A New <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> Strain Producing Laccase. Use in Decolorization of Synthetics Dyes

Laccase activity was detected in a soil bacterium Stenotrophomonas maltophilia AAP56 identified by biochemical and molecular methods. It was produced in cells at the stationary growth phase in Luria Bertani (LB) medium added by 0.4 mM copper sulfate. The addition of CuSO₄ in culture medium improved production of laccase activity. However, one laccase enzyme was detected by native polyacrylamide gel electrophoresis. The enzyme showed syringaldazine (K _m = 53 μM), 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (K _m = 700 μM), and pyrocatechol (K _m = 25 μM) oxidase activity and was activated by addition of 0.1% (v/v) Triton-X-100 in the reaction mixture. Moreover, the laccase activity was increased 2.6-fold by the addition of 10 mM copper sulfate; the enzyme was totally inhibited by ethylenediaminetetraacetic acid (5 mM), suggesting that this laccase is a metal-dependant one. Decolorization activity of some synthetic dyes (methylene blue, methyl green, toluidine blue, Congo red, methyl orange, and pink) and the industrial effluent (SITEX Black) was achieved by the bacteria S. maltophilia AAP56 in the LB growth medium under shaking conditions.     

The Correlations Between TCA-Glyoxalate Metabolite and Antibiotic Production of <Emphasis Type="Italic">Streptomyces</Emphasis> sp. M4018 Grown in Glycerol,Glucose, and Starch Mediums

The alterations of organic acids citrate, α-ketoglutarate, succinate, fumarate, malate production together with isocitrate lyase activity as a glyoxalate shunt enzyme, and antibiotic production of Streptomyces sp M4018 were investigated in relation to changes in the glucose, glycerol and starch concentrations (5–20 g/L) after identification as a strain of Streptomyces hiroshimensis based on phenotypic and genotypic characteristics. The highest intracellular citrate and α-ketoglutarate levels in 20 g/l of glucose, glycerol, and starch mediums were 399.47 ± 4.78, 426.93 ± 6.40, 355.84 ± 5.38 ppm and 444.81 ± 5.12, 192.96 ± 2.26, 115.20 ± 2.87 ppm, respectively. The highest succinate, malate, and fumarate levels were also determined in 20 g/l of glucose medium as 548.9 ± 11.21, 596.15 ± 8.26, and 406.42 ± 6.59 ppm and the levels were significantly higher than the levels in glycerol and starch. Extracellular organic acid levels measured also showed significant correlation with carbon source concentrations by showing negative correlation with pH levels of the growth medium. The antibiotic production of Streptomyces sp. M4018 was also higher in glucose medium as was the case also for organic acids when compared with glycerol. On the other hand, there is no production in starch.     

Gene Cloning,Overexpression, and Characterization of a Xylanase from <Emphasis Type="Italic">Penicillium</Emphasis> sp. CGMCC 1669

A xylanase-encoding gene, xyn11F63, was isolated from Penicillium sp. F63 CGMCC1669 using degenerated polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR techniques. The full-length chromosomal gene consists of 724 bp, including a 73-bp intron, and encodes a 217 amino acid polypeptide. The deduced amino acid sequence of xyn11F63 shows the highest identity of 70% to the xylanase from Penicillium sp. strain 40, which belongs to glycosyl hydrolases family 11. The gene was overexpressed in Pichia pastoris, and its activity in the culture medium reached 516 U ml⁻¹. After purification to electrophoretic homogeneity, the enzyme showed maximal activity at pH 4.5 and 40°C, was stable at acidic buffers of pH 4.5–9.0, and was resistant to proteases (proteinase K, trypsin, subtilisin A, and α-chymotrypsin). The specific activity, K _m, and V _max for oat spelt xylan substrate was 7,988 U mg⁻¹, 22.2 mg ml⁻¹, and 15,105.7 μmol min⁻¹ mg⁻¹, respectively. These properties make XYN11F63 a potential economical candidate for use in feed and food industrial applications.     

