Disproof of the Overall Enzymatic Biosynthesis of Vindoline from Tryptamine and Secologanin by Cell-Free Extracts from the Leaves of Catharanthus roseus (L.) G. DON

A cell-free extract, which was isolated from the leaves of mature Catharanthus roseus plants by a previously published procedure, does not convert a mixture of secologanin and radiolabelled tryptamine to vindoline, as was recently claimed. The radioactivity in the purified alkaloid extract determined by earlier workers is certainly due to ‘impurities’ in the presumed ‘vindoline’. This was shown by extensive purification of the alkaloid extract (which contained added unlabelled vindoline as a carrier) followed by chemical conversion of vindoline to two derivatives and subsequent purification, ultimately giving unlabelled deacetylvindoline.     

Identification of a novel beta-replacement reaction in the biosynthesis of 2,3-diaminobutyric acid in peptidylnucleoside mureidomycin A

2,3-Diaminobutyric acid (DABA) is an unusual di-amino acid component of mureidomycin A, a member of the peptidylnucleoside antibiotic family produced by Streptomyces flavidovirens SANK 60486. Radiochemical assays using cell-free extract from S. flavidovirens revealed that 14C-L-Thr is converted into radiolabelled DABA by an ammonia-dependent beta-replacement activity, and not via oxidation to 3-keto-2-aminobutyric acid. The substrate specificity of partially purified enzyme was assayed using a spectrophotometric assay, and beta-replacement activity was inhibited by known inhibitors of PLP-dependent enzymes. These data imply that DABA is biosynthesised from L-Thr by a PLP-dependent beta-replacement enzyme, using ammonia as a nucleophile. These results are consistent with literature proposals for the biosynthesis of 2,3-diaminopropanoic acid from the viomycin biosynthetic gene cluster.     

Assay of Serum Glutamic-Oxaloacetic Transaminase Using Malate Dehydrogenase Preparation Obtained from Streptomyces aureofaciens

Abstract

A modified spectrophotometric method for serum glutamic-oxaloacetic transaminase (SGOT) assay was developed. A crude cell-free extract from Streptomyces aureofaciens which showed a high level of malate dehydrogenase (MDH) activity (E.C. 1.1.1.37) was used as the enzymatic indicator. The lyophilized microbial preparation was used without previous purification and was quite stable under refrigeration for one year. Serum sample assays using both the method utilizing the crude cell extract and an enzymatic commercial kit showed good correlation.     

Efficient conjugation of peptides to oligonucleotides by "native ligation"

A new strategy has been developed for conjugation of peptides to oligonucleotides. The method is based on the "native ligation" of an N-terminal thioester-functionalized peptide to a 5'-cysteinyl oligonucleotide. Two new reagents were synthesized for use in solid-phase peptide and oligonucleotide synthesis, respectively. Pentafluorophenyl S-benzylthiosuccinate was used in the final coupling step in standard Fmoc-based solid-phase peptide assembly. Deprotection with trifluoracetic acid generated in solution peptides substituted with an N-terminal S-benzylthiosuccinyl moiety. O-trans-4-(N-alpha-Fmoc-S-tert-butylsulfenyl-L-cysteinyl)aminoc yclohe xyl O-2-cyanoethyl-N,N-diisopropylphosphoramidite was used in the final coupling step in standard phosphoramidite solid-phase oligonucleotide assembly. Deprotection with aqueous ammonia solution generated in solution 5'-S-tert-butylsulfenyl-L-cysteinyl functionalized oligonucleotides. Functionalized peptides and oligonucleotides were used without purification in native ligation conjugation reactions in aqueous/organic solution using tris-(2-carboxyethyl)phosphine to remove the tert-butylsulfenyl group in situ and thiophenol as a conjugation enhancer. A range of peptide-oligonucleotide conjugates were prepared by this route and purified by reversed-phase HPLC.     

Purification of salvianolic acid B from the crude extract of Salvia miltiorrhiza with hydrophilic organic/salt-containing aqueous two-phase system by counter-current chromatography

Establishment of hydrophilic organic/salt-containing aqueous two-phase system and purification of salvianolic acid B from crude extract of S. miltiorrhiza by counter-current chromatography with said system were studied. Ethanol and n-propanol were selected to constitute biphasic systems with ammonia sulphate, sodium chloride and phosphate separately, and related system characteristics including phase diagrams, phase ratio, separation time were tested. The partition coefficient of crude salvianolic acid B was also tested in above systems and further finely adjusted by altering the constitution of phosphate in n-propanol/phosphate system. Salvianolic acid B was purified to 95.5% purity by counter-current chromatography in 36% (w/w) n-propanol/8% (w/w) phosphate system with the ratio between dipotassium hydrogen phosphate and sodium dihydrogen phosphate of 94:6. One hundred and eight milligrams of salvianolic acid B was purified from 285 mg crude extract with the recovery of 89%.     

