Direct chemiluminescence determination of penicillin G potassium and a chemometrical optimization approach

A highly selective and simple chemiluminescence (CL) method for determination of penicillin G potassium (PGK) was developed. In the proposed method, CL was elicited from PGK upon its oxidation with H₂O₂. The light emission was enhanced in the presence of N‐cetyl‐N,N,N‐trimethylammonium bromide (CTMAB). An experimental design, central composite design (CCD), was used to realize the optimized variables, including pH, surfactant (CTMAB) and H₂O₂ concentrations. Under optimum condition, the calibration graph was linear in the range 3.3 × 10^?3–3.3 × 10^?1 mmol/L, with a detection limit of 8.8 × 10^?4 mmol/L for PGK. The precision was calculated by analysing samples containing 1.6 × 10^?1 mmol/L PGK (n = 5) and the relative standard deviation (RSD) was 1.40%. The utility of this method was demonstrated by determining PGK in pharmaceutical formulations for injection. The proposed method was validated by a reference method. Copyright © 2011 John Wiley & Sons, Ltd.     

Mannitol Biosynthesis in Pyrenochaeta terrestris I. D-Maunitol-1-Phosphate: NAD Oxidoreductase

Cell-free extracts of mycelial mats of Pgrenochaeta terrestris grown in stationary culture on synthetic glucose or sucrose - salts liquid media contained D-mannitol-1-Phosphate:NAD oxidoreductase (EC 1.1.1.17) activity. Greatest activity occurred early in the growth period. The optimum pH for the reduction of NAD⁺ in the presence of Fru-6-P was 7.4–7.5 while the optimum pH for the oxidation of NADH in the presence of Mtl-1-P was 8.1–8.2. The enzyme was stabilized to some extent in Tris-maleate buffer, pH 7.5, and by the addition of 10% (NH₄)₂SO₄, to this buffer. A 10- to 16-fold purification was attained by a combination of (NH₄)₂SO₄ fractionation and gel filtration on Sephadex G-100. The enzyme was relatively specific in its substrate and coenzyme requirements. The Km values were determined as: Fru-6-P - 3 × 10^?4 M, Mtl-1-P - 1 × 10^?4 M, and NAD⁺ and NADH - 3 × 10^?5 M.     

Determination of dipyridamole using TCPO–H2O2 chemiluminescence in the presence of silver nanoparticles

The method is based on the fact that dipyridamole can enhance the chemiluminescence (CL) emission from the redox reaction of bis (2,4,6‐tricholorophenyl) oxalate (TCPO) with H₂O₂ in the presence of silver nanoparticles (AgNPs). The CL reaction mechanism was discussed. The effect of concentrations of TCPO, H₂O₂, AgNPs and pH value on the CL reaction were investigated. Under the optimum conditions, the linear dynamic range was 1.0–1000 × 10^?9 g/mL and the detection limit (3σ) was 9 × 10^?10 g/mL. The relative standard deviation (RSD) was 4.8% for 1.0 × 10^?9 g/mL dipyridamole (n = 7). The proposed method has been successfully applied to the determination of dipyridamole tablets and the recovery was 99–103%. Copyright © 2011 John Wiley & Sons, Ltd.     

An amylase from fresh fruiting bodies of the monkey head mushroom Hericium Erinaceum

An amylase with a molecular mass of 55 kDa and an N-terminal sequence exhibiting similarity to enzyme from Bacteroides thetaitaomicron was isolated from fruiting bodies of the monkey head mushroom Hericium erinaceum. The purification scheme included extraction with distilled water, ion exchange chromatography on DEAE-cellulose and SP-sepharose, and gel filtration by FPLC on Superdex 75. The amylase of H. erinaceum was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and eluted with 0.2 M NaCl in the same buffer. The enzyme was subsequently adsorbed on SP-Sepharose in 10 mM ammonium acetate buffer (pH 4.5) and eluted with 0.3 M NaCl in the same buffer. This fraction was subsequently subjected to gel filtration on Superdex 75. The first peak eluted had a molecular mass of 55 kDa in SDS-PAGE. The amylase of H. erinaceum exhibited a pH optimum of 4.6 and a temperature optimum of 40°C. The enzyme activity was enhanced by Mn²⁺ and Fe³⁺ ions, but inhibited by Hg²⁺ ions.     