Isolation of Phosphate-Solubilizing Fungi from Phosphate Mines and Their Effect on Wheat Seedling Growth

Three phosphate-solubilizing fungi, identified as Penicillium expansum, Mucor ramosissimus, and Candida krissii, were isolated from phosphate mines (Hubei, People’s Republic of China) and characterized. All the isolates demonstrated diverse levels of phosphate-solubilizing capability in National Botanical Research Institute’s phosphate growth medium containing rock phosphate as sole phosphate source. Acidification of culture medium seemed to be the main mechanism for rock phosphate solubilization. Indeed, citric acid, oxalic acid, and gluconic acid were shown to be present in the culture medium inoculated with these isolates. Moreover, the isolates produced acid and alkaline phosphatases in culture medium, which may also be helpful for RP solubilization. A strong negative correlation between content of soluble phosphorus and pH (r = – 0.89; p < 0.01) in culture medium was observed in this study. All the isolates promoted growth, soil available phosphorus, phosphorus, and nitrogen uptake of wheat seedling in field soil containing rock phosphate under pot culture conditions, thus demonstrating the capability of these isolates to convert insoluble form of phosphorus into plant available form from rock phosphate, and therefore hold great potential for development as biofertilizers to enhance soil fertility and promote plant growth.     

A Process to Produce Penicillin G Acylase by Surface-Adhesion Fermentation Using <Emphasis Type="Italic">Mucor griseocyanus</Emphasis> to Obtain 6-Aminopenicillanic Acid by Penicillin G Hydrolysis

The production of extracellular and mycelia-associated penicillin G acylase (maPGA) with Mucor griseocyanus H/55.1.1 by surface-adhesion fermentation using Opuntia imbricata, a cactus, as a natural immobilization support was studied. Enzyme activity to form 6-aminopencillanic acid (6-APA) from penicillin G was assayed spectrophotometrically. The penicillin G hydrolysis to 6-APA was evaluated at six different times using PGA samples recovered from the skim milk medium at five different incubation times. Additionally, the effect of varying the penicillin G substrate concentration level on the PGA enzyme activity was also studied. The maximum reaction rate, V _max, and the Michaelis constant, K _M, were determined using the Michaelis–Menten model. The maximum levels for maPGA and extracellular activity were found to be 2,126.50 international unit per liter (IU/l; equal to 997.83 IU/g of support) at 48 h and 755.33 IU/l at 60 h, respectively. Kinetics of biomass production for total biomass showed a maximum growth at 60 h of 3.36 and 2.55 g/l (equal to 0.012 g of biomass per gram of support) for the immobilized M. griseocyanus biomass. The maPGA was employed for the hydrolysis of penicillin G to obtain 6-APA in a batch reactor. The highest quantity of 6-APA obtained was 226.16 mg/l after 40-min reaction. The effect of substrate concentration on maPGA activity was evaluated at different concentrations of penicillin G (0–10 mM). K _M and V _max were determined to be 3.0 × 10⁻³ M and 4.4 × 10⁻³ mM/min, respectively.     

Stimulated Production of Triterpenoids of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> by an Ether Extract from the Medicinal Insect, <Emphasis Type="Italic">Catharsius molossus</Emphasis>, and Identification of the Key Stimulating Active Components

The medicinal fungus Ganoderma lucidum was inoculated into the media with and without supplementation of medicinal insect extracts to screen stimulators from Chinese medicinal insects for mycelial growth and triterpenoids production in submerged fermentation. The methanol and ether extracts of the tested insects had no significant stimulatory effect on the mycelial biomass production (P > 0.05), and those of H. remigator and Mylabris phalerata markedly inhibited the mycelial growth. However, the ether extract of Catharsius molossus at a concentration of 200 mg l⁻¹ led to a significant increase in triterpenoids concentration from 231.7 ± 9.77 to 313.7 ± 10.6 mg l⁻¹ (P < 0.01). Analysis of fermentation kinetics of G. lucidum suggests that glucose concentration in the extract of C. molossus-added group decreased more quickly as compared to the control group from day 2 to day 7 of fermentation process, while the triterpenoids biosynthesis was promoted at the same culture period. However, the culture pH profile was not affected by the addition of the extract. Chemical study of the extract show that cis-9,10-methylenehexadecanoic acid (9,10-MEA) and hexadecanoic acid (especially 9,10-MEA) were the key active compounds of the extract responsible for the stimulatory effect on the triterpenoids production.     

