Synthesis of Cholesterol-Conjugated Magnetic Nanoparticles for Purification of Human Paraoxonase 1

Human serum paraoxonase 1 (PON1) is known as an antioxidant and is also involved in the detoxification of many compounds. In this study, a novel purification strategy was employed to purify the PON1 by using cholesterol-conjugated magnetic nanoparticles. Magnetic nanoparticles were synthesized and conjugated with cholesterol through diazotized p-aminohippuric acid. In Fourier transform infrared spectrum of cholesterol-p-aminohippuric acid-Fe₃O₄ nanoparticles, the appearance of peaks at 3,358.3, 1,645 cm⁻¹, and at 2,334.9 cm⁻¹ confirmed the conjugation. The molecular weight of purified PON1 was nearly 45 kDa on sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE), and isoelectric point was 5.3. The specific activity was 438 U mg⁻¹ protein, and the purification fold was 515 with 73% yield. The K _m values were 1.3 and 0.74 mM with paraoxon and phenyl acetate, respectively. Western blot of 2D-PAGE confirmed the homogeneity and stability of the enzyme. Mg⁺², Mn⁺², glycerol, (NH₄)₂SO₄, PEG 6000, Triton X-100, and phenylmethylsulfonyl fluoride did not show any effect on activity. Pb⁺², Co⁺², Zn²⁺, ethanol, β-mercaptoethanol, and acetone reduced the activity while Ni²⁺, Cd²⁺, Cu²⁺, iodoacetic acid, SDS, dimethylformamide, DMSO inhibited the activity. In vitro enzyme activity was slightly reduced by acetyl salicylic and acetaminophen and reduced 50% with amino glycosides and ampicillin antibiotics at concentrations of 0.6 and 30 mg ml⁻¹, respectively. This is the first report for the synthesis of cholesterol-conjugated magnetic nanoparticles for simple purification of PON1 enzyme.     

A Thermostable Alkaline Lipase from a Local Isolate Bacillus subtilis EH 37: Characterization, Partial Purification, and Application in Organic Synthesis

A mesophilic bacterial culture producing a novel thermostable alkaline lipase was isolated from oil rich soil sample and identified as Bacillus subtilis EH 37. The lipase was partially purified by ammonium sulfate precipitation and hydrophobic interaction chromatography with 17.8-fold purification and 41.9 U/ml specific activity. The partially purified enzyme exhibited maximum activity at pH 8.0 and at 60 °C. It retained 100% of activity at 50 °C and 60 °C for 60 min. The presence of Ca⁺², Mg⁺², and Zn²⁺ exhibited stimulatory effect on lipase activity, whereas Fe⁺³ and Co⁺² reduced its activity. The enzyme retained more than 80% of its initial activity upon exposure to organic solvents, exhibited 107% and 115% activity in the presence of 15% isopropyl alcohol and 30% n-hexane, respectively. The EH 37 lipase also proved to be an efficient catalyst in synthesis of ethyl caprylate in organic solvent, thus providing a concept of application of B. subtilis lipase in non-aqueous catalysis.     

Purification and partial characterization of a lipase from Bacillus coagulans ZJU318

An extracellular lipase was purified from the fermentation broth of Bacillus coagulans ZJU318 by CM-Sepharose chromatography, followed by Sephacryl S-200 chromatography. The lipase was purified 14.7-fold with 18% recovery and a specific activity of 141.1 U/mg. The molecular weight of the homogeneous enzyme was (32 kDa), determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The enzyme activity was maximum at pH 9.0 and was stable over a pH range of 7.0–10.0, and the optimum temperature for the enzyme reaction was 45°C. Little activity loss (6.2%) was observed after 1 h of incubation at 40°C. However, the stability of the lipase decreased sharply at 50 and 60°C. The enzyme activity was strongly inhibited by Ag⁺ and Cu²⁺, whereas EDTA caused no inhibition. SDS, Brij 30, and Tween-80 inhibited lipase, whereas Triton X-100 did not significantly inhibit lipase activity.     

