Immobilization of α-amylase from Bacillus circulans GRS 313 on coconut fiber

A simple and inexpensive method for immobilizing alpha-amylase from Bacillus circulans GRS 313 on coconut fiber was developed. The immobilization conditions for highest efficiency were optimized with respect to immobilization pH of 5.5, 30 degrees C, contact time of 4 h, and enzyme to support a ratio of 1:1 containing 0.12 mg/mL of protein. The catalytic properties of the immobilized enzyme were compared with that of the free enzyme. The activity of amylase adsorbed on coconut fiber was 38.7 U/g of fiber at its optimum pH of 5.7 and 48 degrees C, compared with the maximum activity of 40.2 U/mL of free enzyme at the optimum pH of 4.9 and 48 degrees C. The reutilization capacity of the immobilized enzyme was up to three cycles.     

Inhibition of cyclodextrins on the activity of α-amylase

α-amylase activity influences both flour fermentation process and the quality of the fermented products due to its ability of breaking starch into smaller units. The inhibition of cyclodextrins on α-amylase activity was investigated in this paper. Experiment results showed that hydrophobic cavity size was an intrinsic factor during the inhibition processing. Among three types of cyclodextrin (α-, β- and γ-), β-type exhibited the most significant inhibitory activity toward α-amylase. The optimal inhibitory parameters were indicated to be pH 5.9, concentration of β-cyclodextrins 1 mmol/L, reaction temperature 45 °C and reaction time 60 min. Results suggested that the endogenous fluorescence of α-amylase was inhibited by cyclodextrins. Circular dichroism spectrum indicated that the secondary structure of α-amylase, including α-helices, β-sheets and random coils, was changed by cyclodextrins. All the results in this paper aim to provide a further understanding for α-amylase in the industry application.     

Design,synthesis and exploration of in silico α-amylase and α-glucosidase binding studies of pyrrolidine-appended quinoline-constrained compounds

Research on Chemical Intermediates - A series of new pyrrolidine-appended phenoxy-substituted quinoline derivatives were synthesized using 2-chloro-3-formyl quinoline. Initially, the second...     

Crucial role of protein flexibility in formation of a stable reaction transition state in an α-amylase catalysis

Conformational flexibility of proteins provides enzymes with high catalytic activity. Although the conformational flexibility is known to be pivotal for the ligand binding and release, its role in the chemical reaction process of the reactive substrate remains unclear. We determined a transition state of an enzymatic reaction in a psychrophilic α-amylase by a hybrid molecular simulation that allows one to identify the optimal chemical state in an extensive conformational ensemble of protein. The molecular simulation uncovered that formation of the reaction transition state accompanies a large and slow movement of a loop adjacent to the catalytic site. Free energy calculations revealed that, although catalytic electrostatic potentials on the reactive moiety are formed by local and fast reorganization around the catalytic site, reorganization of the large and slow movement of the loop significantly contributes to reduction of the free energy barrier by stabilizing the local reorganization.     

Synthesis and α-amylase inhibitory activity of glucose-deoxynojirimycin conjugates

Inhibitors of α-amylase have attracted attention for their putative effects against diabetes mellitus. Although numerous studies have explored natural small molecule inhibitors, acarbose is currently the only compound with sufficient inhibitory potency and drug-like characteristics to be considered as a potential therapeutic agent. We have synthesized conjugates of the potent glucosidase inhibitor, 1-deoxynojirimycin, and glucose, with the aim of enhancing inhibitory activity against α-amylase. This synthetic conjugate showed increased inhibition of α-amylase compared to 1-deoxynojirimycin alone, suggesting that similar modifications of existing glucosidase inhibitors may yield more potent α-amylase inhibitors.     

Immobilization of Lecitase® ultra on recyclable polymer support: application in resolution of trans-methyl (4-methoxyphenyl)glycidate in organic solvents

Lecitase® Ultra was immobilized on epoxy-activated polymer (DILBEAD-VWR) functionalized with polyethyleneimine via adsorption and crosslinking with glutaraldehyde. The resolution of methyl trans-(±)-3-(4-methoxyphenyl) glycidate was carried out in xylene (e.e. >99%, conversion 50%). The enzyme is not inhibited by the 4-methoxy phenyl acetaldehyde produced during hydrolysis and the immobilized enzyme with 7% moisture content works efficiently in an organic phase. While the immobilized enzyme can be recycled several times, the polymer support can also be recycled after removing the immobilized enzyme by washing with 1?M HCl.     

