Identification and Functional Characterization of an α-Amylase with Broad Temperature and pH Stability from Paenibacillus sp.

Amylases are important industrial enzymes that have been applied widely in the food, detergent, and pulp industries and fermentation processes. In the present study, a gene encoding an alpha-amylase from the genomic DNA library of Paenibacillus sp. was identified and characterized. The amylase gene designated amy1 was shown to consist of 1,980 bp and shared sequence identity towards α-amylase genes from other Bacillus sp. The deduced amino acid sequence for Amy1 indicated 80 % sequence identity with other Bacillus strains. Heterologous expression of recombinant Amy1 in Escherichia coli BL21(DE3) facilitated the recovery of this protein in soluble form. Enzyme kinetic data revealed Amy1 to have a K _m of 23.83 mg/mL and K _cat of 48.74 min^?1 and K _cat /K _m of 2 min^?1 mg^?1 mL^?1 for starch. The activity of this protein was found to be enhanced by Mn²⁺, and furthermore, Amy1 remained active at a broad pH range (4–10) and temperature (30–90 °C). The ability of Amy1 to act on food waste under broad temperature and pH conditions, together with its ability to produce simple sugars, shows many advantages for further application in the food industry.     

Purification, Sequencing, and Biochemical Characterization of a Novel Calcium-Independent α-Amylase AmyTVE from Thermoactinomyces vulgaris

α-Amylase from Thermoactinomyces vulgaris was highly purified 48.9-fold by ammonium sulfate precipitation, gel filtration on Sephadex G-50 column, and ion exchange chromatography column of DEAE-cellulose. The molecular weight of the enzyme was estimated to be 135 and 145 kDa by SDS–PAGE. Its high molecular weight is due to high glycosylation. The purified amylase exhibited maximal activity at pH 6.0 to 7.0 and was stable in the range of pH 4.0 to 9.0. The optimum temperature for its activity was 50 °C. The enzyme half-life time was 120 min at 50 °C, suggesting intermediate temperature stable α-amylase. The enzyme was sensitive to different metal ions, including NaCl, CoCl₂, and CaCl₂, and to different concentrations of EDTA. The enzyme activity was inhibited in the presence of 1 mM CaCl₂, suggesting that it is a calcium-independent α-amylase. The TLC showed that the amylase hydrolyzed starch to produce large maltooligosaccharides as the main products. A 1.1-kb DNA fragment of the putative α-amylase gene (amy TVE) from T. vulgaris was amplified by using two specific newly designed primers. Sequencing analysis showed 56.2 % similarity to other Thermoactinomyces α-amylases with two conserved active sites confirming its function.     

Isolation, Purification, and Properties of α-Amylase from Bacillus subtilis-7A

Cultivation ofBacillus subtilis-7A on waste from alcohol production yielded an active extracellular enzyme -amylase with MW 75 kDa. The enzyme was isolated from the culture medium by 60% saturated ammonium sulfate and purified until homogeneous by gel filtration on Sephadex G-100 and ion-exchange chromatography on DEAE-cellulose. The optimum temperatures for the complex and purified enzyme are 30 and 50°C, respectively. The optimum activity for both preparations occurred at pH 6.5. The substrate specificity of the isolated preparations was studied.     

Recombinant Expression and Characterization of an Organic-Solvent-Tolerant α-Amylase from Exiguobacterium sp. DAU5

The enzyme from halophilic microorganisms often has unique properties such as organic-solvent-tolerance. In this study, a novel organic-solvent-tolerant α-amylase gene was cloned from the mild halophile Exiguobacterium sp. DAU5. The open reading frame (ORF) of the enzyme consisted of 1,545 bp and encoded 514 amino acids, the primary sequence revealed that it belongs to the glycoside hydrolase (GH) family 13. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed an AmyH monomer of 57 kDa. The enzyme exhibited maximal activity at 40 °C in pH?8.5 glycine–NaOH buffer, and the activity was strongly inhibited by Zn²⁺, Cu²⁺, and Fe²⁺. The α-amylase AmyH exhibited high hydrolysis activity toward soluble starch, and the major hydrolysis products were maltose, maltotriose, and maltopentaose; the AmyH could not efficiently hydrolyze oligosaccharides smaller than maltoheptaose, nor could it act on the β-1,4 or α-1,6 glucosidic bonds in xylan or pullulan, respectively. In addition, the α-amylase exhibited better tolerance to organic solvents, as it was stable in the presence of dimethylsulfoxide (DMSO), methanol, ethanol, and acetone. Base on all of these results, the enzyme could be useful for practical application in the bakery industry and in biotechnological processes that occur in the presence of organic solvents.     