Arylsulphatases in human brain: separation, purification, and certain properties of the two soluble arylsulphatases

Abstract— Arylsulphatases (aryl-sulphate sulphohydrolases; E.C. 3.1.6.1) in the soluble subcellular fraction (105000g, 2 h) of human brain were partially purified by ammonium sulphate fractionation, ion exchange chromatography, and Sephadex gel filtration. Potassium-4-methylumbelliferone-sulphatase (MUS-sulphatase) adsorbed on DEAE-cellulose was purified approximately 700-fold over activity in the soluble fraction and the unadsorbed MUS-sulphatase was similarly purified approximately 600-fold. The arylsulphatase adsorbed to DEAE-cellulose exhibited a K_m value for MUS of 12.5 mM and a pH optimum of 5.7, whereas the unadsorbed arylsulphatase exhibited a K_m value for MUS of 8.3 mM and a pH optimum of 5.4. The molecular weights of the two enzymes were approximately 109,600 and 51,300, respectively. Sulphate (0.5 mM) showed pronounced mixed inhibition only of the unadsorbed arylsulphatase. Ag⁺ ions (0.25 mM) showed 96 per cent inhibition of the adsorbed arylsulphatase, whereas an activation of the unadsorbed arylsulphatase was observed.     

Purification and Properties of 2-Ketogluconate Reductase from Gluconobacter liquefaciens.

2-Ketogluconate reductase (2KGR) from the cell free extract of Gluconobacter liquefaciens (IFO 12388) was purified about 1000-fold by a procedure involving ammonium sulfate fractionation and column chromatographies using DEAE-cellulose, hydroxylapatite, and Sephadex gel The purified enzyme gave a single band on polyacrymamide gel electrophoresis. NADP was specifically required for the oxidation reaction of gluconic acid. Using gel filtration a molecular weight of about 110,000 was estimated for the enzyme. The pH optimum for the oxidation of gluconic acid (GA) to 2-ketogluconic acid (2KGA) by the enzyme was 10.5 and for the reduction of 2KGA was 6.5. The optimum temperature of the enzyme was 50 C for both reactions of oxidation and reduction. The enzyme was stable at pH between 5.0 and 11.0 and at temperature under 50°C, The enzyme activity was strongly inhibited with p-chloromercuribenzoate and mercury ions, but remarkably stimulated by manganese ions (1×10^?3 m). Km value of the enzyme for GA was 1.3×10^?2 m and for 2KGA was 6.6×10^?3 _m. Km values for NADP and NADPH₂ were 1.25×10^?5 and 1.52×10^?5 m respectively.     

Flow injection chemiluminescence determination of naphazoline hydrochloride in pharmaceuticals

A simple and sensitive flow injection chemiluminescence (FI‐CL) method was developed for the determination of naphazoline hydrochloride (NPZ). The method is based on the enhancing effect of NPZ on the weak CL signal from the reaction of KIO₄ with H₂O₂. Experimental parameters that affected the CL signal, including the pH of the KIO₄ solution, concentrations of KIO₄, H₂O₂ and disodium‐EDTA and flow rate were optimized. Under the optimum conditions, the increment of CL intensity was linearly proportional to the concentration of NPZ in the range 5.0 × 10^?6 to 70 × 10^?6 mol/L. The detection limit was 1.0 × 10^?6 mol/L and the relative standard deviation for 50 × 10^?6 mol/L NPZ solution was 2.8% (n = 11). In addition, a high throughput of 120 samples/h was achieved. The utility of this method was demonstrated by determining NPZ in pharmaceuticals. Copyright © 2013 John Wiley & Sons, Ltd.     