Characterization of Cyclodextrin Glucanotransferase Produced by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Cyclodextrin glucanotransferase, produced by Bacillus megaterium, was characterized, and the biochemical properties of the purified enzyme were determined. The substrate specificity of the enzyme was tested with different α-1,4-glucans. Cyclodextrin glucanotransferase displayed maximum activity in the case of soluble starch, with a K _m value of 3.4 g/L. The optimal pH and temperature values for the cyclization reaction were 7.2 and 60 °C, respectively. The enzyme was stable at pH 6.0–10.5 and 30 °C. The enzyme activity was activated by Sr²⁺, Mg²⁺, Co²⁺, Mn²⁺, and Cu²⁺, and it was inhibited by Zn²⁺and Ag⁺. The molecular mass of cyclodextrin glucanotransferase was established to be 73,400 Da by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, 68,200 Da by gel chromatography, and 75,000 Da by mass spectrometry. The monomer form of the enzyme was confirmed by the analysis of the N-terminal amino acid sequence. Cyclodextrin glucanotransferase formed all three types of cyclodextrins, but the predominant product was β-cyclodextrin.     

A kinetic study of the displacement reaction between nickel(II) and zinc-ethylenediaminetetraacetate by a radiometric method

A radiometric study of the kinetics of the displacement reaction between nickel(II) and⁶⁵Zn-labeled zinc salt of ethylenediaminetetraacetic acid, which was previously used by the authors for the analysis of trace quantities of nickel, has been carried out under varying conditions of temperature, pH etc. The above reaction was confirmed to be first order with respect to both Ni²⁺ and to^*ZnEDTA. The overall reaction rate constant, k_f, has been shown to be inversely proportional to the concentration of Zn²⁺ and directly proportional to the concentration of H⁺. From the dependence of the rate constant on the concentration of Zn²⁺ and H⁺ a three-step mechanism is proposed for the above reaction. The values for E_a, ΔH^‡, ΔG^‡ and ΔS^‡ for the overall reaction have been computed from the experimental data.     

Purification and Characterization of a Novel Collagenase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> Col-J

The collagenase, produced extracellular by Bacillus pumilus Col-J, was purified by ammonium sulfate precipitation followed by two gel filtrations, involving Sephadex G-100 column and Sepharose Fast Flow column. Purified collagenase has a 31.53-fold increase in specific activity of 87.33 U/mg and 7.00% recovery. The collagenase has a relative molecular weight of 58.64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal temperature for the enzyme reaction was 45 °C. More than 50% of the original activity still remained after 5 min of incubation at 70 °C or 10 min at 60 °C. The maximal enzyme activity of collagenase was obtained at pH 7.5, and it was stable over a pH range of 6.5–8.0. The collagenase activity was strongly inhibited by Mn²⁺, Pb²⁺, ethylenediamine tetraacetic acid, ethylene glycol tetraacetic acid, and β-mercaptoethanol. However, Ca²⁺ and Mg²⁺ greatly increased its activity. The collagenase from B. pumilus Col-J showed highly specific activity towards the native collagen from calf skin. The K _m and V _max of the enzyme for collagen were 0.79 mg/mL and 129.5 U, respectively.     

Purification, characterization, kinetic properties, and thermal behavior of extracellular polygalacturonase produced by filamentous fungus Tetracoccosporium sp

For the first time, a polygalacturonase from the culture broth of Tetracoccosporium sp. was isolated and incubated at 30°C in an orbital shaker at 160 rpm for 48h. The enzyme was purified by ammonium sulfate precipitation and two-step ion-exchange chromatography and had an apparent molecular mass of 36 kDa, as shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Its optimum activity was at pH 4.3 and 40°C, and the K _m and V _max values of this enzyme (for polygalacturonic acid) were 3.23 mg/mL and 0.15 μmol/min, respectively. Ag⁺, Co²⁺, EDTA, Tween-20, Tween-80, and Triton X-100 stimulated polygalacturonase activity whereas Al³⁺, Ba²⁺, Ca²⁺, Fe²⁺, Fe³⁺, Ni²⁺, Mg²⁺, Mn²⁺, and SDS inhibited it. In addition, iodoacetamide and iodoacetic acid did not inhibit enzyme activity at a concentration of 1 mM, indicating that cysteine residues are not part of the catalytic site of polygalacturonase. We studied the kinetic properties and thermal inactivation of polygalacturonase. This enzyme exhibited a t _1/2 of 63 min at 60°C and its specific activity, turnover number, and catalytic efficiency were 6.17 U/mg, 113.64 min⁻¹, and 35.18 mL/(min·mg), respectively. The activation energy (ΔE ^#) for heat inactivation was 5.341 kJ/mol, and the thermodynamic activation parameters ΔG ^#, ΔH ^#, and ΔS ^# were also calculated, revealing a potential application for the industry.     

