Increased synthesis of ajmalicine and catharanthine by cell suspension cultures ofCatharanthus roseus in response to fungal culture-filtrates

The ammonium sulfate-precipitated fraction from mycelia and culture-filtrates and the crude, cell-free culture filtrates from the growth medium of the fungiChrysosporium palmorum, Eurotium rubrum, Micromucor isabellina, andPythium aphanidermatum when aseptically added to cell suspensions ofCantharanthus roseus caused a rapid and dramatic increase in indole alkaloid biosynthesis. Up to 400 μg/L ajmalicine and 600 μg/L catharanthine were detected in C.roseus cell suspension grown in the presence of theM. isabellina fungal culture filtrate for 3 d. Untreated cells produced only trace levels of ajmalicine and catharanthine per liter of cell suspension after 15 d of culture.     

Synthesis and study of a molecularly imprinted polymer for the specific extraction of indole alkaloids from Catharanthus roseus extracts

Two molecularly imprinted polymers (MIP) for catharanthine and vindoline have been synthesized in order to specifically extract these natural indole alkaloids from Catharanthus roseus by solid-phase extraction (SPE). Each MIP was prepared by thermal polymerisation using catharanthine (or vindoline) as template, methacrylic acid (or itaconic acid) as functional monomer, ethylene glycol dimethacrylate (EDMA) as cross-linking agent and acetonitrile (or acetone) as porogenic solvent.For catharanthine-MIP, a SPE protocol (ACN–AcOH 99/1 washing and MeOH–AcOH 90/10 elution) allows a good MIP/NIP selectivity (imprinting factor 12.6). The specificity of catharanthine-MIP versus related bisindole alkaloids was assessed by cross-reactivity study. The catharanthine-MIP specifically retained catharanthine and its N-oxide analogue but displayed a weak cross-reactivity for other Vinca alkaloids (vinorelbine, vincristine, vinblastine, vindoline, vinflunine). It appears that the catharanthine-like unit of these molecules are hardly trapped in catharanthine cavities located in the MIP, probably due to the sterical hindrance of the vindoline moiety. Finally, the MIP-SPE applied to C. roseus extract enabled quantitative recovery of catharanthine (101%) and the total removal of vindoline. Its capacity was determined and was equal to 2.43 μmol g⁻¹.Vindoline is a weaker base than catharanthine, so the vindoline-MIP was achieved with a strong acidic monomer (itaconic acid) to increase vindoline–monomer interactions and a modified washing solvent (ACN–HCOOH 99/1) to reduce non-specific interactions. The influence of the amount of HCOOH (protic modifier) percolated during the washing step upon the elution yield and the imprinting factor for vindoline was investigated. This preliminary optimisation of the washing step, and in particular the number of moles of acid percolated, seems useful to emphasize the use of MIP in conditions of high selectivity or high yield. A compromise was obtained with an imprinting factor equal to 7.6 and an elution recovery of 33%. However MIP-vindoline failed to achieve a specific extraction of vindoline since catharanthine was also extracted probably because of strong non-specific interactions occurring between catharanthine and the sorbent.     

Recombinant 3-Hydroxy 3-Methyl Glutaryl-CoA Reductase from <Emphasis Type="Italic">Candida glabrata</Emphasis> (Rec-CgHMGR) Obtained by Heterologous Expression,as a Novel Therapeutic Target Model for Testing Synthetic Drugs

The enzyme 3-hydroxy-3-methyl-glutaryl CoA reductase (HMGR) is a glycoprotein of the endoplasmic reticulum that participates in the mevalonate pathway, the precursor of cholesterol in human and ergosterol in fungi. This enzyme has three domains: transmembrane, binding, and soluble. In this study, we expressed and purified the soluble fraction of the HMGR enzyme from Candida glabrata (CgHMGR) in an Escherichia coli heterologous system and used it as a model for studying its inhibitory activity. The soluble fraction of CgHMGR was fused to the maltose binding protein (MBP), purified, and characterized. Optimal pH was 8.0, and its optimal temperature activity was 37 °C. The k _m and V _max for the HMG-CoA were 6.5 μM and 2.26 × 10^?3 μM min^?1, respectively. Recombinant CgHMGR was inhibited by simvastatin presenting an IC₅₀ at 14.5 μM. In conclusion, our findings suggest that the recombinant HMGR version from C. glabrata may be used as a study model system for HMGR inhibitors such as statins and newly synthesized inhibitor compounds that might be used in the treatment of hypercholesterolemia or mycosis.     