Antibacterial and preliminary phytochemical and physico-chemical analysis of Eucalyptus citriodora Hk leaf

The present communication deals with some studies on the antibacterial, physico-chemical and phytochemical parameters of different extracts of Eucalyptus citriodora leaf. The antibacterial study was performed using the agar ditch method on some clinically important bacteria, namely Pseudomonas pseudoalcaligenes, Proteus vulgaris, Citrobacter freundii, Staphylococcus subflava, Bacillus megaterium, and Enterobacter aerogenes. Physico-chemical parameters namely water, methanol, 1,4-dioxane, DMF, acetone soluble extractives, total ash, melting point, and pH were determined according to pharmacopoeial procedures. Methanol gave the maximum extract while it was minimum in water. Phytochemical parameters were screened for alkaloids, tannins, cardiac glycosides, saponins, steroids and flavonoids. Tannins and flavonoids gave positive results, while steroids and glycosides were absent. The most susceptible bacteria was C. freundii, while the most resistant was P. vulgaris.     

Effect of Catalase on Biocatalytic Synthesis of Pyruvate by Enzymes from Pseudomonas sp.

Pyruvate was produced from DL-lactate by a kind of green-chemical biocatalyst --cell-free extract from bacterial strain Pseudomonas sp. SM-6. Catalase in cell-free extract, whichcould stabilize the pyruvate formed by lactate oxidase, played an important role in pyruvatepreparation. The effect of catalase in conversion process was evaluated.     

Biocatalytic Synthesis of Pyruvate from DL-lactate with Enzymes in Pseudomonas sp.

Pyruvate has received increasing attention in recent years as a potential precursor for the synthesis of L-amino acids1. Compared with other small non-chiral building blocks, pyruvate is relatively expensive. Conventional processes for preparing pyruvate include a process comprising reacting sodium cyanide and acetyl chloride to synthesize acetyl cyanide and hydrolyzing the acetyl cyanide, and a process comprising reacting tartaric acid and potassium hydrogen sulfate. These processes not on…     

One-step purification of palmatine and its derivative dl-tetrahydropalmatine from Enantia chlorantha using high-performance displacement chromatography

Palmatine and its reduced form, dl-tetrahydropalmatine are a group of isoquinoline alkaloids that have been reported to display a variety of biological and pharmacological activities. Both drugs are hydrophilic and are difficult to be purified by conventional purification methods of natural products. A high-performance displacement chromatography (HPDC) method successfully purified palmatine and its semi-synthetic derivative dl-tetrahydropalmatine from crude extract of the African medicinal plant Enantia chlorantha. The crude extract from the root bark of E. chlorantha was fractionated on an analytical reversed-phase C(18) column by using 0.1% trifluoroacetic acid (TFA) or acetic acid/H2O as a carrier and cetylpyridinium trifluoroacetate (or acetate) (1.9mg/mL) in 0.1% TFA (or acetic acid)/H2O as a displacer. Palmatine was quantitatively purified at >98% purity in the fully developed displacement mode. dl-Tetrahydropalmatine was semi-synthesized by NaBH4 reduction from crude palmatine and directly purified by HPDC. Both palmatine and dl-tetrahydropalmatine were identified by high-resolution electrospray tandem mass spectrometry, (1)H NMR and (13)C NMR. This is the first report of one-step HPDC purification of natural and semi-synthetic products from a complex crude extract.     