Purification and properties of cyclodextrinase fromBacillus stearothermophilus HY-1

A thermostable cyclodextrinase (EC 3.2.1.54) fromBacillus stearothermophilus HY-1 was purified to homogeneity by disc-electrophoresis after sonication disruption, ammonium sulfate fractionation, DEAE-cellulose(DE32) column chromatography, hydroxyapatite chromatography, Sephadex G150 gel-filtration, and α-cyclodextrin-AH-Sepharose 4B affinity chromatography. The enzyme was purified 230-fold with 21.2% of activity recovery. The optimal substrates of the enzyme were α-, Β-, and γ-cyclodextrins and linear maltooligosaccharides, and the final product was mainly maltose. The enzyme could hydrolyze pullulan to produce panose. It could also hydrolyze soluble starch, amylose, and amylopectin, but not glycogen. The Km and Vmax for α-, Β-, and γ-cyclodextrins were 1.79, 1.67, and 2.50 mg/mL, and 336, 185, and 208 Μmol/mg/min, respectively. The molecular weight of the enzyme was 61,000 by SDS-gel-electrophoresis. The isoelectric point was pH 5.0. The enzyme was most active at pH 6.2 and 55‡C, and it was strongly inhibited by Cu²⁺, Hg²⁺, Zn²⁺, Pb²⁺, and slightly by Fe²⁺. The effect of some protein modification reagents on the activity of the enzyme suggested that tryptophan and histidine residue(s) may be located at the active site. The amino acid composition of the enzyme was also determined.     

Highly Thermostable Xylanase of the Thermophilic Fungus Talaromyces thermophilus: Purification and Characterization

A thermostable xylanase from a newly isolated thermophilic fungus Talaromyces thermophilus was purified and characterized. The enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl cellulose anion exchange chromatography, P-100 gel filtration, and Mono Q chromatography with a 23-fold increase in specific activity and 17.5% recovery. The molecular weight of the xylanase was estimated to be 25kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and gel filtration. The enzyme was highly active over a wide range of pH from 4.0 to 10.0. The relative activities at pH5.0, 9.0, and 10.0 were about 80%, 85.0%, and 60% of that at pH7.5, respectively. The optimum temperature of the purified enzyme was 75°C. The enzyme showed high thermal stability at 50°C (7days) and the half-life of the xylanase at 100°C was 60min. The enzyme was free from cellulase activity. K _m and V _max values at 50°C of the purified enzyme for birchwood xylan were 22.51mg/ml and 1.235μmol min⁻¹ mg⁻¹, respectively. The enzyme was activated by Ag⁺, Co²⁺, and Cu²⁺; on the other hand, Hg²⁺, Ba²⁺, and Mn²⁺ inhibited the enzyme. The present study is among the first works to examine and describe a secreted, cellulase-free, and highly thermostable xylanase from the T. thermophilus fungus whose application as a pre-bleaching aid is of apparent importance for pulp and paper industries.     