Heterologous expression of Trametes versicolor laccase in Pichia pastoris and Aspergillus niger

Convenient expression systems for efficient heterologous production of different laccases are needed for their characterization and application. The laccase cDNAs lcc1 and lcc2 from Trametes versicolor were expressed in Pichia pastoris and Aspergillus niger under control of their respective glyceraldehyde-3-phosphate dehydrogenase promoters and with the native secretion signal directing catalytically active laccase to the medium. P. pastoris batch cultures in shake-flasks gave higher volumetric activity (1.3 U/L) and a better activity to biomass ratio with glucose than with glycerol or maltose as carbon source. Preliminary experiments with fed-batch cultures of P. pastoris in bioreactors yielded higher activity (2.8 U/L) than the shake-flask experiments, although the levels remained moderate and useful primarily for screening purposes. With A. niger, high levels of laccase (2700 U/L) were produced using a minimal medium containing sucrose and yeast extract. Recombinant laccase from A. nigher harboring the lcc2 cDNA was purified to homogeneity and it was found to be a 70-kDa homogeneous enzyme with biochemical and catalytic properties similar to those of native T. versicolor laccase A.     

Production of cellulase/β-glucosidase by the mixed fungi culture of Trichoderma reesei and Aspergillus phoenicis on dairy manure

A cellulase production process was developed by growing the fungi Trichoderma reesei and Aspergillus phoenicis on dairy manure. T. reesei produced a high total cellulase titer (1.7 filter paper units [FPU]/mL, filter paper activity) in medium containing 10 g/L of manure (dry basis [w/w]), 2 g/L KH₂PO₄, 2 mL/L of Tween-80, and 2mg/L of CoCl₂. However, β-glucosidase activity in the T. reesei-enzyme system was very low. T. reesei was then cocultured with A. phoenicis to enhance the β-glucosidase level. The mixed culture resulted in a relatively high level of total cellulase (1.54 FPU/mL) and β-glucosidase (0.64 IU/mL). The ratio of β-glucosidase activity to filter paper activity was 0.41, suitable for hydrolyzing manure cellulose. The crude enzyme broth from the mixed culture was used for hydrolyzing the manure cellulose, and the produced glucose was significantly (p<0.01) higher than levels obtained by using the commercial enzyme or the enzyme broth of the pure culture T. reesei.     

Effect of the medium pH on the release of secondary metabolites from roots ofDatura stramonium,Catharanthus roseus,andTagetes patula cultured in vitro

The release of alkaloids from root culturesDatura stramonium andCatharanthus roseus and thiophenes from root cultures ofTagetes patula was found to increase when the pH of the culture media (ranging from 4.8 to 7.0) was reduced to 3.5. The extent of the effect was different in each type of culture. Increases ranged from 4- to 20-fold, which in some cases accounted for 75% of the total secondary metabolite pool produced per flask. When the release of individual metabolites was measured, even larger increases, were observed (nearly 400-fold for ajmalicine). Increased release of alkaloids fromC. roseus roots were also observed in cultures growing in a 14-L fermentor, when the medium pH was reduced. Reduction of the pH of the media did not affect growth of the root cultures in subsequent subcultures. The importance of this treatment as a stategy to improve the recovery of secondary metabolites from producing cultures is discussed.     

Antioxidant potentials and ajmalicine accumulation in Catharanthus roseus after treatment with giberellic acid

Changes in antioxidant potentials and indole alkaloid, ajmalicine, production were studied in Catharanthus roseus (L.) G. Don. plants under treatment with gibberellic acid (GA(3)). The GA(3) treatments were given in two ways, foliar spray and soil drenching methods on 30, 45, 60 and 75 days after planting (DAP). The plants were uprooted randomly on 90 DAP and separated into root, stem and leaves and used for analyses. The antioxidant potential was studied in terms of non-enzymatc antioxidant molecules like ascorbic acid (AA), alpha-tocopherol (alpha-toc) and reduced glutathione (GSH) and activities of antioxidant enzyme, viz., superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT). The alkaloid ajmalicine was extracted and estimated from roots of both control and treated plants. It was found that, GA(3) has a profound effect upon the antioxidant potentials and it caused a significant enhancement in the production of ajmalicine when compared to untreated control as well as foliar-sprayed plants. There was no significant enhancement in GSH and ajmalicine content under GA(3) foliar spray in C. roseus. These preliminary results suggest that, the application of GA(3) may be a useful tool to increase the antioxidant potential and alkaloid production in medicinal plants like C. roseus.     