From gene to HSQC in under five hours: high-throughput NMR proteomics

A simple, rapid, in vitro cell-free protein expression system, Expressway NMR, is introduced and used to express the small ubiquitin-related modifier protein SUMO-1. This 12 kDa molecule is challenging for NMR as it has limited solubility and requires relatively high salt (200 mM) for stability in solution. Starting with the gene, the cell-free system, and milligram amounts of nitrogen-15 isotopically enriched amino acids, sufficient protein is produced in 4 h to obtain a high-resolution 2D HSQC spectrum of the protein in 40 min. This time would be closer to 10 min with the aid of a higher sensitivity salt-tolerant cryogenic NMR probe. With all protein purification steps included, and aggressive data processing using the filter diagonalization method (FDM), it is but 6 h from gene to heteronuclear single quantum coherence (HSQC). As the cell-free system is nearly background-free, it is also possible to work with the crude reaction mixture, in which case only a total of 5 h is required. Sample stability over time, whether crude extract or purified, was notable, with no significant change in the 15N-1H HSQC spectrum over 6 months at 4 degrees C (300 muM, pH 6.1, capped NMR tube). The combination of a turnkey, high-yield, protease-free in vitro protein expression system, an optimized sensitivity-enhanced HSQC pulse sequence, and FDM processing makes this scheme an attractive first step to rapidly assess the suitability of proteins for complete solution structure determination.     

Fluorous affinity purification of oligonucleotides

[structure: see text] Nucleoside phosphoramidites bearing a fluorous dimethoxytrityl (FDMT) group were used to synthesize fluorous-tagged oligonucleotides, which were subjected to solid-phase extraction using a pH-stable fluorinated adsorbent. On-column detritylation afforded the purified oligonucleotides. The fluorous affinity purification method offers one-pass loading without ammonia removal, high selectivity for the removal of failure sequences, high recoveries (typically 70-100%), and the ability to purify long oligonucleotides (e.g., 50-100-mers).     

Effect of Pseudomonas moorei KB4 Cells’ Immobilisation on Their Degradation Potential and Tolerance towards Paracetamol

Pseudomonas moorei KB4 is capable of degrading paracetamol, but high concentrations of this drug may cause an accumulation of toxic metabolites. It is known that immobilisation can have a protective effect on bacterial cells; therefore, the toxicity and degradation rate of paracetamol by the immobilised strain KB4 were assessed. Strain KB4 was immobilised on a plant sponge. A toxicity assessment was performed by measuring the concentration of ATP using the colony-forming unit (CFU) method. The kinetic parameters of paracetamol degradation were estimated using the Hill equation. Toxicity analysis showed a protective effect of the carrier at low concentrations of paracetamol. Moreover, a pronounced phenomenon of hormesis was observed in the immobilised systems. The obtained kinetic parameters and the course of the kinetic curves clearly indicate a decrease in the degradation activity of cells after their immobilisation. There was a delay in degradation in the systems with free cells without glucose and immobilised cells with glucose. However, it was demonstrated that the immobilised systems can degrade at least ten succeeding cycles of 20 mg/L paracetamol degradation. The obtained results indicate that the immobilised strain may become a useful tool in the process of paracetamol degradation.     

类产碱假单胞菌全细胞催化前手性酮对映选择性还原合成西他列汀手性中间体

以苯乙酮为唯一碳源,从土壤中筛选出一株能够将4-氧-4-[3-(三氟甲基)-5,6-二氢[1,2,4]三唑并[4,3-a]吡嗪-7(8H)-基]-1-(2,4,5-三氟苯基)丁-2-酮(2)对映选择性还原为抗Ⅱ型糖尿病药物西他列汀关键手性中间体(S)-3-羟基-1-[3-(三氟甲基)-5,6-二氢[1,2,4]三唑并[4,3-a]吡嗪-7(8H)-基]-4-(2,4,5-,三氟苯基)丁-1-酮((S)-1)的细菌菌株,经鉴定并命名为类产碱假单胞菌(Pseudomonas pseudoalcaligenes)XW-40.系统研究了水溶性辅溶剂、温度、p H、底物浓度、细胞浓度、反应时间等反应条件对生物还原产率和对映选择性的影响.该菌株全细胞能够耐受浓度高达10 g/L的前手性酮2.在最优反应条件下,以制备规模合成了(S)-1,分离收率90%,ee99%.     

Bestimmung von freiem und komplex gebundenem Cyanid, Cyanat und Ammoniak nebeneinander

Zusammenfassung Es wird eine Analysenmethode für Cyanid, komplexe Cyanide, Ammoniak und Cyanat in oxydierende Chlorverbindungen enthaltenden Lösungen beschrieben. Nach unseren Ergebnissen kann auch in solchen Lösungen freies Cyanid enthalten sein. Die Bestimmungen sind nebeneinander nicht durchzuführen, daher werden nach Reduktion mit Arsenitlösung folgende Trennungen durch Wasserdampfdestillation (Wd.) durchgeführt: 1. Wd. bei pH 12; erfaßt wird NH₃ bzw. NH₄ ⁺ und ein kleiner Teil des Cyanates (Hydrolyse zu NH₃). Dieser Teil ist bei kontrollierten Bedingungen proportional der Cyanatkonzentration. 2. Wd. bei pH 7 (Phosphatpuffer). Erfaßt wird freies Cyanid. 3. Wd. bei pH 0 zur Abtrennung des Gesamtcyanids. 4. Verseifung des Cyanats (pH 1, Cyanatkonzentration unter 1,2 g/l) und Wd. bei pH 12. Erfaßt wird die Summe an Ammoniak und Cyanat.