Purification and Characterization of Thermostable α-Galactosidase from <Emphasis Type="Italic">Aspergillus terreus</Emphasis><Subscript>GR</Subscript>

An extracellular thermostable α-galactosidase producing Aspergillus terreus _GR strain was isolated from soil sample using guar gum as sole source of carbon. It was purified to apparent homogeneity by acetone precipitation, gel filtration followed by DEAE-Sephacel chromatographic step. The purified enzyme showed a single band after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme after SDS-PAGE was 108 kDa. The enzyme showed optimum pH and temperature of 5.0 and 65 °C, respectively, for artificial substrate pNPαGal. α-Galactosidase from A. terreus _GR is found to be thermostable, as it was not inactivated after heating at 65 °C for 40 min. The K _m for pNPαGal, oNPαGal, raffinose, and stachyose are 0.1, 0.28, 0.42, and 0.33 mM, respectively. Inhibitors such as 1,10-phenanthroline, phenylmethylsulfonyl fluoride, ethylenediaminetetraacetic acid, mercaptoethanol, and urea have no effect, whereas N-bromosuccinamide inhibited enzyme activity by 100%. Among metal ions tested, Mg²⁺, Ni²⁺, Ca²⁺, Co²⁺, and Mn²⁺ had no effect on enzyme activity, but Ag⁺, Hg²⁺, and Cu²⁺ have inhibited complete activity.     

A Simple Fluorometric Method Using Chlorophyll a for Determination of Hg2+ Ion

A simple and green analytical procedure based on chlorophyll a is presented for the determination of Hg²⁺ ion. Chlorophyll a was extracted and purified from the leaves of pea and is employed as a reagent for analysis of Hg²⁺ ion. It displays remarkable fluorescence emission at 674 nm when excited at 412 nm. The emission intensity decreased significantly on exposure to various concentrations of Hg²⁺ ion. This forms the basis for the determination of Hg²⁺ ion. The proposed method was evaluated for sensitivity and selectivity. The linear concentration range was found to be 2.0–10 μM with r² = 0.997 and the limit of detection for Hg²⁺ ion was 1.3 μM. Ions including Pb²⁺, Cd²⁺, Ag⁺, Zn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Mg²⁺, Mn²⁺, Ru³⁺, Er³⁺, K⁺, Na⁺, NH₄⁺, Cl⁻, NO₃⁻, CH₃COO⁻ and SO₄²⁻ did not interfere with the measurement of Hg²⁺ ion even at 500-fold excess. Since chlorophyll a is widely available in the leaves of most plants, and the extraction and purification process is simple, this technique can provide an alternative, sensitive and economical way to determine Hg²⁺ ion.     

Purification and Characterization of Cell Suspensions Peroxidase from Cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

Two peroxidases, cPOD-I and rPOD-II, have been isolated and purified from cotton cell suspension and their biochemical characteristics studied. rPOD-II from R405-2000, a non-embryogenic cultivar, has higher activity than cPOD-I derived from Coker 312, which developed an embryogenic structure. The cPOD-I and rPOD-II had molecular mass of 39.1 and 64 kDa respectively, as determined by SDS-PAGE. Both enzymes showed high efficiency of interaction with the guaiacol at 25 mM. The optimal pH for cPOD-I and rPOD-II activity was 5.0 and 6.0, respectively. The enzyme had an optimum temperature of 25 °C and was relatively stable at 20–30 °C. The isoenzymes were highly inhibited by ascorbic acid, dithiothreitol, sodium metabisulfite, and β-mercaptoethanol. Their activities were highly enhanced by Al³⁺, Fe³⁺, Ca²⁺, and Ni²⁺, but they were moderately inhibited by Mn²⁺ and K⁺. The enzyme lost 50% to 62% of its activity in the presence of Zn²⁺ and Hg²⁺.     

Isolation and Characterization of a Novel Thermophilic-Organic Solvent Stable Lipase From <Emphasis Type="Italic">Acinetobacter baylyi</Emphasis>