Coconut α-d-galactosidase isoenzymes: isolation,purification and characterization

α-d-Galactosidases (α-d-galactoside galactohydrolase, EC 3.2.1.22) from normal coconut endosperm were isolated and partially purified by a combination of ammonium sulfate fractionation, SP-Sephadex C50–120 ion-exchange chromatography and Sephadex G-200 and G-100 gel filtration. Two molecular forms of the enzyme, designated as A and B, were eluted after SP-Sephadex C50–120 ion-exchange chromatography. α-d-Galactosidase A, which is the major isoenzyme, was partially purified 43-fold on Sephadex G-200 and has a MW of about 23 000 whereas α-d-galactosidase B was partially purified 23-fold on Sephadex G-100 and has a similar MW of about 26 600. Both isoenzymes exhibited optimum activity at pH 7.5. The apparent K_m and V_max of α-d-galactosidase A were obtained at 3.46 × 10^?4M and 1.38 × 10^?3 M p-nitrophenyl α-<d-galactoside, respectively. A distinct substrate inhibition was noted. The enzyme was inhibited strongly by d-galactose and to a lesser extent by myo-inositol, d-glucose-6-phosphate, l-arabinose, melibiose and iodoacetic acid. Similarly, makapuno α-d-galactosidase was localized in the 40–70 % (NH₄)₂SO₄ cut but its optimum activity at pH 7.5 was considerably lower as compared to the normal. Its K_m was obtained at 6.75 × 10^?4 M p-nitrophenyl α-d-galactoside while the V_max was noted at 5.28 × 10^?3 M p-nitrophenyl α-d-galactoside. Based on the above kinetic data, the possible cause(s) of the deficiency of α-d-galactosidase activity in makapuno is discussed.     

SR 140333, a Novel, Selective, and Potent Nonpeptide Antagonist of the NK1 Tachykinin Receptor: Characterization on the U373MG Cell Line

The effects of a novel nonpeptide NK1 tachy-kinin receptor antagonist, SR 140333, on the functional consequences of NK1 receptor activation in a human astrocytoma cell line, U373MG, were investigated. Radioligand binding conducted with ¹²⁵l-Bolton-Hunter substance P revealed a competitive inhibition by SR 140333 and its R enantiomer SR 140603 with K_i values of 0.74 and 7.40 nM, respectively. The NK1-selective agonist, [Sar⁹,Met(O₂)¹¹]-substance P, stimulated the formation of inositol phosphates with an EC₅₀ of 3.8 × 10^?9M. SR 140333 blocked the stimulatory effect of this agonist (10^?7M) with an IC₅₀ of 1.6 × 10^?9M,whereas the effect of another NK1 agonist, septide (EC₅₀= 1.5 × 10^?8M)was antagonized with an IC₅₀ of 2.1 × 10^?10M.Enhancement of [³H]taurine release by [Sar⁹,Met(O₂)¹¹]-substance P (EC₅₀= 7.4 × 10^?9M) was also inhibited by SR 140333 with an IC₅₀ of 1.8 × 10^?9 M. SR 140603 was 10-fold less potent than SR 140333 in inhibiting inositol monophosphate formation and [³H]taurine release. The calcium mobilization induced by [Sar⁹,Met(O₂)¹¹]-substance P (10^?8M) was totally prevented by 10^?8MSR 140333. Patchclamp experiments showed that SR 140333 depressed the outward current evoked by 5 × 10^?8M [Sar⁹, Met(O₂)¹¹]-substance P with an IC₅₀ of 1.3 × 10^?9M. The expression of c-fos was stimulated by [Sar⁹,Met(O₂)¹¹]-substance P with an EC₅₀ of 2.5 × 10^?10M, an effect that was also inhibited by SR 140333 with an IC₅₀ of 1.1 × 10^?9M. The present results illustrate the sequential events of the response elicited by NK1 agonists, which were antagonized by SR 140333, demonstrating its powerful NK1 antagonist activity on a functional basis.     