Electrochemical detection of indole alkaloids of Catharanthus roseus in high-performance liquid chromatography

Hydrodynamic voltammograms for indole and five indole alkaloids with different amino functions were obtained in order to evaluate the applicability of high-performance liquid chromatography (HPLC) with coulometric detection to these compounds. With the exception of serpentine, which has a quaternary nitrogen in its structure, all the compounds were oxidised and gave net signals of greater than 25 nA pmol(-1) at potentials of between +0.2 and +0.85 V versus a solid Pd electrode in an acetate-buffered (pH 6.5) water-methanol-acetonitrile system. An HPLC assay for quantifying picomole amounts of catharanthine and ajmalicine in Catharanthus roseus cell culture samples is described.     

Double Perovskite LaFexNi1−xO3 Nanorods Enable Efficient Oxygen Evolution Electrocatalysis

Perovskite‐based electrocatalysts are one of the most promising materials for oxygen evolution reaction (OER), but their activity and durability are still far from desirable. Herein, we demonstrate that the double perovskite LaFe_xNi_1?xO₃ (LFNO) nanorods (NRs) can be adopted as highly active and stable OER electrocatalysts. The optimized LFNO‐II NRs with Ni/Fe ratio of 8:2 achieve a low overpotential of 302 mV at 10 mA cm^?2 and a small Tafel slope of 50 mV dec^?1, outperforming those of the commercial Ir/C. The LFNO‐II NRs also show high OER stability with slight current decrease after 20 h. The enhanced activity is explained by the improved surface area, tailored electronic structure as well as strong hybridization between O and Ni.     

Does application of cement kiln exhausts affect root nodule biochemistry and soil N2-fixing microbes?

The research work carried out on cement exhausts centers mostly around vegetation and crop productivity (1-3) with little or no work on root nodulation. Soil plus foliar application of exhaust dusts did not affect soil/nodule rhizobial population, nodule initiation, and possible N₂-fixing capacity inCajanus cajan, Vigna radiata, Vigna mungo,Vigna catjung, andGlycine max. The nodular biochemistry was investigated in detail. The heme protein leghemoglobin was higher compared to the control. The levels of intermediary N compounds like total ureides of the nodules, which may serve as indirect evidence of symbiotic N₂ fixation, were higher in the treated plants. There were also increments in free proline, free amino acids, soluble proteins, soluble starch, soluble sugars, total nitrogen, and phenols in the treated plants. The levels of total nitrates, soluble sucrose, and soluble SH compounds of the nodule of the control and treated plants did not show a significant difference. The activities of ascorbate peroxidase, catalase, glutathione reductase, and Superoxide dismutase were significantly higher, possibly indicating their role in alleviation of H₂O₂ and O ₂ ^- damage by the exhausts. Enzymes like nitrate reductase, nitrite reductase, and glutamine synthetase, and also the activities of acid and alkaline phosphatases were not affected. The presence of beneficial soil microbes likeAzotobacter, Azospirillum, and mycorrhizae was not affected at all.     

Partial electro-neutralisation of -α-p-hydroxyphenylglycine in sulphuric acid medium

In the industrial synthesis of -α-p-hydroxyphenylglycine the separation of amino acid is carried out by precipitation. During this process, a mother liquor is produced with a high salt content (2 M phosphates and sulphates) and an amino acid concentration of 0.11–0.12 M. The disposal of this mother liquor causes an environmental problem and an economic loss. The salt content of this mother liquor can be reduced in 70% of the initial by means of an electrodialysis process previously carried out by us, with only an amino acid loss of 15% of the initial. To improve and simplify this process, an electro-electrodialysis process (a membrane electrolysis process; the electrode processes and the transport process across the membrane are used) has been developed in which as a first step, the electro-neutralisation of solutions containing sulphuric acid and -α-p-hydroxyphenylglycine is studied. The sulphuric acid content is reduced to 87% of the initial, without detected loss of amino acid. The final solution is posteriorly neutralised by working up the pH of the solution for precipitating the amino acid, and a mother liquor with approximately 0.10 M -α-p-hydroxyphenylglycine and a low salt content (0.08 M Na₂SO₄) is produced. This mother liquor with low salinity can be recirculated again to a new electro-electrodialysis process.     

