Purification and characterization of an extracellular β-glucosidase with high transglucosylation activity and stability from Aspergillus niger No. 5.1

An extracellular beta-glucosidase was extracted from the culture filtrate of Aspergillus niger No. 5.1 and purified to homogeneity by using ammonium sulfate precipitation, Chitopearl-DEAE chromatography, and Sephadex G-100 chromatography. The specific activity of the enzyme was enriched 6.33-fold, with a recovery of 11.67%. The enzyme was a monomer and the molecular mass was 67.5 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 66.5 kDa by gel-filtration chromatography. The enzyme had optimum activity at pH 6.0 and 60 degrees C and was stable over the pH range of 3.0-9.0. It showed specificity of hydrolysis for p-nitrophenyl-beta-D-glucoside and cellobiose. The Km and Vmax values of the enzyme for cellobiose and salicin were 5.34 mM, 2.57 micromol/(mL.s), and 3.09 mM, 1.34 micromol/(mL.s), respectively. Both amino acid composition and N-terminal amino acid sequence of the enzyme were determined, which provides useful information for cloning of this enzyme.     

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 and Characterization of β-Glucosidase from a Newly Isolated Strain Tolypocladium cylindrosporum Syzx4

A higher β-glucosidase-producing strain was isolated and identified as Tolypocladium cylindrosporum syzx4 based on its morphology and internal transcribed spacer(ITS) rDNA gene sequence.The present study is to ferment,purify and characterize a β-glucosidase from T.cylindrosporum gams.The enzyme was purified to homogeneity by sulfate precipitataion,diethylaminoethyl cellulose anion exchange chromatography and Sephadex G-100 gel filtration with a 9.47-fold increase in specific activity and a recovery of 12.27...     

Purification and Characterization of an Ethanol-Tolerant β-Glucosidase from Sporidiobolus pararoseus and Its Potential for Hydrolysis of Wine Aroma Precursors

An extracellular ethanol-tolerant β-glucosidase from Sporidiobolus pararoseus was purified to homogeneity and characterized, and its potential use for the enhancement of wine aroma was investigated. The crude enzymatic extract was purified in four steps (concentration, dialysis, ultrafiltration, and chromatography) with a yield of around 40 % for total activity. The purified enzyme (designated Sp-βgl-P) showed a specific activity of approximately 20.0 U/mg, an estimated molecular mass of 63 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis, and isoelectric point of 5.0 by isoelectric focusing. Sp-βgl-P has optimal activity at pH 4.0 and at 55 °C. It was stable in a broad pH range at low temperatures and it was tolerant to ethanol and glucose, indicating suitable properties for winemaking. The hydrolysis of glycosidic terpenes was analyzed by adding Sp-βgl-P directly to the wines. The released terpene compounds were evaluated by gas chromatography/mass spectrometry. The enzymatic treatment significantly increased the amount of free terpenes, suggesting that this enzyme could potentially be applicable in wine aroma improvement.     

Lamesticumin G,a new α-glucosidase inhibitor from the fruit peels of Lansium parasiticum

A novel onoceranoid triterpene, named lamesticumin G (1) along with four known compounds (2–5) were isolated from the ethyl acetate extract of the fruit peels of Lansium parasiticum. The structure of lamesticumin G (1) was fully characterised using spectroscopic data. Lamesticumin G (1) inhibited α-glucosidase (maltase) with IC₅₀ value of 2.27 mM, while 2–5 showed no inhibition.     

Purification and Characterization of a Glycoside Hydrolase Family 43 β-xylosidase from Geobacillus thermoleovorans IT-08

The gene encoding a glycoside hydrolase family 43 β-xylosidase (GbtXyl43A) from the thermophilic bacterium Geobacillus thermoleovorans strain IT-08 was synthesized and cloned with a C-terminal His-tag into a pET29b expression vector. The recombinant gene product termed GbtXyl43A was expressed in Escherichia coli and purified to apparent homogeneity. Michaelis–Menten kinetic parameters were obtained for the artificial substrates p-nitrophenyl-β-d-xylopyranose (4NPX) and p-nitrophenyl-α-l-arabinofuranose (4NPA), and it was found that the ratio k _cat/K _m 4NPA/k _cat/K _m 4NPX was ～7, indicting greater catalytic efficiency for 4NP hydrolysis from the arabinofuranose aglycon moiety. Substrate inhibition was observed for the substrates 4-methylumbelliferyl xylopyranoside (muX) and the arabinofuranoside cogener (muA), and the ratio k _cat/K _m muA/k _cat/K _m muX was ～5. The enzyme was competitively inhibited by monosaccharides, with an arabinose K _i of 6.8?±?0.62 mM and xylose K _i of 76?±?8.5 mM. The pH maxima was 5.0, and the enzyme was not thermally stable above 54 °C, with a t _1/2 of 35 min at 57.5 °C. GbtXyl43A showed a broad substrate specificity for hydrolysis of xylooligosaccharides up to the highest degree of polymerization tested (xylopentaose), and also released xylose from birch and beechwood arabinoxylan.     