Optimization of α-amylase production for the green synthesis of gold nanoparticles

Biocompatible gold nanoparticles have received considerable attention in recent years because of their promising applications in bioimaging, biosensors, biolabels, and biomedicine. The generation of gold nanoparticles using extra-cellular α-amylase for the reduction of AuCl₄ with the retention of enzymatic activity in the complex is being reported. The enhanced synthesis of particles has been brought about by optimizing the medium components for α-amylase. Response surface methodology and central composite rotary design (CCRD) were employed to optimize a fermentation medium for the production of α-amylase by Bacillus licheniformis at pH 8. The three variables involved in the study of α-amylase were fructose, peptone and soya meal. Only fructose had a significant effect on α-amylase production. The most optimum medium (medB) containing (%) fructose: 3, peptone: 1, soya meal: 2, resulted in a amylase activity of 201.381 U/ml which is same as that of the central level. The least optimum (medA) and most optimum (medB) media were compared for the synthesis of particles indicated by difference in color formation. Spectrophotometric analysis revealed that the particles exhibited a peak at 582 nm and the A₅₈₂ for the Med B was 8-fold greater than that of the Med A. The TEM analysis revealed that the particle size ranged from 10 to 50 nm.     

Determination of α-amylase activity using Fourier transform infrared spectroscopy

A new method for the determination of -amylase activity in aqueous solutions and human serum with FTIR-spectroscopy is proposed. The chemical reaction catalyzed by the enzyme under study can be followed directly when applying FTIR-spectroscopic detection also in the case, where no colored or electrochemical active species are generated or consumed during the course of the reaction of -amylase with simple starch. Therefore the determination of the -amylase activity could successfully be performed by recording two FTIR-spectra, one immediately after mixing the sample and a substrate (starch-) solution and the other after a 20 min reaction time. From these two FTIR-spectra a difference spectrum was calculated hereby eliminating an unspecific absorption of the matrix. The intensities of the resulting difference spectra corresponded to the extent of the reaction which took place during the investigated time interval and hence could be related to the activity of the enzyme in the sample. The developed method is linear from 80 to 1400 U/l (r.s.d.=5% for 700 U/l) in aqueous solutions and was also successfully applied to the determination of -amylase activity in human serum where a linear working range from 100 to 800 U/l (r.s.d.=11% for 150 U/l) was achieved.     

Time-dependent nanogel aggregation for naked-eye assays of α-amylase activity

This work designs an enzyme-stimulated nanogel aggregation system for the naked-eye assays of α-amylase activity. The visible aggregation of the starch-stabilized CdTe nanogels may be accelerated by α-amylase through its efficient cleavage of glycosidic bonds in the starch network, which has been verified by the evidences from transmission electron microscopy and dynamic light scattering spectra. The required aggregation time, as validated by both the theoretical deduction and the experimental results, is inversely proportional to the enzymatic activity. Therefore a facile method has been proposed for the detection of enzyme activity, with an excellent linear range and a low detection limit. This nanogel-based protocol can be successfully applied in the fast and accurate assays of α-amylase activity in saliva samples with a satisfactory correlation with the standard protocol, suggesting its promising applications in the biomedical and clinical fields, especially in point-of-care testing.     

Thermal behaviour of corn starch granules under action of fungal α-amylase

Corn starch, partially hydrolyzed by fungal α-amylase was investigated by using thermal analysis, microscopy and X-ray diffraction. After enzymatic treatment lower degradation onset temperatures were observed. DSC analysis showed almost similar range of gelatinization temperature, however, the enthalpies of gelatinization increased for the partially hydrolyzed starch granules. According to the X-ray diffraction analysis, stronger cereal pattern peaks were recognized after enzymatic digestion. The results suggested that the hydrolysis was more pronounced in the amorphous part of the starch granules.     