Synthesis and characterization of novel aromatic polyamides containing 3-trifluoromethyl-substituted triphenylamine

A new 3-trifluoromethyl-substituted triphenylamine-containing aromatic diacid monomer, N,N-bis(4-carboxyphenyl)-3-trifluoromethylaniline, was prepared by the substitution reaction of 3-trifluoromethylaniline with 4-fluorobenzonitrile, followed by alkaline hydrolysis of the dinitrile intermediate. Novel aromatic polyamides with 3-trifluoromethyl-substituted triphenylamine moieties were prepared from the diacid and various aromatic diamines via the direct phosphorylation polycondensation. All the polyamides were amorphous and readily soluble in many polar organic solvents such as N,N-dimethylacetamide and N-methyl-2-pyrrolidone, and could be solution-cast into transparent, tough, and flexible films with good mechanical properties. They exhibited good thermal stability with relatively high glass-transition temperatures (258–327°C), 10% weight-loss temperatures above 500°C, and char yields higher than 60% at 800°C in nitrogen. These polymers had low dielectric constants of 3.22–3.70 (100 Hz), low moisture absorption in the range of 1.75–2.58%, and high transparency with an ultraviolet–visible absorption cut-off wavelength in the 375–395 nm range. Cyclic voltammograms of the polyamide films cast onto an indium tin oxide coated glass substrate exhibited a reversible oxidation redox couple with oxidation half-wave potentials (E_1/2) of 0.95–1.00 V vs. Ag/AgCl in an acetonitrile solution.     

Isolation and GC-MS determination of flavonoids from Glycyrrhiza glabra root

Components of the ethylacetate extract of Glycyrrhiza glabra root were separated based on their acid-base properties. The major components of the fraction soluble in aqueous NaOH were identified by GC-MS as glabridin, the principal component of the ethylacetate extract of licorice root (Glycyrrhiza glabra L.), 4-O-methylglabridin, and hispaglabridin B. The isoflavene glabrin was identified in addition to the isoflavan derivatives. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 236–240, May–June, 2006.     

Stress Response Kinetics of Two Nisin Producer Strains of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> spp. <Emphasis Type="Italic">lactis</Emphasis>

The purpose of this study is to determine the survival and nisin production behaviors of two strains of Lactococcus lactis under different stress conditions that represent the food ecosystem. In this respect, the survival ratios of two nisin producers were determined under different pH, temperature, NaCl, and bile salt concentrations. Then, nisin production levels of the strains were determined at each stress conditions. Both strains had similar growth or inactivation patterns under the same stress conditions. NaCl and bile salt stresses on the survival ratio of the strains could be successfully described by the exponential decay function, whereas Gaussian function produced good fits for temperature and pH stresses. The nisin activity of two nisin producers (in their mid-exponential and/or early stationary phase) decreased dramatically under all stress conditions, except osmotic (NaCl) and low temperature applications. The results of this study showed that two nisin producers had similar adaptive responses under severe stress conditions, which could be described by appropriate mathematical equations. Moreover, the effect of harsh environment on the nisin activity of L. lactis strains depends on the stress factors applied.     

Preparation and properties of glucose isomerase immobilized on Indion 48-R

Partially purified glucose isomerase fromStreptomyces thermonitrificans when coupled to glutaraldehyde-activated Indion 48-R, retained 30–40% activity of the soluble enzyme. However, an approximately twofold increase in the activity could be achieved by binding the enzyme in the presence of glucose. Binding the enzyme to matrices presaturated with either glucose or fructose and influence of lysine modification on the activity of the soluble enzyme revealed that the comparatively low activity observed in case of the enzyme bound in the absence of substrate is the result of the nonspecific binding of either substrate or product to the matrix. Immobilization did not affect the pH and temperature optima of the enzyme, but it lowered the temperature stability. Immobilization resulted in a marginal increase in theK _m and a threefold decrease in theV _max . Substrate concentrations as high as 36% glucose could be converted to 18.5% fructose in 5 h, at pH 7.0 and 70‡C. The bound enzyme, however, showed inferior stability to repeated use and lost approx 40% of its initial activity after five cycles of use. Indion 48-R bound glucose isomerase could be stored, in wet state, for 30 d without any apparent loss in its initial activity.     