Striking Features of DPH Electronic Spectra as a Function of Multicomponent Solvent Nature

1,6-Diphenyl-1,3,5-hexatriene is one of the most used fluorescent labels in cell membrane fluidity studies. However, its solubility in aqueous biological media is low and an initial solubilizing step is necessary. In this study, the authors present spectroscopic and computational results in order to explain the striking displacement observed in electronic spectra (absorption and fluorescence) in different solvents. The experimental results were correlated with those obtained by molecular dynamics simulations of multicomponent solutions, which revealed non-homogenous microscopic regions formation in the ternary aqueous DPH solutions.     

Structure of an Extracellular Polysaccharide from a Strain of Lactic Acid Bacteria

A new extracellular polysaccharide (EPS-I) isolated and purified from Z222, a strain of Lactic acid bacteria has been investigated. Sugar composition analysis, methylation analysis and ^1H NMR and ^13C NMR spectroscopy reveal that the EPS-I is composed of a pentasaccharide repeating unit. The sequence of sugar residue was determined by using two-dlmensional NMR spectroscopy, including heteronudear multiple-bond correlation(HMBC) and nuclear overhauser effect spectroscopy (NOESY).     

Synthesis of Nitriles and Symmetrical Organic Sulphides from Biprotic Carbothioamides and an α-Halogenated Ketone,Ester or Nitrile

Thioacetamide has been used as an elegant sulphur transfer reagent for C-S-C coupling of reactive halides.^1–4 Sulphur extrusion of biprotic carbothioamides to form corresponding nitriles has been reported with the help of (i) dichlorocarbene;⁵ (ii) triphenylphosphine, carbon tetrachloride, triethylamine;⁶ (iii) diethyl azocarboxylate, triphenylphosphine;⁷ and (iv) soft metal ions.⁸ Here, we report that under non-hydrolytic conditions viz. DMF/OEt, EtOH/OEt and DMF/Et₃N, biprotic carbothioamides (1, R=CH₃, C₆H₅, CH₂C₆H₅) react with bromoacetophenone (2, Z=COC₆H₅), ethyl bromoacetate (2, Z=COOEt).     

Estimates of Internal Pressure and Molar Refraction of Imidazolium Based Ionic Liquids as a Function of Temperature

Estimates of the internal pressure (∂ U/∂ V) _T of the ionic liquids (ILs) 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF₄], 1-butyl-3-methylimidazolium hexafluorophosphate [BMIM][PF₆], and 1-methyl-3-octylimidazolium tetrafluoroborate [OMIM][BF₄] were made from experimentally determined densities and speeds of sound in the temperature range 283.15 to 343.15 K. Values (∂ U/∂ V) _T for all the ILs studied are higher than those of water and molecular organic liquids. We also measured the refractive indices n _D in the temperature range 288.15 to 343.15 K and estimated the molar refraction R _M. Refractive indices of ILs were also higher than those of normal organic liquids but were comparable to those of long hydrocarbon chain organic solvents.     

Instrumented Macroindentation Techniques for Polymers and Composites – Mechanical Properties,Fracture Toughness and Time-Dependent Behaviour as a Function of the Temperature

An innovative cooling and heating device has been successfully applied to an instrumented macrohardness testing machine in close collaboration with the company Zwick/Roell. The prototype allows the local time-dependent analysis of mechanical properties such as Martens hardness and indentation modulus, as well as fracture toughness and creep and relaxation behaviour at temperatures ranging from −100 °C to +100 °C. On the basis of load–indentation depth, load–time or indentation depth–time diagrams, the indentation behaviour as a function of test speed and/or temperature (which has rarely been done for polymers in the macro-range of loading) depending on matrix and materials composition (amorphous/semicrystalline thermoplastics, epoxy resins, micro- and nanocomposites) has been analysed. Martens-hardness, indentation modulus on the one hand and creep compliance and relaxation modulus on the other have been found to be strongly temperature dependent. Adequate methods of indentation fracture mechanics have been enhanced for polymers and applied to determine the fracture toughness of very different polymer-based materials.     