Production, Purification, Immobilization, and Characterization of a Thermostable β-Galactosidase from Aspergillus alliaceus

A fungal strain isolated from rotten banana and identified as Aspergillus alliaceus was found capable of producing thermostable extracellular ??-galactosidase enzyme. Optimum cultural conditions for ??-galactosidase production by A. alliaceus were as follows: pH?4.5; temperature, 30?°C; inoculum age, 25?h; and fermentation time, 144?h. Optimum temperature, time, and pH for enzyme substrate reaction were found to be 45?°C, 20?min, and 7.2, respectively, for crude and partially purified enzyme. For immobilized enzyme?Csubstrate reaction, these three variable, temperature, time, and pH were optimized at 50?°C, 40?min, and 7.2, respectively. Glucose was found to inhibit the enzyme activity. The K _m values of partially purified and immobilized enzymes were 170 and 210?mM, respectively. Immobilized enzyme retained 43?% of the ??-galactosidase activity of partially purified enzyme. There was no significant loss of activity on storage of immobilized beads at 4?°C for 28?days. Immobilized enzyme retained 90?% of the initial activity after being used four times.     

Resveratrol and arctigenin production from polydatin and arctiin respectively by a thermostable β-glucosidase from Thermotoga maritima

Abstract

For preparation of resveratrol and arctigenin from peanut hulls and arctium lappa fruits, respectively, a recombinant β-glucosidase (TmBglA) from hyperthermophile Thermotoga maritima was purified and characterized. The hydrolytic activity was the highest at 90?°C and pH 6.2 for arctiin with K_m of 1.61?mM and k_cat of 197.4?s^?1, and 90?°C and 5.8 for polydatin with K_m of 0.38?mM and k_cat of 47.6?s^?1. The enzyme produced 215.4?mg L^?1 resveratrol and 355.7?mg L^?1 arctigenin from 400?mg L^?1 polydatin and 540?mg L^?1 arctiin after 60?min of incubation at 80?°C, with capable of hydrolyzing up to 92.1 and 94.9% of polydatin and arctiin, respectively. The enzymatic hydrolysis of peanut hulls and fructus arctii displayed a conversion yield of 3.8 and 0.33?mg resveratrol and arctigenin per gram of substrate material flour, respectively. Of the reported β-glucosidase, TmBglA exhibited the highest thermostability, k_cat, k_cat/K_m, and conversion productivity for hydrolyzing polydatin and arctiin, and has great potential applications in functional food and medicine production.     

Purification and Characterization of a Low Molecular Weight of β-Mannanase from Penicillium occitanis Pol6

The highest β-mannanase activity was produced by Penicillium occitanis Pol6 on flour of carob seed, whereas starch-containing medium gave lower enzymes titles. The low molecular weight enzyme was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography procedures. The purified β-mannanase (ManIII) has been identified as a glycoprotein (carbohydrate content 5%) with an apparent molecular mass of 18 kDa. It was active at 40 °C and pH 4.0. It was stable for 30 min at 70 °C and has a broad pH stability (2.0–12.0). ManIII showed K _m, V _max, and K _cat values of 17.94 mg/ml, 93.52 U/mg, and 28.13 s⁻¹ with locust bean gum as substrate, respectively. It was inhibited by mannose with a K _I of 0.610⁻³ mg/ml. ManIII was activated by CuSO₄ and CaCl₂ (2.5 mM). However, in presence of 2.5 mM Co²⁺, its activity dropped to 60% of the initial activity. Both N-terminal and internal amino acid sequences of ManIII presented no homology with mannanases of glycosides hydrolases. During incubation with locust bean gum and Ivory nut mannan, the enzyme released mainly mannotetraose, mannotriose, and mannobiose.     