Cathodoluminescence Depth Profiling in SiO2:Ge Layers

 For investigation of the luminescent center profile cathodoluminescence measurements are used under variation of the primary electron energy E ₀ = 2…30 keV. Applying a constant incident power regime (E ₀·I ₀ = const), the depth profiles of luminescent centers are deduced from the range of the electron energy transfer profiles dE/dx. Thermally grown SiO₂ layers of thickness d = 500 nm have been implanted by Ge⁺-ions of energy 350 keV and doses (0.5–5)10¹⁶ ions/cm². Thus Ge profiles with a concentration maximum of (0.4 – 4) at% at the depth of d_m≅240 nm are expected. Afterwards the layers have been partially annealed up to T _a = 1100 °C for one hour in dry nitrogen. After thermal annealing, not only the typical violet luminescence (λ = 400 nm) of the Ge centers is strongly increased but also the luminescent center profiles are shifted from about 250 nm to 170 nm depth towards the surface. This process should be described by Ge diffusion processes, precipitation and finally Ge nanocluster formation. Additionally, a Ge surface layer is piled-up extending to a depth of roughly 25 nm.     

Probing of ascorbic acid by CdS/dendrimer nanocomposites: a spectroscopic investigation

The gamma irradiation method has provided a route for synthesis of highly water-soluble, good-quality luminescent CdS/dendrimer nanocomposites with amino- or carboxyl-terminated PAMAM dendrimer. An attempt has been made to probe ascorbic acid with the as-synthesized CdS/dendrimer nanocomposites (DNC). Ascorbic acid (AA) is an important biological antioxidant and marker for different diseases in clinical chemistry as well as in quality control in the food industry. Micromolar concentrations of AA significantly quenched the photoluminescence (PL) of both amino (–NH₂) and carboxylic (–COOH) functionalized semiconductor nanocomposites. The quenching followed a linear Stern–Volmer equation and time-resolved photoluminescence spectroscopy confirmed its static nature. A strong size dependence of the quenching pattern was observed. The binding constants, and the corresponding thermodynamic parameters ΔG ^θ, ΔH ^θ, ΔS ^θ at different temperatures were calculated. CdS DNC showed selectivity towards ascorbic acid even in the presence of possible interfering molecules, such as uric acid, tartaric acid and citric acid. Nanocomposites-based assay techniques could override the complications involved in multitudes of assay procedures, providing a simple and fast new strategy for the quantification of Ascorbic acid in the range of 16.6 to 100 μM (R = 0.998, n = 9). The proposed method was applied to the detection of ascorbic acid in Vitamin C tablets with satisfactory results.     

Xylanase Production by <Emphasis Type="Italic">Penicillium canescens</Emphasis> on Soya Oil Cake in Solid-State Fermentation

There is an increasing interest for the organic residues from various sectors of agriculture and industries over the past few decades. Their application in the field of fermentation technology has resulted in the production of bulk chemicals and value-added products such as amino acid, enzymes, mushroom, organic acids, single-cell protein, biologically active secondary metabolites, etc. (Ramachandran et al., Bioresource Technology 98:2000–2009, 2007). In this work, the production of extracellular xylanase by the fungus Penicillium canescens was investigated in solid-state fermentation using five agro-industrial substrates (soya oil cake, soya meal, wheat bran, whole wheat bran, and pulp beet). The best substrate was the soya oil cake. In order to optimize the production, the most effective cultivation conditions were investigated in Erlenmeyer flasks and in plastic bags with 5 and 100 g of soya oil cake, respectively. The initial moisture content, initial pH, and temperature of the culture affected the xylanase synthesis. The optimal fermentation medium was composed by soya oil cake crushed to 5 mm supplemented with 3% and 4% (w/w) of casein peptone and Na₂HPO₄.2H₂O. After 7 days of incubation at 30 °C and under 80% of initial moisture, a xylanase production level of 18,895 ± 778 U/g (Erlenmeyer flasks) and 9,300 ± 589 U/g (plastic bags) was reached. The partially purified enzyme recovered by ammonium sulfate fractionation was completely stable at freezing and refrigeration temperatures up to 6 months and reasonably stable at room temperature for more than 3 months.     