Physiological activities of chitosan and N‐trimethyl chitosan chloride in U937 and 3T3‐L1 cells

Chitosan (CH) is a biopolymer with biocompatible, biodegradable, and bioactive properties. N,N,N‐trimethyl chitosan chloride (TMC) is a quaternized form of CH that is highly cationic and more water soluble than unmodified CH. The physiological activities of CHs with different molecular weights (M_w) and degrees of TMCs quaternization were investigated in U937 and 3T3‐L1 cell lines. ¹H‐NMR spectrometry and size exclusion chromatography were used for characterization of the biopolymers. The half inhibition concentration (IC₅₀) of DPPH‐radical‐scavenging activity was below 0.9 mg/m with quaternized CHs. The IC₅₀ values of chitooligosaccharides, low‐ and medium‐molecular‐weight CHs were 8.4, 10.9, and 13.9 mg/ml, respectively. High‐molecular‐weight CHs and TMCs showed apoptotic activity on U937 cells. T41, a TMC of 549 kDa with a 41% degree of quaternization (DQ), yielded 30.7% apoptotic cell death in U937 at 20 µg/ml and effectively repressed cell differentiation and triglyceride accumulation in 3T3‐L1. Depolymerized CHs reduced triglyceride accumulation but also caused cell differentiation. TMCs showed repressor activity to both cell differentiation and triglyceride accumulation. Increasing the molecular weight of CHs and TMCs generally resulted in increased physiological activity. Copyright © 2010 John Wiley & Sons, Ltd.     

Cloning and expression of L-asparaginase gene in Escherichia coli

The L-asparaginase (ASN) from Escherichia coli AS1.357 was cloned as a DNA fragment generated using polymerase chain reaction technology and primers derived from conserved regions of published ASN gene sequences. Recombinant plasmid pASN containing ASN gene and expression vector pBV220 was transformed in different E. coli host strains. The activity and expression level of ASN in the engineering strains could reach 228 IU/mL of culture fluid and about 50% of the total soluble cell protein respectively, more than 40-fold the enzyme activity of the wild strain. The recombinant plasmid in E. coli AS1.357 remained stable after 72h of cultivation and 5h of heat induction without selective pressure. The ASN gene of E. coli AS1.357 was sequenced and had high homology compared to the reported data.     

The Bioluminescence Resonance Energy Transfer from Firefly Luciferase to a Synthetic Dye and its Application for the Rapid Homogeneous Immunoassay of Progesterone

The sensitive BRET system for the homogeneous immunoassay of a low‐molecular weight antigen was developed using progesterone as an example. Two thermostable mutants of the Luciola mingrelica firefly luciferase (Luc)—the “red” mutant with λ_max.em = 590 nm (RedLuc) and the “green” mutant with λ_max.em = 550 nm (GreenLuc)—were tested as the donors. The water‐soluble Alexa Fluor 610× (AF) dye was selected as the acceptor because its two absorption maxima, located at 550 and 610 nm, are close to the bioluminescence maxima of the GreenLuc and RedLuc, respectively. The methods for the synthesis of the luciferase–progesterone (Luc–Pg) conjugate and the conjugate of the dye and the polyclonal antiprogesterone antibody (AF–Ab) were developed. Both conjugates retained their functional properties, had high antigen–antibody binding activity, and demonstrated a high BRET signal. The homogeneous immunoassay system based on the BRET from the firefly luciferase to the synthetic dye was established to assay progesterone as a model antigen. Optimization of the assay conditions, the composition of the reaction mixture, and the concentrations of the donor and the acceptor made it possible to reach the minimum detectable progesterone concentration of 0.5 ng mL^?1.     

Cellular association of glucosyltransferases in Leuconostoc mesenteroides and effects of detergent on cell association

Most glucosyltransferase (GTF) activity in sucrose-grown cultures of some strains of Leuconostoc mesenteroides is found with the cell pellet after centrifugation. GTFs are known to bind to dextrans, and it was traditionally assumed that cell-associated GTFs were bound to those dextrans that cosedimented with the cells. We used a mutant strain (LC-17), derived from strain NRRL B-1355, which produced dextransucrase in the absence of dextrans, to investigate the extent to which GTFs were bound to cells or dextrans. Much of the GTF activity in glucose-grown cultures of strain LC-17, which do not produce dextran, was located in the cell pellets. Soluble enzyme activity increased when cell suspensions from glucose- or sucrose-grown cultures were incubated with mild nonionic detergents or zwitterionic reagents. Alternansucrase produced by the parent strain B-1355 was almost entirely associated with cells under conditions in which dextrans were or were not produced. Alternansucrase, but not dextransucrase, tended to be enriched in the particulate fraction of B-1355 cells that had been broken in a French press. The distribution of alternansucrase and the effects of detergents on the distribution of GTFs suggest that soluble GTFs sequestered in the cytoplasm, and GTFs bound or adsorbed to the cell membrane are probably the major contributors to the cell-associated GTF activity.