Purification and Characterization of an Antibacterial Peptide from Rana Temporaria Chensinensis Skin

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First synthesis and absolute configuration of a β-farnesene-trimethoxystyrene conjugate isolated from Pachypodanthium confine

(E)-1-[3′-(4″,8″-Dimethylnona-3″,7″-dienyl)cyclohex-3′-enyl]-2,4,5-trimethoxybenzene (1), a β-farnesene-trimethoxystyrene conjugate, was isolated from Pachypodanthium confine. Its first synthesis was accomplished, and absolute configuration was determined to be R.     

Antioxidant activity of α and β-amyrin isolated from Myrcianthes pungens leaves

Abstract

Brazil has a great variety of native plants which could be explored and among them guabiju (Myrcianthes pungens) stands out. Thus, this study consisted of isolating pentacyclic triterpenes, α and β-amyrin from guajibu leaves, and determine the antioxidant activity.The leaves were dried, pulverized and submitted to a dynamic maceration process with ethanol 96° GL, concentrated to obtain crude leaf extract (CLE). CLE was eluted with dichloromethane until total depletion, resulting in a dichloromethane fraction (FRDicl) which was concentrated and analyzed by GC/MS and NMR. The result of the chemical analysis revealed the presence of pentacyclic triterpenes of oleanane (β-amyrin), and ursane (α-amyrin) groups. The β-carotene bleaching method revealed a high antioxidant activity for the CLE as well as for FRDicl. The antioxidant protection equivalent to the trolox was of 137 and 129%, respectively at 500 µg/mL. The antioxidant potential of FRDicl can be explained by the presence of α and β-amyrins.     

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.     

Influence of carbon and nitrogen sources and temperature on hyperproduction of a thermotolerant β-glucosidase from synthetic medium by Kluyveromyces marxianus

The effect of carbon source and its concentration, inoculum size, yeast extract concentration, nitrogen source, pH of the fermentation medium, and fermentation temperature on β-glucosidase production by Kluyveromyces marxianus in shake-flask culture was investigated. These were the independent variables that directly regulated the specific growth and β-glucosidase production rate. The highest product yield, specific product yield, and productivity of β-glucosidase occurred in the medium (pH 5.5) inoculated with 10% (v/v) inoculum of the culture. Cellobiose (20 g/L) significantly improved β-glucosidase production measured as product yield (Y _P/S ) and volumetric productivity (Q _P ) followed by sucrose, lactose, and xylose. The highest levels of productivity (144 IU/[L·h]) of β-glucosidase occurred on cellobiose in the presence of CSL at 35°C and are significantly higher than the values reported by other researchers on almost all other organisms. The thermodynamics and kinetics of β-glucosidase production and its deactivation are also reported. The enzyme was substantially stable at 60°C and may find application in some industrial processes.     

Synthesis and characterization of a novel amphiphilic copolymer containing β-cyclodextrin

In this article, maleic anhydride was used to attain the modified β-cyclodextrin (MAH-β-CD), and the MAH-β-CD was copolymerized with acrylamide (AM), acrylic acid (AA), and 2-acrylamido-2-methyl propane sulfonic acid (AMPS) via free radical copolymerization. The polymerization conditions were optimized through single-variable method. Subsequently, the copolymer structure was characterized through Fourier transform infrared (FTIR) spectroscopy, proton nuclear magnetic resonance (¹HNMR) spectroscopy, and scanning electron microscopy (SEM). The three-dimensional network structure formed in aqueous solutions was observed via experimental results of SEM. Subsequently, several aspects of the properties of the copolymer, such as temperature tolerance, shear tolerance, salt resistance, and oil displacement ability, were investigated by comparing with hydrolyzed polyacrylamide (HPAM). Results indicate that the copolymer showed better performances than HPAM; thus, it could be concluded that the introduction of β-cyclodextrin in the polymer chain was helpful in enhancing the oil displacement efficiency in high-temperature and high-salinity oil field.     

Phenolic compounds isolated from Morus nigra and their α-glucosidase inhibitory activities

Abstract

A novel benzofuranone unprecedentedly with a prenyl group at C-3, nigranol A (1), a new flavonol, nigranol B (2), and three known compounds, sanggenon M (3), nigrasin C (4), and nigrasin A (5) were isolated from the twigs of Morus nigra Linn. Their structures were elucidated on the basis of the analysis of multiple spectroscopic data. All of the compounds, along with eight previously isolated ones (6–13) were investigated for their α-glucosidase inhibitory activities. The result showed that compounds 1–13 except 4 exhibited prominent inhibitory activities against α-glucosidase. Among them, compounds 2 and 7 were the best α-glucosidase inhibitory candidates with IC₅₀ values at 1.63 and 1.43?μM, respectively. Furthermore, the structure-activity relationships of the sanggenon-type flavanones were summarized.     