Keratinase Production by Endophytic <Emphasis Type="Italic">Penicillium</Emphasis> spp. Morsy1 Under Solid-State Fermentation Using Rice Straw

Among all endophytic keratinolytic fungal isolates recovered from marine soft coral Dendronephthya hemprichii, Penicillium spp. Morsy1 was selected as the hyperactive keratinolytic strain under solid substrate fermentation of different agriculture and poultry wastes. The optimization of extraction process, physicochemical parameters affecting the keratinase production in solid-state fermentation, and the purified keratinase parameters were studied. Maximum keratinase activity (1,600 U g⁻¹, initial dry substrate) was recovered from moldy bran with 0.1% Tween 80. The optimized production conditions were rice straw as carbon source, pH of medium 6, growth temperature 26 °C, initial moisture content of 80% (v/w), inoculum size of 10⁵ spores ml⁻¹, and an average particle size of the substrate 0.6 mm (3,560 U g⁻¹, initial dry substrate after 5 days of fermentation). Two types of keratinase (Ahm1 and Ahm2) were purified from the culture supernatant through ammonium sulfate precipitation, DEAE-Sepharose, and gel filtration chromatography. Enzyme molecular weights were 19 kDa (Ahm1) and 40 kDa (Ahm2). The kinetic parameters of purified keratinases were optimized for the hydrolysis of azokeratin by Ahm1 (pH 7.0–8.0, stable in pH range of 6.0 to 8.0 at 50 °C) and Ahm2 enzymes (pH 10.0–11.0, stable in pH range of 6.0 to 11.0 at 60–65 °C). Whereas inhibitors of serine (phenylmethylsulfonyl fluoride) and cysteine (iodoacetamide) proteases had minor effects on both Ahm1 and Ahm2 activity, both keratinases were strongly inhibited by chelating agents EDTA and EGTA. These findings suggest that serine and cysteine residues are not involved in the catalytic mechanisms, and they are metalloproteases.     

Cage Structure Formation of Singly Doped Aluminum Cluster Cations Al<Subscript><Emphasis Type="BoldItalic">n</Emphasis></Subscript><Emphasis Type="BoldItalic">TM</Emphasis><Superscript>+</Superscript> (<Emphasis Type="BoldItalic">TM</Emphasis> = Ti,V, Cr)

Structural information on free transition metal doped aluminum clusters, Al _n TM ⁺ (TM = Ti, V, Cr), was obtained by studying their ability for argon physisorption. Systematic size (n = 5 – 35) and temperature (T = 145 – 300 K) dependent investigations reveal that bare Al _n ⁺ clusters are inert toward argon, while Al _n TM ⁺ clusters attach one argon atom up to a critical cluster size. This size is interpreted as the geometrical transition from surface-located dopant atoms to endohedrally doped aluminum clusters with the transition metal atom residing in an aluminum cage. The critical size, n _crit , is found to be surprisingly large, namely n _crit = 16 and n _crit = 19 – 21 for TM = V, Cr, and TM = Ti, respectively. Experimental cluster–argon bond dissociation energies have been derived as function of cluster size from equilibrium mass spectra and are in the 0.1–0.3 eV range.     

