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.     

Purification, characterization, and structural investigation of a new moderately thermophilic and partially calcium-independent extracellular α-amylase from Bacillus sp. TM1

A new α-amylase was extracted from a recently found strain of Bacillus sp. and purified by ion-exchange chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a single band for the purified enzyme with an apparent molecular weight of 59 kDa. The optimum temperature and pH range of the enzyme were 40–60°C and 4.5–7.5, respectively, and its activation energy was 1.974 kcal/mol. The K _m value for the enzyme activity on solubie starch was 4 mg/mL, and the T _m values obtained from the circular dichroism (CD) results of thermal unfolding were 78.7 and 80.2°C in the absence and presence of the calcium, respectively. The enzyme was almost completely inhibited by the addition of Fe³⁺, Mn²⁺, and Zn²⁺ and was activated by EDTA, Cr³⁺, and Al³⁺. Moreover, it was partially inhibited by Ca²⁺, Ba²⁺, Ni²⁺, and Co²⁺. Proteolytic digestion of the enzyme using trypsin combined with results from T _m using CD and irreversible thermoinactivation suggests that this enzyme can be considered a moderate thermophile with both mild flexibility and rigidity.     

Purification and characterisation of α-amylase produced by mutant strain of Aspergillus oryzae EMS-18

α-Amylase produced by a mutant strain of Aspergillus oryzae EMS-18 has been purified to homogeneity as judged by sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified by using 70% ammonium sulphate precipitation followed by anion exchange chromatography on DEAE-Sephadex column and gel filtration on Sephadex G-100. An enzyme purification factor of 9.5-fold was achieved with a final specific activity of 1987.7 U/mg protein and overall yield of 23.8%. The molecular weight of purified α-amylase was estimated to be 48 kDa by SDS-PAGE. The purified enzyme revealed an optimum assay temperature and pH 40°C and 5.0, respectively. Except Ca⁺⁺ all other metal ions such as Mg, Mn, Na, Zn, Ni, Fe, Cu, Co and Ba were found to be inhibitory to enzyme activity.     

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.     

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.     

Controlled release of α-amylase from microchamber arrays containing carbon nanoparticle aggregates

Microchamber arrays have been prepared from a composite of polylactic acid and carbon nanoparticles. Due to the presence of the nanoparticles, the microchambers are sensitive to infrared laser irradiation. α-Amylase has been encapsulated in the microchambers and released under controlled laser irradiation at 1064 nm with retention of the enzyme activity.     

Identification and functional characterization of a novel α-conotoxin (EIIA) from Conus ermineus

Nicotinic acetylcholine receptors (nAChRs) are one of the most important families in the ligand-gated ion channel superfamily due to their involvement in primordial brain functions and in several neurodegenerative pathologies. The discovery of new ligands which can bind with high affinity and selectivity to nAChR subtypes is of prime interest in order to study these receptors and to potentially discover new drugs for treating various pathologies. Predatory cone snails of the genus Conus hunt their prey using venoms containing a large number of small, highly structured peptides called conotoxins. Conotoxins are classified in different structural families and target a large panel of receptors and ion channels. Interestingly, nAChRs represent the only subgroup for which Conus has developed seven distinct families of conotoxins. Conus venoms have thus received much attention as they could represent a potential source of selective ligands of nAChR subtypes. We describe the mass spectrometric-based approaches which led to the discovery of a novel α-conotoxin targeting muscular nAChR from the venom of Conus ermineus. The presence of several posttranslational modifications complicated the N-terminal sequencing. To discriminate between the different possible sequences, analogs with variable N-terminus were synthesized and fragmented by MS/MS. Understanding the fragmentation pathways in the low m/z range appeared crucial to determine the right sequence. The biological activity of this novel α-conotoxin (α-EIIA) that belongs to the unusual α4/4 subfamily was determined by binding experiments. The results revealed not only its selectivity for the muscular nAChR, but also a clear discrimination between the two binding sites described for this receptor.     

Kinetic profiling of prolinate-catalyzed α-amination of aldehydes

Deconvolution of the role of off-cycle species from the desired catalytic cycle leads to an optimized protocol for the prolinate-catalyzed amination of aldehydes. The scope of complex reaction networks will be greatly broadened by understanding ancillary rate processes that influence the productive catalytic pathway.     

Purification of extracellular α-amylase and proteinase from <Emphasis Type=Italic>Bacillus subtilis</Emphasis> SKB 256

The enzymes α-amylase and proteinase with specific activity 310 and 68-72 units/mg, respectively, were isolated from Bacillus subtilis SKB 256 culture medium. The enzyme preparations were purified and a-amylase and proteinase were separated using biospecific chromatography. Gel filtration of a-amylase was performed over a column (20 × 600 mm) of TSK HW-55F gel (Toyopearl, Japan).     

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.     

Comparative studies on a mesophilic and a thermophilic α-amylase

A comparative study was performed on thermal stability of mesophilic and thermophilic α-amylases, and the effects of various denaturing agents, organic solvents, and stabilizers were investigated. As expected, the thermophilic enzyme showed higher resistance toward denaturation in water as its natural medium, but such a difference could not be detected in nonaqueous environments. Furthermore, stability of these molecules was improved by including various stabilizing agents. Of the compounds tested, sorbitol provided the highest degree of protection, which was found to be owing to its effect on increasing T _m and its ability in totally preventing deamidation of amino acid residues in the protein molecules.     

