α‐Amylase Modulation: Discovery of Inhibitors Using a Multi‐Pharmacophore Approach for Virtual Screening

Better control of postprandial hyperglycemia can be achieved by delaying the absorption of glucose resulting from carbohydrate digestion. Because α‐amylase initiates the hydrolysis of polysaccharides, the design of α‐amylase inhibitors can lead to the development of new treatments for metabolic disorders such as type II diabetes and obesity. In this study, a rational computer‐aided approach was developed to identify novel α‐amylase inhibitors. Three‐dimensional pharmacophores were developed based on the binding mode analysis of six different families of compounds that bind to this enzyme. In a stepwise virtual screening workflow, seven molecules were selected from a library of 1.4 million. Five out of seven biologically tested compounds showed α‐amylase inhibition, and the two most potent compounds inhibited α‐amylase with IC₅₀ values of 17 and 27 μm . The scaffold benzylideneacetohydrazide was shared by four of the discovered inhibitors, emerging as a novel drug‐like non‐carbohydrate fragment and constituting a promising lead scaffold for α‐amylase inhibition.     

Bioartificial dextran and poly[ethylene‐co‐(vinyl alcohol)] hybrid sponges as support for proteins

A biological–synthetic hybrid material was prepared as a support for proteinic molecules. To control the conformational stability and ‘vitality’ of the entrapped compound, an enzyme, which catalyses a chemical reaction, was chosen. The physical immobilisation of α‐amylase on a novel biological–synthetic hybrid material was realised using a synthetic (poly[ethylene‐co‐(vinyl alcohol)]) and a biological (dextran) component. The supports were obtained in the form of porous sponges using the phase inversion process. The kinetic parameters of enzymatic hydrolysis of soluble starch were obtained by determining the maltose (reaction product) concentration in the test solution by a spectrophotometric method. A high amount of enzyme (10 and 15 mg per g of polymer matrix) was successfully entrapped in the polymer support and a rather good enzyme activity of entrapped α‐amylase and a constant activity for repeated use, compared with native α‐amylase, were observed. The results obtained clearly indicated that the bioartificial polymer sponges, showing an immobilisation of α‐amylase with maintenance of the catalytic function of the enzyme, can be used as suitable supports for proteins. Copyright © 2005 Society of Chemical Industry     

Stabilization of α‐amylase by chemical modification with carboxymethylcellulose

Carboxymethylcellulose activated by periodate oxidation was covalently linked to porcine pancreatic α‐amylase (EC 3.2.1.1). The specific activity of the conjugate prepared was 54% of the native enzyme. The carbohydrate content was estimated to be 62% by weight as a result of the modification of 67% of the amino groups from the protein. In comparison with the native enzyme, the thermostability and pH stability were improved for α‐amylase by this modification. The conjugate was also more resistant to the action of denaturant agents such as urea and sodium dodecylsulfate. We conclude that modification of enzymes by the anionic polysaccharide carboxymethylcellulose might be a useful method for improving enzyme stability under various denaturing conditions. © 1999 Society of Chemical Industry     

Immobilization of malto‐oligosaccharide forming α‐amylase from Bacillus subtilis KCC103: properties and application in starch hydrolysis

BACKGROUND: A malto‐oligosaccharide forming α‐amylase from Bacillus subtilis KCC103 immobilized in calcium alginate beads was repeatedly used in batch processes of starch hydrolysis. The degree of starch degradation and operational stability of the immobilized system were optimized by varying the physical characteristics and composition of the beads. The products formed from hydrolysis of various starches by α‐amylase immobilized in different supports were analyzed. RESULTS: Immobilized beads prepared from 3% (w/v) alginate and 4% (w/v) CaCl₂ were suitable for up to 10 repeated uses, losing only 25% of their efficiency. On addition of 1% silica gel to alginate prior to gelation, the operational stability of the immobilized enzyme was enhanced to 20 cycles of operation, retaining > 90% of the initial efficiency. Distribution of malto‐oligosaccharides in the starch hydrolyzate depended on the type of starch, reaction time and mode of immobilization. Soluble starch and potato starch formed a wide range of malto‐oligosaccharides (G1–G5). Starches from wheat, rice and corn formed a narrow range of smaller oligosaccharides (G1–G3) as the major products. CONCLUSION: The immobilized beads of α‐amylase from KCC103 prepared from alginate plus silica gel showed high efficiency and operational stability for hydrolysis of starch. This immobilized system is useful for production of malto‐oligosaccharides applied in the food and pharmaceutical industries. Since this KCC103 amylase can be produced at low cost utilizing agro‐residues in a short time and immobilized enzyme can be recycled, the overall cost of malto‐oligosaccharide production would be economical for industrial application. Copyright © 2008 Society of Chemical Industry     