Production of Lipid and Fatty Acids during Growth of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> on Hydrocarbon Substrates

An Aspergillus terreus, isolated from oil contaminated soil, could degrade a wide range of petroleum hydrocarbons including the immediate oxidation products of hydrocarbons, like alkanols and alkanals. Among all the linear chain carbon substrates, highest growth of 39.1 ± 3.8 g l⁻¹ (wet weight) was observed when n-hexadecane was used as the sole source of carbon. The growth of the fungus on this highly hydrophobic substrate was associated with the morphological change of the hyphae and increase production of lipid in the cells. The lipid production in the hydrocarbon (n-hexadecane) grown cells was sevenfold higher than the corresponding glucose grown cells. The fatty acid profile of the lipid content formed in the hydrocarbon grown cells was significantly different from the glucose grown cells and was composed of fatty acids with chain length C₁₄ to C₃₃ as revealed from the liquid chromatography electrospray ionization mass spectrometry analyses. Among the ranges, the fatty acids with chain lengths C₁₄ to C₁₈ were predominant in the profile. Considering the fatty acid profile and the high level of lipid production, this A. terreus mediated production of lipid is envisaged to have potential application in the oleochemical industries including the production of biodiesel.     

The electrochemical polymerization of <Emphasis Type="Italic">o</Emphasis>-phenylenediamine on <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine functionalized glassy carbon electrode and its application

The polymerization of o-phenylenediamine (OPD) on l-tyrosine (Tyr) functionalized glassy carbon electrode (GCE) and its electro-catalytic oxidation towards ascorbic acid (AA) had been studied in this report. l-Tyrosine was first covalently grafted on GCE surface via electrochemical oxidation, which was followed by the electrochemical polymerization of OPD on the l-tyrosine functionalized GCE. Then, the poly(o-phenylenediamine)/l-tyrosine composite film modified GCE (POPD-Tyr/GCE) was obtained. X-ray photo-electron spectroscopy (XPS), field emission scanning electron microscope (SEM), and electrochemical techniques have been used to characterize the grafting of l-tyrosine and the polymerization and morphology of OPD film on GCE surface. Due to the doping of the carboxylic functionalities in l-tyrosine molecules, the POPD film showed good redox activity in neutral medium, and thus, the POPD-Tyr/GCE exhibited excellent electrocatalytic response to AA in 0.1 mol l⁻¹ phosphate buffer solution (PBS, pH 6.8). The anode peak potential of AA shifted from 0.58 V at GCE to 0.35 V at POPD-Tyr/GCE with a greatly enhanced current response. A linear calibration graph was obtained over the AA concentration range of 2.5 × 10⁻⁴–1.5 × 10^–3 mol l⁻¹ with a correlation coefficient of 0.9998. The detection limit (3δ) for AA was 9.2 × 10⁻⁵ mol l⁻¹. The modified electrode showed good stability and reproducibility and had been used for the determination of AA content in vitamin C tablet with satisfactory results.     

Simultaneous determination of harpagoside and cinnamic acid in rat plasma by high-performance liquid chromatography: application to a pharmacokinetic study

Radix Scrophulariae (Xuanshen) is one of the famous Chinese herbal medicines widely used to treat rheumatism, tussis, pharyngalgia, arthritis, constipation, and conjunctival congestion. Harpagoside and cinnamic acid are the main bioactive components of Xuanshen. The purpose of this study was to develop an HPLC–UV method for simultaneous determination of harpagoside and cinnamic acid in rat plasma and investigate pharmacokinetic parameters of harpagoside and cinnamic acid after oral administration of Xuanshen extract (760 mg kg⁻¹). After addition of syringin as internal standard, the analytes were isolated from plasma by liquid–liquid extraction. Separation was achieved on a Kromasil C₁₈ column, and detection was by UV absorption at 272 nm. The described assay was validated in terms of linearity, accuracy, precision, recovery, and limit of quantification according to the FDA validation guidelines. Calibration curves for both analytes were linear with the coefficient of variation (r) for both was greater than 0.999. Accuracy for harpagoside and cinnamic acid ranged from 100.7–103.5% and 96.9–102.9%, respectively, and precision for both analytes were less than 8.5%. The main pharmacokinetic parameters found for harpagoside and cinnamic acid after oral infusion of Xuanshen extract were as follows: C _max 1488.7 ± 205.9 and 556.8 ± 94.2 ng mL⁻¹, T _max 2.09 ± 0.31 and (1.48 ± 0.14 h, AUC_0–24 10336.4 ± 1426.8 and 3653.1 ± 456.4 ng h mL⁻¹, 11276.8 ± 1321.4 and 3704.5 ± 398.8 ng h mL⁻¹, and t _1/2 4.9 ± 1.3 and 2.5 ± 0.9 h, respectively. These results indicated that the proposed method is simple, selective, and feasible for pharmacokinetic study of Radix Scrophulariae extract in rats. Figure Radix Scrophulariae     