Isolation and identification of novel α-amylase inhibitors from Euonymus laxiflorus Champ.

Research on Chemical Intermediates - Euonymus laxiflorus Champ. (ELC) has been reported to possess potential antidiabetic activity. The aim of this study is to isolate and identify the active...     

Purification and characterization of α-amylase from <Emphasis Type="Italic">Trichoderma pseudokoningii</Emphasis>

Background

Previous studies have demonstrated that members of Trichoderma are able to generate appreciable amount of extracellular amylase and glucoamylase on soluble potato starch. In this study the α-amylase was purified and characterized from Trichoderma pseudokoningii grown on orange peel under solid state fermentation (SSF).

Results

Five α-amylases A1-A5 from Trichodrma pseudokoningii were separated on DEAE-Sepharose column. The homogeneity of α-amylase A4 was detected after chromatography on Sephacryl S-200. α-Amylase A4 had molecular weight of 30 kDa by Sephacryl S-200 and SDS-PAGE. The enzyme had a broad pH optimum ranged from 4.5 to 8.5. The optimum temperature of A4 was 50 °C with high retention of its activity from 30 to 80 °C. The thermal stability of A4 was detected up to 50 °C and the enzyme was highly stable till 80 °C after 1 h incubation. All substrate analogues tested had amylase activity toward A4 ranged from 12 to 100% of its initial activity. The Km and Vmax values of A4 were 4 mg starch/ml and 0.74 μmol reducing sugar, respectively. The most of metals tested caused moderate inhibitory effect, except of Ca²⁺ and Mg²⁺ enhanced the activity. Hg²⁺ and Cd^+?2 strongly inhibited the activity of A4. EDTA as metal chelator caused strong inhibitory effect.

Conclusions

The properties of the purified α-amylase A4 from T. pseudokoningii meet the prerequisites needed for several applications.

     

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.     

Inhibition mechanism of human salivary α-amylase by lipid transfer protein from Vigna unguiculata

VuLTP1.1, a LTP1 from Vigna unguiculata, inhibits 78.1 % of the human salivary α-amylase (HSA) activity at 20 μM. We had performed a correlation study between VuLTP1.1 structure and HSA inhibitory activity and showed that two VuLTP1.1 regions are responsible for HSA inhibition. In one of them we had characterized the crucial importance of an Arg₃₉ for inhibition. In this work, we analyzed the VuLTP1.1-HSA interaction by protein-protein docking to understand the most probable interaction model and the mechanism of HSA inhibition by VuLTP1.1. The VuLTP1.1 tertiary structure quality and refinement as well as the docking assay between VuLTP1.1 and HSA were done by bioinformatic programs. HSA inhibition occurs by direct interaction of the VuLTP1.1 with the HSA causing the obstruction of the carbohydrate biding cleft with Gibbs free energy of -18.5 Kcal/mol and the dissociation constant of 2.6E⁻¹⁴ M. The previously identified Arg₃₉ of VuLTP1.1 is burrowed into the active site of the HSA and there it interacts with the Asp₃₀₀ of HSA catalytic site by a hydrogen bond. We had confirmed the importance of the Arg₃₉ of VuLTP1.1 for the HSA inhibition which interacts with the Asp₃₀₀ at the HSA active site. I-2, a LTP-like peptide, presents the same HSA inhibition pattern that VuLTP1.1, which indicates that the inhibition mechanism of the LTPs towards α-amylase is very similar. For the best of our knowledge, it is the first time that the HSA inhibition mechanism was understood and described for the LTP1s using VuLTP1.1 and I-2 as prototype inhibitors.     

Immobilization of α-amylase in vinyl-polymer-based interpenetrating polymer networks

Semi-interpenetrating polymer networks (IPNs) of poly(ethylene glycol), poly(vinyl alcohol) and polyacrylamide were prepared as a support for enzyme immobilization and kinetic studies were performed for the immobilization of -amylase. The effect of IPN composition on the extent of immobilization was investigated and the percentage of relative activity of the immobilized enzyme was evaluated as a function of the chemical architecture of the IPNs, pH and temperature, taking starch as a substrate. The kinetic constants and the maximum reaction velocity were also evaluated. The IPNs were characterized by IR spectral analysis.     

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.     

Isothiourea-Catalyzed Acylative Kinetic Resolution of Tertiary α-Hydroxy Esters

A highly enantioselective isothiourea-catalyzed acylative kinetic resolution (KR) of acyclic tertiary alcohols has been developed. Selectivity factors of up to 200 were achieved for the KR of tertiary alcohols bearing an adjacent ester substituent, with both reaction conversion and enantioselectivity found to be sensitive to the steric and electronic environment at the stereogenic tertiary carbinol centre. For more sterically congested alcohols, the use of a recently-developed isoselenourea catalyst was optimal, with equivalent enantioselectivity but higher conversion achieved in comparison to the isothiourea HyperBTM. Diastereomeric acylation transition state models are proposed to rationalize the origins of enantiodiscrimination in this process. This KR procedure was also translated to a continuous-flow process using a polymer-supported variant of the catalyst.