectively, were prepared and their solubility in alcohol (95%) determined. Both complexes were only slightly soluble in alcohol, the former being less soluble than the latter. The solubility decreased with decreasing concentration of alcohol, and also on storage. Complexes of malachite green, basic fuchsin and Victoria blue B with iodine were also prepared, and complexes of each dye with picric acid. Two complexes of different composition could be obtained from crystal violet and picric acid, and from Victoria blue and iodine. Complexes with basic fuchsin were much more soluble in alcohol (95%) than complexes with the other dyes tested. Dye-mordant complexes have some of the properties of organic charge-transfer complexes.     

Determination of depolymerization kinetics of amylose, amylopectin, and soluble starch by Aspergillus oryzae alpha-amylase using a fluorimetric 2-p-toluidinylnaphthalene-6-sulfonate/flow-injection analysis system

This study reports on the determination of the depolymerization kinetics of amylose, amylopectin, and soluble starch by Aspergillus oryzae alpha-amylase using flow-injection analysis with fluorescence detection and 2-p-toluidinylnaphthalene-6-sulfonate as the fluorescent probe. The experimental data points, corresponding to the evolution of the concentration of "detectable" substrate with depolymerization time, were fit to a single exponential decay curve in the case of amylose and to a double exponential decay curve in the cases of amylopectin and soluble starch. For all the assayed substrates, the determined depolymerization rates at time zero correlated well with the initial enzyme and substrate concentrations through the usual Michaelis-Menten hyperbola. Therefore, this methodology allows the determination of alpha-amylase activity using any of these substrates. For amylopectin and soluble starch, the value of the total depolymerization rate at any depolymerization time was the result of the additive contribution of two partial depolymerization rates. In contrast, the total depolymerization rate for amylose was always a single value. These results, in conjunction with the relative time evolution of the two partial depolymerization rates (for amylopectin and soluble starch), are in good agreement with a linear molecular structure for amylose, a "grape-like" cluster molecular structure for amylopectin, and an extensively degraded grape-like cluster structure for soluble starch.     

Purification and properties of midgut alpha-amylase isolated from Morimus funereus (Coleoptera: Cerambycidae) larvae

Using soluble starch as a substrate five isoforms of alpha-amylase were identified in a crude extract of Morimus funereus larvae. The main alpha-amylase (termed AMF-3) was purified by gel filtration chromatography and anion exchange chromatography to obtain a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Its enzymatic purity was confirmed by an in-gel activity assay after SDS-PAGE. The purity of AMF-3 was increased 112-fold with a 15.4% yield. AMF-3 had apparent molecular masses of 33 and 31 kDa when analysed using SDS-PAGE and Superdex 75 FPLC gel filtration chromatography, respectively and a calculated isoelectric point of 3.2. Purified AMF-3 showed maximal activity at pH 5.2 and had an optimum activity temperature of 45 degrees C. AMF-3 retained over 90% of its maximum activity at temperatures from 45 to 60 degrees C. AMF-3 exhibited a high affinity towards soluble starch with a K(m) value of 0.43 mg/mL. Maximal AMF-3 activity was achieved in the presence of 0.1 mM CaCl(2), while at higher concentrations its activity decreased. AMF-3 activity increased with increasing NaCl concentration. AMF-3 activity was significantly inhibited by alpha-amylase wheat inhibitor. Using a number of raw starch substrates maximum AMF-3 activity was achieved with horse-radish starch, in contrast to undetectable activity towards potato starch.     

Glucose oxidase from Aspergillus niger: the mechanism of action with molecular oxygen, quinones, and one-electron acceptors

Glucose oxidase from the mold Aspergillus niger (EC 1.1.3.4) oxidizes beta-D-glucose with a wide variety of oxidizing substrates. The substrates were divided into three main groups: molecular oxygen, quinones, and one-electron acceptors. The kinetic and chemical mechanism of action for each group of substrates was examined in turn with a wide variety of kinetic methods and by means of molecular modeling of enzyme-substrate complexes. There are two proposed mechanisms for the reductive half-reaction: hydride abstraction and nucleophilic attack followed by deprotonation. The former mechanism appears plausible; here, beta-D-glucose is oxidized to glucono-delta-lactone by a concerted transfer of a proton from its C1-hydroxyl to a basic group on the enzyme (His516) and a direct hydride transfer from its C1 position to the N5 position in FAD. The oxidative half-reaction proceeds via one- or two-electron transfer mechanisms, depending on the type of the oxidizing substrate. The active site of the enzyme contains, in addition to FAD, three amino acid side chains that are intimately involved in catalysis: His516 with a pK(a)=6.9, and Glu412 with pK(a)=3.4 which is hydrogen bonded to His559, with pK(a)>8. The protonation of each of these residues has a strong influence on all rate constants in the catalytic mechanism.