The benzene tolerant Acinetobacter baylyi isolated from marine sludge in Angsila, Thailand could constitutively secrete lipolytic enzymes. The enzyme was successfully purified 21.89-fold to homogeneity by ammonium sulfate precipitation and gel-permeable column chromatography with a relative molecular mass as 30 kDa. The enzyme expressed maximum activity at 60°C and pH 8.0 with p-nitrophenyl palmitate as a substrate and found to be stable in pH and temperature ranging from 6.0-9.0 to 60-80°C, respectively. A study on solvent stability revealed that the enzyme was highly resisted to many organic solvents especially benzene and isoamyl alcohol, but 40% inhibited by decane, hexane, acetonitrile, and short-chain alcohols. Lipase activity was completely inhibited in the presence of Fe²⁺, Mn²⁺, EDTA, SDS, and Triton X-100 while it was suffered detrimentally by Tween 80. The activity was enhanced by phenylmethylsulfonyl fluoride (PMSF), Na⁺, and Mg²⁺ and no significant effect was found in the presence of Ca²⁺ and Li⁺. Half of an activity was retained by Ba²⁺, Ag⁺, Hg⁺, Ni²⁺, Zn²⁺, and DTT. The enzyme could hydrolyze a wide range of p-nitrophenyl esters, but preferentially medium length acyl chains (C₈-C₁₂). Among natural oils and fats, the enzyme 11-folds favorably catalyzed the hydrolysis of rice bran oil, corn oil, sesame oil, and coconut oil in comparison to palm oil. Moreover, the transesterification activity of palm oil to fatty acid methyl esters (FAMEs) revealed 31.64 ± 1.58% after 48 h. The characteristics of novel A. baylyi lipase, as high temperature stability, organic solvent tolerance, and transesterification capacity from palm oil to FAMEs, indicate that it could be a vigorous biocatalyzer in the prospective fields as bioenergy industry or even in organic synthesis and pharmaceutical industry.     

Purification and Characterization of Extracellular Inulinase from a Marine Yeast <Emphasis Type="Italic">Cryptococcus aureus</Emphasis> G7a and Inulin Hydrolysis by the Purified Inulinase

The extracellular inulinase in the supernatant of the cell culture of the marine yeast Cryptococcus aureus G7a was purified to homogeneity with a 7.2-fold increase in specific inulinase activity compared to that in the supernatant by ultrafiltration, concentration, gel filtration chromatography (Sephadex™ G-75), and anion exchange chromatography (DEAE sepharose fast flow anion exchange). The molecular mass of the purified enzyme was estimated to be 60.0 kDa. The optimal pH and temperature of the purified enzyme were 5.0 and 50 °C, respectively. The enzyme was activated by Ca²⁺, K⁺, Na⁺, Fe²⁺, and Zn²⁺. However, Mg²⁺, Hg²⁺, and Ag⁺ acted as inhibitors in decreasing the activity of the purified inulinase. The enzyme was strongly inhibited by phenylmethanesulphonyl fluoride (PMSF), iodoacetic acid, EDTA, and 1,10-phenanthroline. The K _m and V _max values of the purified enzyme for inulin were 20.06 mg/ml and 0.0085 mg/min, respectively. A large amount of monosaccharides were detected after the hydrolysis of inulin with the purified inulinase, indicating the purified inulinase had a high exoinulinase activity.     

Production and properties of 3-cyanopyridine hydratase inRhodococcus equi SHB-121

A strain ofRhodococcus equi SHB-121 forming 3-cyanopyridine hydratase was screened from nitrile-polluted soil. The optimum conditions for the formation of 3-cyanopyridine hydratase by the strain SHB-121 have been studied. Under the optimum conditions, the specific activity of the enzyme reached 5.32 U/mg of dry cell, 95 times higher than that cultured in screening medium. In addition, the activity of coexistent amidase was very low. 3-Cyanopyridine hydratase was purified from methylacrylamide-induced cells ofRh. equi SHB-121 by procedures including ultrasonic oscillation, ammonium sulfate precipitation, and column chromatographies on DEAE-cellulose DE52, hydroxyapatite, and Sephadex G-25. The overall purification was 31-fold. The molecular weight of the enzyme was about 30 kDA by SDS-PAGE. The pI value was 4.1. The transition temperature and pH were 7.0°C and 6.0, respectively, resulting from the differential spectra. The optimum pH and temperature for the enzyme reaction were 8.0 and 30°C. The enzyme activity was strongly inhibited by Ag⁺, Hg²⁺, Cu²⁺, and NH₄ ⁺, whereas it was enhanced by Fe³⁺ slightly. The enzyme catalyzed the hydration of 3-cyanopyridine to nicotinamide, and itsKm value was 0.1 mol/L. Uncompetitive inhibitor sodium cyanide has a K, value of 5 mmol/L.     