Characterization of trinucleotide SSR motifs in wheat

Length differences among trinucleotide-based microsatellite alleles can be more easily detected and frequently produce fewer ”stutter bands” as compared to dinucleotide-based microsatellite markers. Our objective was to determine which trinucleotide motif(s) would be the most-polymorphic and abundant source of trinucleotide microsatellite markers in wheat (Triticum aestivum L.). Four genomic libraries of cultivar ’Chinese Spring’ were screened with nine trinucleotide probes. Based on the screening of 28550 clones, the occurrences of (CTT/GAA) _n , (GGA/CCT) _n , (TAA/ATT) _n , (CAA/GTT) _n , (GGT/CCA) _n , (CAT/GTA) _n , (CGA/GCT) _n , (CTA/GAT) _n , and (CGT/GCA) _n repeats were estimated to be 5.4×10⁴, 3.5×10⁴, 3.2×10⁴, 1.2×10⁴, 6.3×10³, 4.9×10³, 4.5×10³, 4.5×10³ and 3.6×10³, i.e., once every 293 kbp, 456 kbp, 500 kbp, 1.3 Mbp, 2.6 Mbp, 3.2 Mbp, 3.6 Mbp, 3.6 Mbp and 4.5 Mbp in the wheat genome, respectively. Of 236 clones selected for sequencing, 38 (93%) (TAA/ATT) _n , 30 (43%) (CTT/GAA) _n , 16 (59%) (CAA/GTT) _n , 3 (27%) (CAT/GTA) _n and 2 (4%) (GGA/CCT) _n clones contained microsatellites with eight or more perfect repeats. From these data, 29, 27 and 16 PCR primer sets were designed and tested to the (TAA/ATT) _n , (CTT/GAA) _n and (CAA/GTT) _n microsatellites, respectively. A total of 12 (41.4%) primers designed to (TAA/ATT) _n , four (14.8%) to (CTT/GAA) _n , and two (12.5%) to (CAA/GTT) _n resulted in polymorphic markers. The results indicated that (TAA/ATT) _n microsatellites would provide the most-abundant and the most-polymorphic source of trinucleotide microsatellite markers in wheat. Received: 17 February 2001 / Accepted: 31 May 2001     

Biosorption of Cr, Cu and Al bySargassum biomass

The biosorption and desorption of Cr, Cu and Al were carried out using brown marine algaeSargassum fluitans biomass, known as the good biosorbent of heavy metals. The content of alginate bound to light metals could be changed by physical and chemical pretreatment. The maximum uptake of Cr, Cu and Al was independent of the alginate content. The maximum uptake of Al was two times(mole basis) than those of Cu and Cr. The aluminum-alginate complex was found in the sorption solution of raw and protonated biomass. Most of Cu, Al and light metals sorbed in the biomass were eluted at pH 1.1. However, only 5 to 10% of Cr sorbed was eluted at pH 1.1. The stoichometric ion exchange between Cu and Ca ion was observed on Cu biosorption with Ca-loaded biomass. A part of Cr ion was bound to biomass as Cr(OH)₂ ⁺ or Cr(OH)²⁺. Al was also bound to biomass as multi-valence ion and interfered with the desorbed Ca ion. The behavior of rawS. fluitans in ten consecutive sorption-desorption cycles has been investigated in a packed bed flow-through-column during a continuous removal of copper from a 35 mg/L aqueous solution at pH 5. The eluant used was a 1%(w/v) CaCl/HCl solution at pH3.     

Helix promotion in polypeptides by polyols

The helix content of [L -Lys(Me₃)]_n · ClO₄, and [L -Lys(Me₃)⁵⁰, L -Ala⁵⁰]_n · ClO₄ in water is markedly increased by the presence of sucrose and glycerol. For [L -Lys(Me₃)]_n · ClO₄ the ellipticity at 222 nm changes from +2 × 10³ deg cm² dmole^?1 in water to ?44 × 10³ in 50% glycerol. Sucrose does not promote helix formation in melittin at pH 7.2, but glycerol does. At pH 5.5 sucrose and, more so, glycerol, induce helix in melittin. Glycerol induces some helix in methylated melittin, but less than in melittin. The results are discussed in relation to excluded volume effects, ΔG of transfer of peptide and hydrophobic groups from water to mixed solvents, electrostatic effects, and preferential hydration. © 1993 John Wiley & Sons, Inc.     