Immobilization of α‐amylase in polyelectrolyte complexes

Herein, we report the formation of α‐amylase containing polyelectrolyte complexes (PECs). The method for the encapsulation of α‐amylase is based on interactions between two oppositely charged polyelectrolytes, poly(acrylic acid) (PAA) and polyethylenimine (PEI). We could show that electrostatic interactions ensure the incorporation of the enzyme into the formed polyelectrolyte complexes. The encapsulation has no negative effect on enzyme activity and protects against denaturation of the enzyme initiated by low pH values. The resulting PECs are 150–250 nm in size with narrow size distribution, appear in a spherical shape and are colloidally stable. The complexation of both polyelectrolytes and the immobilization of α‐amylase are investigated using fractionating techniques mainly the analytical ultracentrifugation and asymmetrical‐flow field‐flow fractionation. The formation of PECs represents a simple method for the encapsulation of α‐amylase without the use of organic solvents and requires no additional purifications steps. This one‐step approach, yielding high encapsulation efficiencies, shows the potential as a drug delivery system for sensitive hydrophilic actives in future. α‐amylase is immobilized in polyelectrolyte complexes made of polyethylenimine and poly(acrylic acid). Optimized encapsulation conditions and the resulting polyelectrolyte complexes are investigated via determination of IEP, α‐amylase activity assays, nanoDSC measurements, zeta potential values, dynamic light scattering, microscopy, and fractionating techniques. The encapsulated enzyme is protected against denaturation initiated by low pH values. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 45036.     

Production of α‐amylase under solid‐state fermentation utilizing coffee waste

Coffee industry substrates such as coffee pulp, coffee cherry husk, silver skin, spent coffee and mixtures of these coffee wastes (MC) were evaluated for their efficacy as sole carbon source for the synthesis of α‐amylase in solid‐state fermentation (SSF) using a fungal strain of Neurospora crassa CFR 308. For SSF with coffee pulp and with MC, α‐amylase activity of 3908 U g^?1 ds (units per gram of dry substrate) and 3870 U g^?1 ds, respectively, was observed. Parameters such as moisture (60%), pH (4.6), temperature (28 °C), particle size (1.0 mm), inoculum size (10⁷ spores g^?1 ds), and fermentation time (5 days) were optimized for enzyme synthesis, wherein 4981 and 4324 U g^?1 U g^?1 ds of α‐amylase activity was obtained in SSF with coffee pulp and MC, respectively. The enzyme production was further improved when the substrates were subjected to pre‐treatment by steaming. Accordingly, maximum α‐amylase activity of 7084 U g^?1 ds and 6342 U g^?1 ds was obtained with steam‐pretreated coffee pulp and MC, respectively, demonstrating them to be excellent sole carbon sources for synthesis of α‐amylase production. Copyright © 2009 Society of Chemical Industry     

Kinetics of the thermal inactivation of Bacillus subtilis α‐amylase and its application on the desizing of cotton fabrics

The thermal inactivation of Bacillus subtilis α‐amylase was studied in the presence and in the absence of Ca²⁺ at various temperatures. Inactivation rate constant (k), half‐life time (t_1/2), and activation energy (E_a) were determined to characterize the inactivation of the enzyme. Results obtained showed that the thermal inactivation of Bacillus subtilis α‐amylase followed a first‐order kinetics. The addition of Ca²⁺ had a good thermostabilizing effect on the enzyme. The stabilizing effect of Ca²⁺ is reflected by the increased values of the activation energy, which is about two times higher in the presence than that in the absence of 20 mM Ca²⁺, and the decreased values of the inactivation rate constants. The desizing of the cotton fabrics was performed through steaming at 100°C with Bacillus subtilis α‐amylase. The desizing efficiency seemed to be dependent on the concentration and pH value of the enzyme solution. It was found that through the steaming process with α‐amylase, the desizing ratio of the cotton fabrics could be beyond 98% and little damage happened to the fibers of the fabrics. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Optimization of medium and process parameters for a constitutive α‐amylase production from a catabolite derepressed Bacillus subtilis KCC103