The Comparison of Lutein Production by <Emphasis Type="Italic">Scenesdesmus</Emphasis> sp. in the Autotrophic and the Mixotrophic Cultivation

The results of this study indicate that an increase in CO₂ percentage to 30% can enhance Scenedesmus sp. growth in autotrophic cultivation to a maximum of 0.85 g/l as compared with 0.6 g/l obtained in the batch with air (after 6 days of cultivation). However, while the CO₂ was higher than 30%, it showed a negative impact on cell growth. A mixotrophic cultivation with 3 g/l of glycerol can achieve 0.38 g l⁻¹ day⁻¹ of the maximum biomass productivity compared with that of 0.21 g l⁻¹ day⁻¹ in autotrophic cultivation. Nevertheless, the lutein content of the mixotrophic cultivation was 0.08–0.1% lower than 0.2–0.25% obtained in autotrophic cultivation, which led to a lower lutein productivity of 0.36 mg l⁻¹ day⁻¹ in the mixotrophic batch compared with 0.44 mg l⁻¹ day⁻¹ obtained in the autotrophic batch. The limitation of cell growth in the mixotrophic cultivation would be the contributing factor regarding the lower lutein productivity. The mixotrophic cultivation of repeated batch to remove potential inhibitive metabolic products from glycerol catabolism does not show an obvious improvement on biomass. Conclusively, mixotrophic cultivation achieves higher biomass productivity with lower lutein content than that of autotrophic cultivation, which leads to lower lutein productivity. Therefore, the autotrophic cultivation is preferred in the lutein production.     

Comparison between Different Hydrolysis Processes of Vine-Trimming Waste to Obtain Hemicellulosic Sugars for Further Lactic Acid Conversion

Trimming vine shoot samples were treated with water under selected operational conditions (autohydrolysis reaction) to obtain a liquid phase containing hemicellulose-decomposition products. In a further acid-catalyzed step (posthydrolysis reaction), xylooligosaccharides were converted into single sugars for the biotechnological production of lactic acid using Lactobacillus pentosus. A wide range of temperatures, reaction times, and acid concentrations were tested during the autohydrolysis–posthydrolysis process to investigate their influence on hemicellulose solubilization and reaction products. The maximum concentration of hemicellulosic sugars was achieved using autohydrolysis at 210 °C followed by posthydrolysis with 1% H₂SO₄ during 2 h. Data from autohydrolysis–posthydrolysis were compared with the results obtained at the optima conditions assayed for prehydrolysis (3% H₂SO₄ at 130 °C during 15 min) based on previous works. Prehydrolysis extracted more hemicellulosic sugars from trimming vine shoots; however, the protein content in the hydrolysates from autohydrolysis–posthydrolysis was higher. The harsher conditions assayed during the autohydrolysis process and the higher content of protein after this treatment could induce Maillard reactions decreasing consequently the concentration of hemicellulosic sugars in the hydrolysates. Therefore, despite the several advantages of autohydrolysis (less equipment caused by the absence of mineral acid, less generation of neutralized sludges, and low cost of reagents) the poor results obtained in this work with no detoxified hydrolysates (Q _P = 0.36 g/L h, Q _S = 0.79 g/L h, Y _P/S = 0.45 g/g, Y _P/Sth = 61.5 %) or charcoal-treated hydrolysates (Q _P = 0.76 g/L h, Q _S = 1.47 g/L h, Y _P/S = 0.52 g/g, Y _P/Sth = 71.5 %) suggest that prehydrolysis of trimming vine shoots with diluted H₂SO₄ is more attractive than autohydrolysis-posthydrolysis for obtaining lactic acid through fermentation of hemicellulosic sugars with L. pentosus. Besides the higher hemicellulosic sugars concentration achieved when using the prehydrolysis technology, no detoxification steps are required to produce efficiently lactic acid (Q _P = 1.14 g/L h; Q _S = 1.64 g/L h; Y _P/S = 0.70 g/g; Y _P/Sth = 92.6 %), even when vinification lees are used as nutrients (Q _P = 0.89 g/L h; Q _S = 1.54 g/L h; Y _P/S = 0.58 g/g; Y _P/Sth = 76.1 %).     