Purification of α-Amylase from Bacillus sp. GHA1 and its partial characterization

Bacillus sp. GHA1 was isolated from water samples and screened for the production of α-amylase. Maximum production of amylase by this strain occurs at 42 °C, pH 6.5 and 72 h after cultivation in production medium. The enzyme was purified through successive applications of ammonium sulfate precipitation, ion exchange and hydrophobic interaction chromatography, resulting in a single band with an apparent molecular weight of 66 kDa, as judged by SDS-PAGE. Calcium analysis of the purified enzyme revealed that it contained three metal ions per molecule. The new extracellular α-amylase is active in a wide range of pH with its maximum activity at pH values 5.5–8.0. The optimum temperature for enzyme activity is 57 °C and the presence of calcium has relatively low influence on its activity and thermostability. The Bacillus sp. GHA1 α-amylase with these properties may be suitable for use in detergent and food industries.     

Synthesis and Inhibitory Effect of Some Indole-Pyrimidine Based Hybrid Heterocycles on α-Glucosidase and α-Amylase as Potential Hypoglycemic Agents

The Michael addition reaction of barbituric acid with chalcones incorporating the indole scaffold was achieved by using a highly efficient bimetallic Iron–palladium catalyst in the presence of acetylacetone (acac). This catalytic approach produced the desired products in a simple operation and low catalyst loading with acceptable yield of the new hybrids. All tested compounds were subjected for biological activity on α-glucosidase and α-amylase. The results revealed that all synthesized compounds exhibited very good activity against both enzymes when compared to positive control (acarbose). Moreover, compound 5o showed the best activity whereas its IC₅₀ (μM) are 13.02+0.01 and 21.71+0.82 for α-glucosidase and α-amylase respectively. Both compounds 5o and 5l exhibited high similarity in binding mode and pose with amylase protein (4UAC). The obtained data may be used for developing potential hypoglycemic agents.     

Thiazolium Salt Immobilized on Ionic Liquid: An Efficient Catalyst and Solvent for Preparation of α-Hydroxyketones

Aldehydes were efficiently converted to acyloins and benzoins using a new ionic liquid, 3-[2-(1-butyl-1H-imidazol-1,3-ium-3-yl)ethyl]-4,5-dimethyl-1,3-thiazol-3-ium dibromide 1 . This ionic liquid is introduced as a catalyst and a solvent. Acyloins and benzoins were easily isolated from the reaction mixture via simple extraction, and the ionic liquid could be recycled for further use. Also, α-hydroxy ketones with an aromatic and aliphatic substituent were prepared starting from aromatic and aliphatic aldehydes in the presence of ionic liquid 1 .     

Evidence for a Molten Globule State in Cicer α-Galactosidase Induced by pH, Temperature, and Guanidine Hydrochloride

Physiologically as well as industrially, α-galactosidases are very important enzymes, but very little is known about the stability and folding aspect of enzyme. In the present study, we have investigated the temperature, pH, and guanidine hydrochloride (GuHCl) induced unfolding of Cicer α-galactosidase using circular dichroism and fluorescence spectroscopy. Strong negative ellipticities at 208, 215, and 222 nm indicate the presence of both α and β structures in Cicer α-galactosidase and showed that its secondary structure belongs to α?+?β class of proteins with 31 % α-helicity. For Cicer α-galactosidase the emission maximum was found to be 345 nm which suggests that tryptophan residues are less exposed to solvent. However, at pH?2.0, protein showed blue-shift. This state of protein lacked activity but it retained significant secondary structure. Enhanced binding of ANS at pH?2.0 indicated significant unfolding and exposure of hydrophobic regions. The unfolded state of Cicer α-galactosidase showed a red-shift of 15 nm with a concomitant decrease in the fluorescence intensity. The enzyme maintained its native structure and full activity up to 40 °C; however, above this temperature, denaturation was observed.     