Properties of Phenylalanyl-tRNA Synthetase and Phenylalanine Ammonia-lyase of Pine Suspension Cultures

Phenylalanyl-tRNA synthetase and phenylalanine ammonia-lyase activities were demonstrated in partially purified extracts of pine (Pinus elliottii) suspension cultures. The optimum pH for the phenylalanyl-tRNA synthetase reaction was 7.5 and the optimum ATP and Mg²⁺ concentrations were 1.0 and 15 mM respectively. Pine, calf liver and yeast tRNA were inadequate substitutes for pea tRNA in the synthetase reaction mixtures. The optimum pH for the phenylalanine ammonia-lyase reaction was 9.0. The K_m for phenylalanine was approximately 6.6 × 10^?5M. The activity of both enzymes in the partially purified extracts was unstable on storage.     

The properties of subtilisin,Novo type,immobilized on porous glass

Studies of the properties of subtilisin, Novo type, immobilized on porous glass with the aid of hexamethylene diisocyanate were carried out. The immobilized proteinase preparation shows optimum activity at a pH value of 10.7 and at a temperature between 60–65°C. It was stable in a wider range of pH and temperature values than the native subtilisin. The K_M values for hemoglobin and BAEE were 9.2 × 10^?5 [M] and 139 × 10^?5 [M], respectively. Under relatively non-aqueous conditions, immobilised subtilisin was able to synthesize phenylacetic acid ethyl ester.     

Behaviour of Endomycopsis fibuligera glucoamylase towards raw starch

An extracellular glucoamylase [exo-1,4-α-d-glucosidase, 1,4-α-d-glucan glucohydrolase, EC 3.2.1.3] of Endomycopsis fibuligera has been purified and some of its properties studied. It had a very high debranching activity (0.63). The enzyme was completely adsorbed onto raw starch at all the pH values tested (pH 2.0–7.6). Amylase inhibitor from Streptomyces sp. did not prevent the adsorption of glucoamylase onto raw starch although the enzyme did not digest raw starch in the presence of amylase inhibitor. Sodium borate (0.1 m) eluted only 35% of the adsorbed enzyme from raw starch. The optimum pH for raw starch digestion was 4.5 whereas that of boiled soluble starch hydrolysis was 5.5. Waxy starches were more easily digested than non-waxy starches, and root starches were slowly digested by this enzyme.     

Preparation of Large Oligonucleotides by RNase T1 Partial Hydrolysis of MS2 Virus Treated with Succinic Nhydride

Intact MS2 virus was reacted with succinic anhydride to modify the protein coat and then treated with RNase Tl to obtain controlled hydrolysis of the viral RNA. Viral protein and enzyme were removed by phenol extraction. The RNA fraction was adsorbed on DEAE-cellulose and eluted stepwise with 0.1, 0.5, and 1.0 M NH₄HCO₃, pH 8.6. tRNA^arg, used as a marker, was eluted in the 1.0 M NH₄HCO₃ fraction. The oligomers in the 1.0 M fraction isolated from the viral derivative were further examined by paper chromatography, polyacrylamide gel electrophoresis, and sucrose gradient cestrifuga-tion. Yields of the large oligomer fraction as defined by elution with 1.0 MNH₄HCO₃ from DEAE-cellulose ranged from 51–86% of the amount applied.     