BACKGROUND: In Bacillus subtilis KCC103 α‐amylase is hyper‐produced and not catabolite repressed by glucose. Various sugars, raw starches and nitrogen sources were tested for their repression effect on α‐amylase synthesis. Enhancement of α‐amylase production by supplementing micronutrients and surfactants was studied. Using optimized medium, process parameters were optimized for improved α‐amylase production. RESULTS: α‐Amylase was produced from KCC103 utilizing simple sugars indicating the absence of catabolite repression. Raw potato and yeast extract were best carbon and nitrogen sources for α‐amylase production. α‐Amylase synthesis was enhanced by micronutrients cysteine, thiamine, Mg²⁺ and SDS. Maximum α‐amylase (394 IU mL^?1) was produced in the optimized medium consisting of (in g L^?1) raw potato (30.0), yeast extract (20.0), cysteine (0.3), thiamine (0.2), SDS (0.2) and MgSO₄ (0.5 mmol L^?1) at 36–48 h under optimal conditions (pH 7.0, 37 °C, 200 rpm). The α‐amylase production was further enhanced to 537.7 IU mL^?1 with shorter time (15–18 h) in a bioreactor with optimized agitation rate of 700 rpm at 30% dissolved oxygen. CONCLUSION: Since there was no carbon catabolite repression of α‐amylase synthesis, sugar mixture from various agro‐residues hydrolysates could be utilized for α‐amylase production. The study showed the feasibility of utilization of raw potato for α‐amylase production from the KCC103, which would lead to a significant reduction in process cost. Copyright © 2008 Society of Chemical Industry     

Optimization of growth medium for the production of α‐amylase from Bacillus amyloliquefaciens using response surface methodology

The optimization of nutrient levels for the production of α‐amylase by Bacillus amyloliquefaciens was carried out using response surface methodology (RSM) based on the 2³ factorial central composite design (CCD). This procedure limited the number of actual experiments performed while allowing for possible interactions between three components. RSM was adopted to derive a statistical model for the effect of starch, peptone and yeast extract (YE) on α‐amylase production. The P‐value of the coefficient for linear effects of starch and YE concentration was <0.0001, suggesting that this was the principal experimental variable, having the greatest effect on the production of α‐amylase. The optimal combinations of media constituents for maximum α‐amylase production were determined as 12.61 g L^?1 starch, 2.83 g L^?1 peptone and 1.25 g L^?1 YE. The optimization of the medium resulted not only in a 34% higher enzyme activity than unoptimized medium but also in a reduced amount of the required medium constituents. Copyright © 2006 Society of Chemical Industry     

Characterization and application of grafted polypropylene and polystyrene treated with epichlorohydrin coupled with cellulose or starch for immobilization processes

The direct‐irradiation‐induced grafting of acrylamide onto polypropylene films and polystyrene films with crosslinking epichlorohydrin (Epi) as a spacer was studied. The reaction of polyamide–Epi with cellulose and starch was carried out. Fourier transform infrared spectroscopy, scanning electron microscopy, and thermogravimetric analysis were carried out before and after the enzyme was immobilized to confirm the characterization of the polymer and the α‐amylase enzyme‐immobilized one. The physicochemical parameters of the immobilized enzymes were measured, and the kinetics of the coenzyme–polymer‐catalyzed reactions and stability were investigated. A direct comparison was made between the activities of the free and immobilized preparations. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Blends of poly(vinyl chloride) with α‐methylstyrene‐acrylonitrile‐butadiene‐styrene copolymer: Thermal properties,mechanical properties,and morphology