Enhancement of Human γ-Interferon Production in Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Using Batch Cultivation

Development of inexpensive and simple culture media and appropriate induction conditions are always favorable for industry. In this research, chemical composition and stoichiometric data for γ-interferon production and recombinant Escherichia coli growth were used in order to achieve a simple medium and favorable induction conditions. To achieve this goal, the effects of medium composition and induction conditions on the production of γ-interferon were investigated in batch culture of E. coli BL21 (DE3) [pET3a-ifnγ]. These conditions were considered as suitable conditions for the production of γ-interferon: 2.5× M9 medium, supplemented with a mixture of amino acids (milligram per liter), including glutamic acid 215, aspartic acid 250, lysine 160, and phenylalanine 90, and induction at late-log phase (OD₆₀₀ = 4.5). Under these conditions, dry cell weight of 6 ± 0.2 g/l and γ-interferon concentration of 2.15 ± 0.1 g/l were obtained. Later, without changing the concentration ratio of amino acids and glucose, the effect of increase in the primary glucose concentration on productivity of γ-interferon was investigated. It was found that 25 g/l glucose will result in maximum attainable biomass and recombinant human γ-interferon. At improved conditions, a dry cell weight of 14 ± 0.2 g/l, concentration and overall productivity of γ-interferon 4.2 ± 0.1 g/l and 420 ± 10 mg/l h, respectively, were obtained.     

Determination of the primary structure and carboxyl p<Emphasis Type="Italic">K</Emphasis><Subscript>A</Subscript>s of heparin-derived oligosaccharides by band-selective homonuclear-decoupled two-dimensional <Superscript>1</Superscript>H NMR

Determination of the structure of heparin-derived oligosaccharides by ¹H NMR is challenging because resonances for all but the anomeric protons cover less than 2 ppm. By taking advantage of increased dispersion of resonances for the anomeric H¹ protons at low pD and the superior resolution of band-selective, homonuclear-decoupled (BASHD) two-dimensional ¹H NMR, the primary structure of the heparin-derived octasaccharide ∆UA(2S)-[(1 → 4)-GlcNS(6S)-(1 → 4)-IdoA(2S)-]₃-(1 → 4)-GlcNS(6S) has been determined, where ∆UA(2S) is 2-O-sulfated ∆^4,5-unsaturated uronic acid, GlcNS(6S) is 6-O-sulfated, N-sulfated β-d-glucosamine and IdoA(2S) is 2-O-sulfated α-l-iduronic acid. The spectrum was assigned, and the sites of N- and O-sulfation and the conformation of each uronic acid residue were established, with chemical shift data obtained from BASHD-TOCSY spectra, while the sequence of the monosaccharide residues in the octasaccharide was determined from inter-residue NOEs in BASHD-NOESY spectra. Acid dissociation constants were determined for each carboxylic acid group of the octasaccharide, as well as for related tetra- and hexasaccharides, from chemical shift–pD titration curves. Chemical shift–pD titration curves were obtained for each carboxylic acid group from sub-spectra taken from BASHD-TOCSY spectra that were measured as a function of pD. The pK _As of the carboxylic acid groups of the ∆UA(2S) residues are less than those of the IdoA(2S) residues, and the pK _As of the carboxylic acid groups of the IdoA(2S) residues for a given oligosaccharide are similar in magnitude. Relative acidities of the carboxylic acid groups of each oligosaccharide were calculated from chemical shift data by a pH-independent method.     