Alkaline Catalase Produced by Bacillus No. Ku-1

Bacillus No. Ku-1 isolated from soil produced and alkaline catalase in alkaline media. The characteristic point of this bacteria was especially good growth in alkaline media. The alkaline catalase in the culture fluid was purified by DEAE-cellulose and Sephadex columns. The enzyme was most active at pH 10.0 and was stable at pH 7.0 to 8.5. The sedimentation constant was about 12.5 S. The enzyme was strongly inhibited by NaN₃, KCN, FeSO₄ and Fe₂ (SO₄)₃. Properties of the enzyme are almost same as those of catalases so far reported except optimum pH for enzyme action and Kat.f. value (4.4×10⁴).     

Purification and Characterization of Xylose Isomerase of a Methanol Yeast,Candida boidinii,Which is Involved in Sorbitol Production from Glucose

d-Xylose (xylose) isomerase was extracted from xylose-grown cells of a methanol yeast, Candida boidinii (Kloeckera sp.) No. 2201. The enzyme was purified 70-fold, over the original cell- free extract, with a yield of 2.4% in a homogeneous state, as judged on sodium dodecyl sulfate- polyacrylamide gel electrophoresis and high performance liquid chromatography. The molecular weight of the enzyme was determined to be 130,000, the enzyme being composed of two subunits of 65,000. The optimum pH and temperature for activity were 4.5 and 37~45°C, respectively. The enzyme activity was markedly enhanced by Mn²⁺, Mg²⁺ and Co²⁺, and the enzyme isomerized aldopentoses and aldohexoses. The Km values for xylose and d-glucose were 5.6 × 10?¹m and 4.1 × 10?¹m, and the V_max values were 5.8 × 10² and 3.3 × 10² µmol/min/mg, respectively. NaHAsO₄ 7H₂O and NaCN strongly inhibited the activity, but HgCl₂, NaN₃, dithiothreitol, monoiodoacetate and polyols such as d-sorbitol, xylitol and d-mannitol did not inhibit the activity.     

Flow injection–chemiluminescence determination of amoxycillin using potassium permanganate and formaldehyde system

It was found that amoxycillin can react with potassium permanganate in an acidic medium to produce chemiluminescence, which is greatly enhanced by formaldehyde. The optimum conditions for this chemiluminescent reaction were studied in detail using a flow‐injection system. The experimental results indicate that, under optimum conditions, the chemiluminescence intensity is linearly related to the concentration of amoxycillin in the range 5.48 × 10^?8–2.74 × 10^?6 mol[sol ]L, with a detection limit (3σ) of 4.1 × 10^?8 mol[sol ]L. The relative standard deviation was 1.0% at 1.1 × 10^?6 mol[sol ]L amoxycillin (n = 11 measurements). This method has the advantages of high sensitivity, fast response and ease of operation. The method was successfully applied to the determination of amoxycillin in raw medicines and capsules. Copyright © 2005 John Wiley & Sons, Ltd.     

Production of Pseudomonas Cells Having a High Fumarate Hydratase Activity

An extracellular 45 kDa endochitosanase was purified and characterized from the culture supernatant of Bacillus sp. P16. The purified enzyme showed an optimum pH of 5.5 and optimum temperature of 60°C, and was stable between pH 4.5-10.0 and under 50°C. The K _m and V _max were measured with a chitosan of a D.A. of 20.2% as 0.52 mg/ml and 7.71×10^?6 mol/sec/mg protein, respectively. The enzyme did not degrade chitin, cellulose, or starch. The chitosanase digested partially N-acetylated chitosans, with maximum activity for 15-30% and lesser activity for 0-15% acetylated chitosan. The chitosanase rapidly reduced the viscosity of chitosan solutions at a very early stage of reaction, suggesting the endotype of cleavage in polymeric chitosan chains. The chitosanase hydrolyzed (GlcN)₇ in an endo-splitting manner producing a mixture of (GlcN)_2-5. Time course studies showed a decrease in the rate of substrate degradation from (GlcN)₇ to (GlcN)₆ to (GlcN)₅, as indicated by the apparent first order rate constants, k ₁ values, of 4.98×10^?4, 2.3×10^?4, and 9.3×10^?6 sec^?1, respectively. The enzyme hardly catalyzed degradation of chitooligomers smaller than the pentamer.