Binary blends of poly(vinyl chloride) (PVC) with α‐methylstyrene‐acrylonitrile‐butadiene‐styrene copolymer (AMS‐ABS) were prepared via melt blending. A single glass transition temperature (T_g) was observed by differential scanning calorimetry, thus indicating that PVC is miscible with the α‐methylstyrene‐acrylonitrile‐styrene in AMS‐ABS. The results from attenuated total reflection Fourier transform infrared spectra indicated that specific strong interactions were not available in the blends. With increasing amounts of AMS‐ABS, both heat distortion temperature and thermal stability were increased considerably. With regard to mechanical properties, flexural and tensile properties decreased with increasing AMS‐ABS content. A synergism was observed in impact strength. The morphology of both impact‐fractured and tensile‐fractured surfaces, observed by scanning electron microscopy, correlated well with the mechanical properties. It is suggested that there was a transition of fracture mechanisms with the changing composition of the binary blends—from shear yielding for blends rich in PVC to cavitation for blends rich in AMS‐ABS. J. VINYL ADDIT. TECHNOL., 19:1–10, 2013. © 2013 Society of Plastics Engineers     

Optimum Production and Characterization of Thermostable Amylolytic Enzymes from B. stearothermophilus GRE1

Optimum production of extracellular, thermostable amylolytic enzymes (α and β‐amylase) by a newly isolated bacterium, Bacillus stearothermophilus, was investigated in a batch bioreactor. Starch and lactose at 1.0% and 3.0% (w/v) respectively were found to be optimum for maximum enzyme production. Optimization of cultural conditions (pH 7.0 and temperature 45°C) resulted in high bacterial specific growth rate (0.64 h^?1), yielding 2.20 gL^?1 biomass, 11.43 UmL^?1 α‐amylase and 10.04 UmL^?1 of β‐amylase. Hydrolysis of native starches from wheat, cassava, corn and potato at 60°C using the crude enzyme showed 60‐80% saccharification with potato starch showing the least and wheat starch showing the greatest hydrolysis. The K_m and V_max values of the crude α‐amylase for starch were 4.78 mg starch/mL and 6.67 mg/mL.min respectively.     

Morphology,crystallization and melting properties of isotactic polypropylene blended with lignin

The influence of lignin (L) on the thermal properties and kinetics of crystallization of isotactic polypropylene (PP) is reported in this article. PP blends containing 5 and 15 wt % of L were prepared by mixing the components in a screw mixer. An increase of the thermal degradation temperature of the blends was observed as a function of L content. The crystallization and thermal behavior of the pure PP and of the PP/L blends were analyzed by differential scanning calorimetry (DSC). Isothermal crystallization kinetics were described by means of the Avrami equation, which suggests a three‐dimensional growth of crystalline units, developed by heterogeneous nucleation. The isothermal growth rate of PP spherulites was studied using a polarizing optical microscope. The enhancement of PP crystallization rate for the PP/L blends was observed and ascribed to the nucleating action of lignin particles. Non‐isothermal crystallization kinetics were applied, according to the results elaborated by Ziabicki and the method modified by Jeziorny. The kinetic crystallizability of the PP is not influenced by the L present in the blend. In the presence of L, PP can simultaneously crystallize in both the α and β crystalline forms, and the ratio between the α and β forms was determined by X‐ray diffraction analysis. Two melting peaks relative to the two crystalline form of PP were observed for the PP/L blends, for all isothermal crystallization temperatures investigated by means of DSC. The equilibrium melting temperature for α‐form of pure PP was obtained. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 91: 1435–1442, 2004     

Thermal characterization and morphological study of polyphenylene sulfide–polycarbonate blends

The thermal behavior and phase morphology of binary blends of poly(phenylene sulfide) (PPS) with polycarbonate (PC) have been investigated. Differential scanning calorimetry and dynamic mechanical thermal analysis indicate the blends are immiscible, but the glass transition temperature of PC in the blends was found to be decreased due to the degradation of the PC. The PC degradation was investigated by measuring the molecular weight of PC extracted from the blends. Rheological properties of the blends were also studied using a rheodynamic spectrometer. An inversion of the phase morphology was observed from the scanning electron microscopy and dynamic mechanical thermal analysis. The increase of crystallinity of the PPS in the blends was found from a DSC study.     