Purification and Characterization of a Hyperthermostable and High Maltogenic α-Amylase of an Extreme Thermophile <Emphasis Type="Italic">Geobacillus thermoleovorans</Emphasis>

The purified α-amylase of Geobacillus thermoleovorans had a molecular mass of 26 kDa with a pI of 5.4, and it was optimally active at 100 °C and pH 8.0. The T _1/2 of α-amylase at 100 °C increased from 3.6 to 5.6 h in the presence of cholic acid. The activation energy and temperature quotient (Q ₁₀) of the enzyme were 84.10 kJ/mol and 1.31, respectively. The activity of the enzyme was enhanced strongly by Co²⁺ and Fe²⁺; enhanced slightly by Ba²⁺, Mn²⁺, Ni²⁺, and Mg²⁺; inhibited strongly by Sn²⁺, Hg²⁺, and Pb²⁺, and inhibited slightly by EDTA, phenyl methyl sulfonyl fluoride, N-ethylmaleimide, and dithiothreitol. The enzyme activity was not affected by Ca²⁺ and ethylene glycol-bis (β-amino ethyl ether)-N,N,N,N-tetra acetic acid. Among different additives and detergents, polyethylene glycol 8000 and Tween 20, 40, and 80 stabilized the enzyme activity, whereas Triton X-100, glycerol, glycine, dextrin, and sodium dodecyl sulfate inhibited to a varied extent. α-Amylase exhibited activity on several starch substrates and their derivatives. The K _m and K _cat values (soluble starch) were 1.10 mg/ml and 5.9 × 10³ /min, respectively. The enzyme hydrolyzed raw starch of pearl millet (Pennisetum typhoides) efficiently.     

Glycosidases of turnip leaf tissues

Four myrosinase (β-thioglucosidase EC. 3.2.3.1) and seven disaccharase (β-fructofuranosidase, EC. 3.2.1.26) isoenzymes were isolated from turnip leaves. The most active enzymes were isolated in pure form. Myrosinase and disaccharase mol wt was 62.0 × 10³ and 69.5 × 10³ dalton, respectively, on the basis of gel filtration on Sephadex G-200. Myrosinase pH profile showed high activity between pH 5 and 7 with the optimum at pH 5.5. The purified enzyme was heat-stable for 60 min at 30°C with only loss of 24% of activity. Its activity is strongly inhibited (100%) by Pb²⁺, Ba²⁺, Cu²⁺ and Ca²⁺ ions, and activated (70%) by EDTA at 0.04M. The pure enzyme failed to hydrolyze amylose, glycogen, lactose, maltose, and sucrose. TheK _m andV _max values of myrosinase using sinigrin as specific substrate was 0.045 mM and 2.5 U, respectively. The maximal activity of disaccharase enzyme was obtained at pH 4–5 and 35–37°C. The enzyme was heat-stable at 30°C for 30 min with only 10% loss of its activity. Its activity is strongly activated (70–240%) by Ca²⁺, Ba²⁺, Cu²⁺, and EDTA at 0.01M. The enzyme activity is specific to the disaccharide sucrose and failed to hydrolyze other disaccharides (maltose and lactose). TheK _m andV _max of disaccharase were 0.123 mM and 3.33 U, respectively.     

New ionic liquid for inorganic cations analysis by capillary electrophoresis: 2-hydroxy- N , N , N -trimethyl-1-phenylethanaminium bis(trifluoromethylsulfonyl)imide (phenylcholine NTf2)