Purification and Characterisation of a β-1,4-Xylanase from Remersonia thermophila CBS 540.69 and Its Application in Bread Making

This work reports the first investigation of Remersonia thermophila hemicellulosic hydrolytic enzyme production, with subsequent purification of an extracellular endo-β-1,4-xylanase (RtXyl) and its application in bread making. The research describes RtXyl purification from sorghum-induced submerged liquid cultures of this moderately thermophilic, aerobic, ascomycete fungus. The purified enzyme is a single subunit protein with a molecular mass of 42 kDa and exhibits glycosyl hydrolase family-10-like activity over a broad pH and temperature range. Optimal activity was measured at pH 6.0 and 65 °C respectively, which is suitable for bread making applications. Substrate specificity studies revealed that RtXyl is purely xylanolytic with no side-activities against other plant polysaccharides. The RtXyl catalytic efficiency (K _cat/K _m) was highest with oats spelt xylan (810.90 mg mL^?1 s^?1), wheat arabinoxylan (809.52 mg mL^?1 s^?1) and beechwood xylan (417.40 mg mL^?1 s^?1) with less efficiency towards insoluble oats spelt xylan (236.40 mg mL^?1 s^?1). Hydrolysis products analysed by thin layer chromatography yielded a range of xylosaccharides, predominantly xylotriose and xylobiose. RtXyl application in a basic wheat bread recipe at low dosages (0.297 XU/g) showed its suitability to increase loaf volume by 8.0 % compared with the control bread. RtXyl increased loaf softness by 19.6 % while reducing bread staling by 20.4 % up to 4 days of storage.     

Pyrrole β-amides: Synthesis and characterization of a dipyrrinone carboxylic acid and an N-Confused fluorescent dipyrrinone

Pyrrole β-amides are useful building blocks for the preparation of novel molecular architectures that can be used in supramolecular chemistry and sensor development. Under basic conditions, pyrrole β-amides with an α-aldehyde produce different condensation products when reacted with pyrrolinones depending on the amide substitution. Secondary amides form the expected dipyrrinones, but unexpectedly undergo a subsequent trans-amidation with the pyrrolinone nitrogen to produce an unsymmetrical imide (an N-confused fluorescent dipyrrinone). Under the same conditions, tertiary amides produce the expected dipyrrinone carboxylic acids, which have been shown to have strong self-association properties as determined by vapor pressure osmometry measurements, NMR studies, and X-ray crystal structure determination. Furthermore, an N-confused fluorescent dipyrrinone was produced from the same trans-amidation reaction during attempts to decarboxylate a dipyrrinone amide with a 9-carboxylic moiety.     

In silico design, synthesis and evaluation of 3'-O-benzylated analogs of salacinol, a potent α-glucosidase inhibitor isolated from an Ayurvedic traditional medicine "Salacia"

With the aid of an in silico method, α-glucosidase inhibitors with far more potent activities than salacinol (1), a potent natural α-glucosidase inhibitor isolated from an Ayurvedic traditional medicine Salacia reticulata, have been developed.     

Determination of acetylcholinesterase and α-glucosidase inhibition by electrophoretically-mediated microanalysis and phenolic profile by HPLC-ESI-MS/MS of fruit juices from Brazilian Myrtaceae Plinia cauliflora (Mart.) Kausel and Eugenia uniflora L.

Abstract

Alzheimer’s disease and diabetes mellitus are contemporary diseases of great concern. Phenolic compounds are linked to several health benefits and could lead to novel strategies to combat these ailments. The objective of this study was to evaluate by electrophoretically-mediated microanalysis the potential inhibitory activity of the fruit juices from Plinia cauliflora (“jaboticaba”) and Eugenia uniflora (“pitanga”) toward acetylcholinesterase (AChE) and α-glucosidase, target enzymes in strategies for the treatment of these diseases. The phenolic profiles of the samples were also investigated. Jaboticaba and pitanga juices inhibited 85.90?±?1.73 and 52.67?±?1.24% of AChE activity at 5?mg mL^?1, and 57.91?±?2.60 and 69.47?±?2.89% of α-glucosidase activity at 1?mg mL^?1, respectively. Total phenolic content of the juices were 303.54?±?28.28 and 367.00?±?11.42 mgGA L^?1, respectively. The observed inhibitory activity can be explained, at least in part, by the presence of the phenolic compounds.