Effect of surfactants on α‐amylase production in a solid substrate fermentation process

Solid substrate fermentation (SSF) is a process where the substrate is a moist solid, which is insoluble in water but not suspended in water. In this study SSF of Bacillus subtilis (ATCC 21556) was used to produce an enzyme of commercial importance, α‐amylase, using as a substrate potato peel. To enhance the production of this enzyme, two nonionic synthetic surfactants were used, Tween 80 and Tween 20, one anionic surfactant, SDS at concentrations of 0.05% and 0.10% (v/w) and a biosurfactant produced by Bacillus subtilis (ATCC 21332), known as surfactin, at concentrations of 0.003%, 0.007%, 0.013% and 0.03% (w/w). The results have shown that surfactants significantly increase the production of α‐amylase. Tween 80 at 0.10% and surfactin at 0.013% provided the highest enzyme activity when compared with the control. © 1999 Society of Chemical Industry     

Optimization of agro‐residual medium for α‐amylase production from a hyper‐producing Bacillus subtilis KCC103 in submerged fermentation

BACKGROUND: Although submerged fermentation (SmF) is the conventional method in industry, use of low‐cost agro‐residues for α‐amylase production in SmF has not been well established. Here we optimized agro‐residue‐based medium and culture conditions for α‐amylase production in SmF using a hyper‐producing Bacillus subtilis KCC103. RESULTS: B. subtilis KCC103 produced α‐amylase in SmF by utilizing agro‐residues. Wheat bran (WB) and sunflower oil cake (SFOC) were selected as the best substrates using shake flasks. Medium containing WB (carbohydrate rich) and SFOC (rich in protein and free amino acids) at 1:1 (w/w) ratio produced high levels (90 IU mL⁻¹) of α‐amylase at 30–36 h in a shake flask. The α‐amylase yield was 14‐fold enhanced (1258 IU mL⁻¹) by optimizing process parameters and medium composition following response surface methodology in a bioreactor. The optimal conditions were: WB 1.27%, SFOC 1.42%, pH 7, 37 °C and 10–12 h. Both in shake flask and bioreactor α‐amylase synthesis was not repressed by the release of simple sugars into the medium. CONCLUSION: KCC103 with catabolite derepression and hyperproducing ability is useful for economic α‐amylase production using low‐cost agro‐residual substrates in conventional SmF. Since the production time (10–12 h) is much shorter than other strains this would improve productivity and further reduce the cost of α‐amylase production. Copyright © 2008 Society of Chemical Industry     

Hydrolytic degradation of poly(l‐lactic acid)/poly(methyl methacrylate) blends

The hydrolytic degradation of poly(l ‐lactic acid)/poly(methyl methacrylate) (PLLA/PMMA) blends was carried out by the immersion of thin films in buffer solutions (pH = 7.24) in a shaking water bath at 60 °C for 38 days. The PLA/PMMA blends (0/100; 30/70; 50/50; 70/30; 100/0) were obtained by melt blending using a Brabender internal mixer and shaped into thin films of about 150 µm in thickness. Considering that PMMA does not undergo hydrolytic degradation, that of PLLA was followed via evolution of PLA molecular weight (recorded by size exclusion chromatography), thermal parameters (differential scanning calorimetry (DSC)) and morphology of the films (scanning transmission electron microscopy). The results reveal a completely different degradation pathway of the blends depending on the polymethacrylate/polyester weight ratio. DSC data suggest that, during hydrolysis at higher PMMA content, the polyester amorphous chains, more sensitive to water, are degraded before being able to crystallize, while at higher PLLA content, the crystallization is favoured leading to a sample more resistant to hydrolysis. In other words, and quite unexpectedly, increasing the content of water‐sensitive PLLA in the PLLA/PMMA blends does not mean de facto faster hydrolytic degradation of the resulting materials. © 2018 Society of Chemical Industry     

Miscibility and rheological properties of poly(vinyl chloride)/styrene–acrylonitrile blends prepared by melt extrusion