The potentialities of new ionic liquids (ILs) based on choline were evaluated as an electrophoretic medium in capillary electrophoresis for the analysis of alkaline and alkaline earth cations (Li⁺, K⁺, Na⁺, Cs⁺, Mg²⁺, Ba²⁺, Ca²⁺, and Sr²⁺) with indirect UV detection. Two types of capillaries were tested: an untreated fused silica and fused silica coated with a film of polyvinylalcohol. The coated capillary proved to be the best adapted for the metal ions studied. Moreover, it appeared that the nature of the ionic liquid anion influenced the baseline stability, and the bis(trifluoromethylsulfonyl) imide (NTf₂ ⁻) anion seemed to be the most efficient. These preliminary studies led us to synthesize a new ionic liquid, 2-hydroxy-N,N,N-trimethyl-1-phenylethanaminium NTf₂ (phenylcholine NTf₂). This liquid was able to act as the running electrolyte and probe, generating the background signal in indirect UV light and consequently simplifying the electrophoretic medium. Excellent baseline stability, good reproducibility, as well as good sensitivity of detection were obtained with this new ionic liquid. Thus, 510,000 plates/meter for Li⁺ with 40 mM IL were successfully obtained. The optimal concentration of IL was 20 mM with a detection limit ranging from 28 μg L⁻¹ for Li⁺ to 1,000 μg L⁻¹ for Cs⁺. This method (phenylcholine NTf₂ with polyvinylalcohol capillary) was applied to analyze different commercial source and mineral waters. Finally, the potentiality of this ionic liquid in nonaqueous capillary electrophoresis was explored. The use of phenylcholine NTf₂ with a fused silica capillary, in pure methanol medium and in the presence of acetic acid, made it possible to obtain separation selectivity different from that obtained in aqueous medium.     

An Oxidant- and Organic Solvent-Resistant Alkaline Metalloprotease from Streptomyces olivochromogenes

Organic solvent- and detergent-resistant proteases are important from an industrial viewpoint. However, they have been less frequently reported and only few of them are from actinomycetes. A metalloprotease from Streptomyces olivochromogenes (SOMP) was purified by ion exchange with Poros HQ and gel filtration with Sepharose CL-6B. Apparent molecular mass of the enzyme was estimated to be 51 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and gelatin zymography. The activity was optimum at pH 7.5 and 50 °C and stable between pH 7.0 and 10.0. SOMP was stable below 45 °C and Ca²⁺ increased its thermostability. Ca²⁺ enhanced while Co²⁺, Cu²⁺, Zn²⁺, Mn²⁺, and Fe²⁺ inhibited the activity. Ethylenediaminetetraacetic acid and ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, but not phenylmethylsulfonyl fluoride, aprotinin, and pefabloc SC, significantly suppressed the activity, suggesting that it might be a metalloprotease. Importantly, it is highly resistant against various detergents, organic solvents, and oxidizing agents, and the activity is enhanced by H₂O₂. The enzyme could be a novel protease based on its origin and peculiar biochemical properties. It may be useful in biotechnological applications especially for organic solvent-based enzymatic synthesis.     

Dissociation kinetics of singly protonated leucine enkephalin investigated by time-resolved photodissociation tandem mass spectrometry

The yields of post-source decay (PSD) and time-resolved photodissociation (PD) at 193 and 266 nm were measured for singly protonated leucine enkephalin ([YGGFL + H]⁺), a benchmark in the study of peptide ion dissociation, by using tandem time-of-flight mass spectrometry. The peptide ion was generated by matrix-assisted laser desorption ionization (MALDI) using 2,5-dihydroxybenzoic acid as the matrix. The critical energy (E₀) and entropy (ΔS‡ at 1000 K) for the dissociation were determined by Rice-Ramsperger-Kassel-Marcus fit of the experimental data. MALDI was done for a mixture of YGGFL and Y₆ and the plume temperature determined by the kinetic analysis of [Y₆ + H]⁺ data were used to improve the precision of E₀ and ΔS‡ for [YGGFL + H]⁺. E₀ and ΔS‡ thus determined (E₀ = 0.67 ± 0.08 eV, ΔS‡=−24.4 ± 3.2 eu with 1 eu = 4.184 J K⁻¹mol⁻¹) were significantly different from those determined by blackbody infrared radiative dissociation (BIRD) (E₀ = 1.10 eV, ΔS‡ = −14.9 eu), and by surface-induced dissociation (SID) (E₀ = 1.13 eV, ΔS‡ = −10.3 eu). Analysis of the present experimental data with the SID kinetics (and BIRD kinetics also) led to an unrealistic situation where not only PSD and PD but also MALDI-TOF signals could not be detected. As an explanation for the discrepancy, it was suggested that transition-state switching occurs from an energy bottleneck (SID/BIRD) to an entropy bottleneck (PSD/PD) as the internal energy increases.