Synthetic, structural, and biosynthetic studies of an unusual phospho-glycopeptide derived from α-dystroglycan

Aberrant glycosylation of α-dystroglycan (α-DG) results in loss of interactions with the extracellular matrix and is central to the pathogenesis of several disorders. To examine protein glycosylation of α-DG, a facile synthetic approach has been developed for the preparation of unusual phosphorylated O-mannosyl glycopeptides derived from α-DG by a strategy in which properly protected phospho-mannosides are coupled with a Fmoc protected threonine derivative, followed by the use of the resulting derivatives in automated solid-phase glycopeptide synthesis using hyper-acid-sensitive Sieber amide resin. Synthetic efforts also provided a reduced phospho-trisaccharide, and the NMR data of this derivative confirmed the proper structural assignment of the unusual phospho-glycan structure. The glycopeptides made it possible to explore factors that regulate the elaboration of critical glycans. It was established that a glycopeptide having a 6-phospho-O-mannosyl residue is not an acceptor for action by the enzyme POMGnT1, which attaches β(1,2)-GlcNAc to O-mannosyl moietes, whereas the unphosphorylated derivate was readily extended by the enzyme. This finding implies a specific sequence of events in determining the structural fate of the O-glycan. It has also been found that the activity of POMGnT1 is dependent on the location of the acceptor site in the context of the underlying polypeptide/glycopeptide sequence. Conformational analysis by NMR has shown that the O-mannosyl modification does not exert major conformational effect on the peptide backbone. It is, however, proposed that these residues, introduced at the early stages of glycoprotein glycosylation, have an ability to regulate the loci of subsequent O-GalNAc additions, which do exert conformational effects. The studies show that through access to discrete glycopeptide structures, it is possible to reveal complex regulation of O-glycan processing on α-DG that has significant implications both for its normal post-translational maturation, and the mechanisms of the pathologies associated with hypoglycosylated α-DG.     

Purification and characterization of an extracellular α-l-arabinofuranosidase from Fusarium oxysporum

An α-l-arabinofuranosidase from Fusarium oxysporum F3 was purified to homogeneity by a two-step ion exchange intercalated by a gel filtration chromatography. The enzyme had a molecular mass of 66 kDa and was optimally active at pH 6.0 and 60°C. It hydrolyzed aryl α-l-arabinofuranosides and cleaved arabinosyl side chains from arabinoxylan and arabinan. There was a marked synergistic effect between the α-l-arabinofuranosidase and an endo-(1 →4)-β-d-xylanase produced by F. oxysporum in the extensive hydrolysis of arabinoxylan.     

Darzens reaction as a convenient method for the synthesis of α-chloroketones, α-chloroepoxides,and symmetrically substituted dioxines

Summary TheDarzens reaction of dichloroacetophenone (DCAP) with substituted benzaldehydes has been studied. The structure of the products was shown to depend on the phenyl group substituents. Reaction of benzaldehyde, 4-bromo-, and 2,4-dichlorobenzaldehydes results in 1-phenyl-3-aryl-3-chloropropane-1,2-diones (2a–c), reaction ofpara- ormeta-nitrobenzaldehydes yields 1-phenyl-2-chloro-3-aryl-2,3-epoxypropane-1-ones (3a, b). Upon the introduction of an alkoxy group into the phenyl ring of benzaldexyde and/or dichloroacetophenone, symmetrically substituted dioxines were obtained (6a–c). The structure of the reaction products has been established by single crystal X-ray investigations.

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Darzens-Reaktion als bequemes Verfahren zur Synthese von -Chloroketonen, -Chloroepoxiden und symmetrisch substituierten Dioxinen

Zusammenfassung DieDarzens-Reaction von Dichloracetophenon (DCAP) mit substituierten Benzaldehyden wurde untersucht. Die Struktur der Produkte ist von den Substituenten an der Phenylgruppe abhängig. Die Umsetzung mit Benzaldehyd, 4-Brom- und 2,4-Dichlorbenzaldehyd liefert 1-Phenyl-3-aryl-3-chloropropan-1,2-dione (2a–c), die Reaktion vonpara- odermeta-Nitrobenzaldehyd 1-Phenyl-2-chloro-3-aryl-2,3-epoxipropan-1-one (3a,b). Durch Einführung einer Alkoxygruppe in den Phenylring des Benzaldehyds und/oder des Dichloracetophenons erhält man symmetrisch substituierte Dioxine (6a–c). Die Struktur der Reaktionsprodukte wurde mittels Röntgenstrukturanalyse bestätigt.

     

Synthesis,In Vitro α-Amylase Activity,and Molecular Docking Study of New Benzimidazole Derivatives

Russian Journal of Organic Chemistry - New benzimidazole derivatives were synthesized by reacting substituted phenacyl bromides with 1H-benzimidazole-2-thiols. The synthesized compounds were...