Styrene–acrylonitrile (SAN) with acrylonitrile (AN) concentrations of 11.6–26 wt % and α‐methylstyrene acrylonitrile (αMSAN) with a wide range of AN concentrations are miscible with poly(vinyl chloride) (PVC) through solution blending. Here we examine the rheological properties and miscibility of PVC/SAN and PVC/αMSAN blends prepared by melt extrusion for commercial applications. We have investigated the rheological properties of the blends with a rheometer and a melt indexer. The PVC/SAN and PVC/αMSAN blends have a low melting torque, a long degradation time, and a high melt index, and this means that they have better processability than pure PVC. The miscibility of the blends has been characterized with differential scanning calorimetry, dynamic mechanical thermal analysis, and advanced rheometrics expansion system analysis. The miscibility of the blends has also been characterized with scanning electron microscopy. The SAN series with AN concentrations of 24–31 wt % is immiscible with PVC by melt extrusion, whereas αMSAN with 31 wt % AN is miscible with PVC, even when they are blended by melt extrusion, because of the strong interaction between PVC and αMSAN. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007     

Preparation and characterization of biodegradable polymer blends from poly(3‐hydroxybutyrate)/poly(vinyl acetate)‐modified corn starch

Biodegradable polymer blends prepared by blending poly(3‐hydroxybutyrate) (PHB) and corn starch do not form intact films due to their incompatibility and brittle behavior. For improving their compatibility and flexibility, poly(vinyl acetate) (PVAc) was grafted from the corn starch to prepare the PVAc‐modified corn starch (CSV). The resulting CSV consisted of 47.2 wt% starch‐g‐PVAc copolymer and 52.8 wt% PVAc homopolymer and its structure was verified by FT‐IR analysis. In comparison with 35°C of the neat PVAc, the glass transition temperature (T_g) of the grafted PVAc chains on starch‐g‐PVAc was higher at 44°C because of the hindered molecular mobility imposed from starch on the grafted PVAc. After blending PHB with the CSV, structure and thermal properties of the blends were investigated. Only a single T_g was found for all the PHB/CSV blends and increased with increasing the CSV content. The T_g‐composition dependence of the PHB/CSV blends was well‐fitted with the Gordon‐Taylor equation, indicating that the CSV was compatible with the PHB. In addition, the presence of the CSV could raise the thermal stability of the PHB component. It was also found that the presence of the PHB and PVAc components would not hinder the enzymatic degradation of the corn starch by α‐amylase. POLYM. ENG. SCI., 55:1321–1329, 2015. © 2015 Society of Plastics Engineers     

Evaluation of the effect of ammonium carbamate on the stability of proteins

BACKGROUND: The use of the volatile salt ammonium carbamate in protein downstream processing has recently been proposed. The main advantage of using volatile salts is that they can be removed from precipitates and liquid effluents through pressure reduction or temperature increase. Although previous studies showed that ammonium carbamate is efficient as a precipitant agent, there was evidence of denaturation in some enzymes. In this work, the effect of ammonium carbamate on the stability of five enzymes was evaluated. RESULTS: Activity assays showed that α‐amylase (1,4‐α–D‐glucan glucanohydrolase, EC 3.2.1.1), lysozyme (1,4‐β‐N‐acetylmuramoylhydrolase, EC 3.2.1.17) and lipase (triacyl glycerol acyl hydrolase, EC 3.1.1.3) did not undergo activity loss in ammonium carbamate solutions with concentrations from 1.0 to 5.0 mol kg^?1, whereas cellulase complex (1,4‐(1,3:14)‐β‐D‐glucan 4‐glucano‐hydrolase, EC 3.2.1.4) and peroxidase (hydrogen peroxide oxidoreductase, EC 1.11.1.7) showed an average activity loss of 55% and 44%, respectively. Precipitation assays did not show enzyme denaturation or phase separation for α‐amylase and lipase, while celullase and peroxidase precipitated with some activity reduction. Analysis of similar experiments with ammonium and sodium sulfate did not affect the activity of enzymes. CONCLUSION: Celullase and peroxidase were denatured by ammonium carbamate. While more systematic studies are not available, care must be taken in designing a protein precipitation with this salt. The results suggest that the generally accepted idea that salts that denature proteins tend to solubilize them does not hold for ammonium carbamate. Copyright © 2010 Society of Chemical Industry