Kinetic study for adsorption of α-naphtholphthalein onto silica gel surface-imprinted polymer and non-imprinted polymer

In this study, the molecular imprinting polymer (MIP) was prepared using α-naphtholphthalein as a template, 2-(diethylamino)ethyl acrylate as a functional monomer and ethylene glycol dimethacrylate as a crosslinking agent by aid of free radical polymerization onto surfaces of vinyltrimethoxysilane modified silica gel. The MIP was extracted with acetonitrile for overnight to remove the template molecule from the MIP. Non-imprinted polymer (NIMP) was synthesized using the same materials except α-naphtholphthalein as template molecule. α-Naphtholphthalein adsorption on surfaces of the both polymer was studied at three different temperatures (19°C, 25°C and 35°C). It was observed that the adsorption capacity increased with increasing temperature and with time. The time required to reach the equilibrium for two polymers and all temperature was accepted to be nearly 6 h. The saturated adsorption amounts at the equilibrium were found as 120 mg/g, 123 mg/g and 127 mg/g at 19°C, 25°C and 35°C, respectively, for MIP, and 78 mg/g, 98 mg/g and 120 mg/g at 19°C, 25°C and 35°C, respectively, for NIMP. The mechanism of adsorption of α-naphtholphthalein onto MIP and NIMP is nearly appropriate to pseudo-first-order kinetic model with an activation energy of 11.63 kJ/mol for MIP, and 23.69 kJ/mol for NIMP. Thermodynamic parameters of activated complex in the adsorption process showed that the adsorption was carried out with an endothermic activation enthalpy, large negative entropy changes and the positive values of ΔG* that the adsorption processes is not favorable.     

Magnetic Bead Cellulose as a Suitable Support for Immobilization of α-Chymotrypsin

Magnetic bead cellulose was prepared by a suspension method from the mixture of viscose and magnetite using thermal sol?Cgel transition and regeneration of cellulose. The prepared magnetic particles after their activation with divinyl sulfone were shown to be suitable magnetic carrier for immobilization of ??-chymotrypsin and for its application in proteomic studies. The specific activity of the immobilized proteinase was high; its activity did not change in the course of storage. The following properties of the immobilized proteinase were compared with those of the soluble enzyme: pH and temperature dependence of the activity, self-cleavage activity, and possibility of repeated use. ??-Chymotrypsin immobilized to magnetic bead cellulose was used for the proteolytic digestion of porcine pepsin A and human gastric juice and a possibility of direct use of enzyme reaction products for matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis was shown.     

The effect of pectinase on the bubble fractionation of invertase from α-amylase

Fermentation broth normally contains many extracellular enzymes of industrial interest. To separate such enzymes on-line could be useful in reducing the cost of recovery as well as in keeping their yield at a maximum level by minimizing enzyme degradation from broth proteases (either the desired enzymes or the proteases could be removed selectively or both removed together and then separated). Several large-scale separation methods are candidates for such on-line recovery such as ultrafiltration, precipitation, and two-phase partitioning. Another promising technique for on-line recovery is adsorptive bubble fractionation, the subject of this study. Bubble fractionation, like ultrafiltration, does not require contaminating additives and can complement ultrafiltration by preconcentrating the enzymes using the gases normally present in a fermentation process. A mixture of enzymes in an aqueous bubble solution can, in principle, be separated by adjusting the pH of that solution to the isoelectric point (pI) of each enzyme as long as the enzymes have different pIs. The model system investigated here is comprised of three enzyme separations and the problem is posed as the effect of pectinase (a charged enzyme) on the bubble fractionation of invertase (a relatively hydrophilic enzyme) from α-amylase (a relatively hydrophobic enzyme). The primary environmental variable studied, therefore, is the pH in the batch bubble fractionation column. Air was used as the carrier gas. This prototype mixture exemplifies an aerobic fungal fermentation process for producing enzymes. The enzyme concentration here is measured as total protein concentration by the Coomassie Blue (Bradford) solution method (1), both as a function of time and column position for each batch run. Since, from a previous study (2), it was found that invertase and α-amylase in a two-enzyme system can be partially separated in favor of one vs the other at two different pHs (pH 5.0 and 9.0) with significant separation ratios, emphasis is placed on the effect of pectinase at these pHs. In this study, the addition of pectinase reduced the total separation ratio of the α-amylase-invertase mixture at both pHs.     