A rapid and universal bacteria-counting approach using CdSe/ZnS/SiO2 composite nanoparticles as fluorescence probe

In this paper, a rapid, simple, and sensitive method was described for detection of the total bacterial count using SiO₂-coated CdSe/ZnS quantum dots (QDs) as a fluorescence marker that covalently coupled with bacteria using glutaraldehyde as the crosslinker. Highly luminescent CdSe/ZnS were prepared by applying cadmium oxide and zinc stearate as precursors instead of pyrophoric organometallic precursors. A reverse-microemulsion technique was used to synthesize CdSe/ZnS/SiO₂ composite nanoparticles with a SiO₂ surface coating. Our results showed that CdSe/ZnS/SiO₂ composite nanoparticles prepared with this method possessed highly luminescent, biologically functional, and monodispersive characteristics, and could successfully be covalently conjugated with the bacteria. As a demonstration, it was found that the method had higher sensitivity and could count bacteria in 3 × 10² CFU/mL, lower than the conventional plate counting and organic dye-based method. A linear relationship of the fluorescence peak intensity (Y) and the total bacterial count (X) was established in the range of 3 × 10²–10⁷ CFU/mL using the equation Y = 374.82X − 938.27 (R = 0.99574). The results of the determination for the total count of bacteria in seven real samples were identical with the conventional plate count method, and the standard deviation was satisfactory.     

LC–MS–MS determination of ibuprofen, 2-hydroxyibuprofen enantiomers,and carboxyibuprofen stereoisomers for application in biotransformation studies employing endophytic fungi

The purpose of this study was the development and validation of an LC–MS–MS method for simultaneous analysis of ibuprofen (IBP), 2-hydroxyibuprofen (2-OH-IBP) enantiomers, and carboxyibuprofen (COOH-IBP) stereoisomers in fungi culture medium, to investigate the ability of some endophytic fungi to biotransform the chiral drug IBP into its metabolites. Resolution of IBP and the stereoisomers of its main metabolites was achieved by use of a Chiralpak AS-H column (150 × 4.6 mm, 5 μm particle size), column temperature 8 °C, and the mobile phase hexane–isopropanol–trifluoroacetic acid (95: 5: 0.1, v/v) at a flow rate of 1.2 mL min⁻¹. Post-column infusion with 10 mmol L⁻¹ ammonium acetate in methanol at a flow rate of 0.3 mL min⁻¹ was performed to enhance MS detection (positive electrospray ionization). Liquid–liquid extraction was used for sample preparation with hexane–ethyl acetate (1:1, v/v) as extraction solvent. Linearity was obtained in the range 0.1–20 μg mL⁻¹ for IBP, 0.05–7.5 μg mL⁻¹ for each 2-OH-IBP enantiomer, and 0.025–5.0 μg mL⁻¹ for each COOH-IBP stereoisomer (r ≥ 0.99). The coefficients of variation and relative errors obtained in precision and accuracy studies (within-day and between-day) were below 15%. The stability studies showed that the samples were stable (p > 0.05) during freeze and thaw cycles, short-term exposure to room temperature, storage at −20 °C, and biotransformation conditions. Among the six fungi studied, only the strains Nigrospora sphaerica (SS67) and Chaetomium globosum (VR10) biotransformed IBP enantioselectively, with greater formation of the metabolite (+)-(S)-2-OH-IBP. Formation of the COOH-IBP stereoisomers, which involves hydroxylation at C3 and further oxidation to form the carboxyl group, was not observed.