Welding dynamics in an atomistic model of an amorphous polymer blend with polymer–polymer interface

We consider an atomistic model of thermal welding at the polymer-polymer interface of a polyetherimide/polycarbonate blend, motivated by applications to 3D manufacturing in space. We follow diffusion of semiflexible chains at the interface and analyze strengthening of the samples as a function of the welding time t_w by simulating the strain–stress and shear viscosity curves. The time scales for initial wetting, and for fast and slow diffusion, are revealed. It is shown that each component of the polymer blend has its own characteristic time of slow diffusion at the interface. Analysis of strain–stress demonstrates saturation of the Young's modulus at t_w = 240 ns, while the tensile strength continues to increase. The shear viscosity is found to have a very weak dependence on the welding time for t_w > 60 ns. It is shown that both strain–stress and shear viscosity curves agree with experimental data.     

A new potentiometric sensor for the determination of α-amylase activity

A platinum redox sensor for the direct potentiometric determination of α-amylase concentration has been described. The sensor measured the amount of triiodide released from a starch-triiodide complex, which was correlated with the α-amylase activity after biocatalytic starch degradation. The composition and stability of the potassium triiodide solution was optimized. The starch-triiodide complex was characterized potentiometrically at variable starch and triiodide concentrations. The response mechanism of the platinum redox sensor towards α-amylase was proposed and the appropriate theoretical model was elaborated. The results obtained using the redox sensor exhibited satisfactory accuracy and precision and good agreement with a standard spectrophotometric method and high-sensitive fully automated descret analyser method. The sensor was tested on pure α-amylase (EC 3.2.1.1, Fluka, Switzerland), industrial granulated α-amylase Duramyl 120 T and an industrial cogranulate of protease and α-amylase Everlase/Duramyl 8.0 T/60 T. The detection limit was found to be 1.944 mU for α-amylase in the range of 0-0.54 U (0-15 μg), 0.030 mKNU for Duramyl 120 T in the range of 0-9.6 mKNU (0-80 μg) and 0.032 mKNU for Everlase/Duramyl 8.0 T/60 T in the range of 0-9.24 mKNU (0-140 μg).     

Preparation of conjugates of α-amylase with a protease inhibitor and their stability

A method for obtaining conjugates of -amylase with a trypsin inhibitor and separating them into fractions has been developed. Two fractions have been obtained—thermostable and thermolabile. The thermostable fraction retained about 80% of its amylase activity after incubation at 50°C for 2 h, with activation of the enzyme during the first 30 min. In the presence of trypsin the conjugated enzyme, retained 91% of its initial activity after incubation for 1 h, although the activity of the native enzyme fell to 35% under the same conditions.Mirzo Ulugbek [Ulugh-Beg] Tashkent State University. Translated from Khimiya Prirodnykh Soedimenii, No. 2, pp. 201–204, Marhc–April, 1998.     

Isolation of polyphenol compounds from olive waste and inhibition of their derivatives for α-glucosidase and α-amylase

Abstract

Olive waste was used as a sustainable resource because it contained a variety of valuable compounds. The polyphenols active fraction from enrichment by microporous resin and extraction with ethyl acetate were analysed by different chromatographic methods. A total of 14 polyphenolic compounds were isolated and identified by structure elucidation. Based on the above obtained compounds, tyrosol was selected as a characteristic polyphenol and participated in transesterification reaction to synthesise β-ketoester using Yb(OTf)₃. Then the Biginelli reaction with benzaldehyde, urea and ketoester (1:1.2:1.2) was performed at 90?°C for 3.0?h under the acidic condition. In addition, the β-ketoester prepared using tyrosol with benzyl had a greater inhibitory effect on α-glucosidase and α-amylase, and the inhibition of enzyme activity for 3, 4-dihydropyrimidinone derivatives prepared using abovementioned β-ketoester was improved significantly. Meanwhile, fluorine-containing dihydropyrimidinone derivatives were considerable inhibitors for both enzymes.     

Applications of self-consistent field theory in polymer systems

1Introduction Owing to the specificity of the long chain,polymers present complexity and versatility.These molecules in the system can be various in their topological struc-tures,such as linear,star,comb or circle structures;meanwhile they can be polymeri…