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Determination of free and complex cyanide, cyanate and ammonia in presence of each other

A procedure is described for the analysis of solutions containing cyanide, complex cyanides, ammonia and oxidizing chlorine compounds. Our results show the possible existence of free cyanide ions in such solutions. Simultaneous determination is impossible; therefore, after reduction with arsenite solution, the following separations are made by steam distillation (s. d.): (1) S.d. at pH 12 gives the sample ammonia and some ammonia produced by hydrolysis of cyanate (this is under controlled conditions proportional to the cyanate concentration). (2) S.d. at pH 7 (phosphate buffer) gives the free cyanide. (3) S.d. at pH 0 separates complex and free cyanide. (4) Hydrolysation of cyanate at pH 1 (cyanate conc. below 1.2 g/l) and following s.d. at pH 12 gives the sum of sample ammonia and the ammonia equivalent to the cyanate content.

     

UV-ABC screens of luteolin derivatives compared to edelweiss extract

Pure luteolin is a remarkably heat (200°C/6 days) and UV stable UV-A screen, however, native luteolin enriched to 37% in an edelweiss extract lost its UV-A screen properties upon UV irradiation (～4MJm(-2)). This contrasting behavior led to the examination of a series of purified luteolin derivatives as UV screen candidates. 3',4',5,7-Tetralipoyloxyflavones were synthesized from luteolin (3',4',5,7-tetrahydroxyflavone) and fatty acid chlorides. These acylated semi-biomolecules show a hypsochromic shift in UV-Vis spectra of about Δλ(A→B)=58nm and absorbed in the centre of the harmful UV-B band (λ(max)=295nm). Luteolin was also hydroxyethylated with Br(CH(2))(2)OH. This substitution has no effect on the λ(max)=330nm absorption of luteolin (UV-A band). Finally the natural 4'-O-β-glucosyl-3',5,7-trihydroxyflavone was extracted from edelweiss and used as a purified natural benchmark. Glycosylated and hydroxyethylated luteolin are both UV stable. Fully acylated luteolin derivatives degrade upon UV exposure to a stable UV-C screen with a hypsochroic shift Δλ(B→C)=35nm. All in all, three molecular structures based on luteolin with sunscreen properties were found, distinguishable in: UV-A, UV-B, and UV-C filters. The natural product based UV-absorbers show promise as alternatives to synthetic molecules and nanoparticles in sunscreen products.     

Isotachophoretic determination of the ionic mobilities and ionization constants of weak monoacidic bases by a simple computer-aided slope-intercept method

Shrimp abdomenal muscle NADP-dependent malic enzyme (E.C.1.1.1.40) was purified about 1500-fold to a specific activity of 48 units (mumol/min)/mg at 30 degrees C with good quantitative recovery in three chromatographic steps, including affinity chromatography on 2',5'-ADP-Sepharose 4B, a "substrate activation" method using malate substrate plus manganese chloride. In addition to the malate-manganese chloride substrate pair, succinate or glutamate plus manganese chloride or magnesium chloride could be used in this "substrate activation" method for crustacean NADP-malic enzyme purification on 2',5'-ADP-Sepharose 4B. Affinity chromatography alone purified malic enzyme almost 43 fold, and the overall method resulted in homogeneous enzyme since polyacrylamide gel electrophoresis of the native purified enzyme revealed only a single band staining for protein and enzyme activity.     

Dissociation rates of urea in the presence of NiOOH catalyst: a DFT analysis

Single molecule reactions have been studied between nickel oxyhydroxide, urea, and the hydroxide ion to understand the process of urea dissociation into ammonia, isocyanic acid, cyanate ion, carbon dioxide, and nitrogen. In the absence of hydroxide ions, nickel oxyhydroxide will catalyze urea to form ammonia and isocyanic acid with the rate-limiting step being the formation of ammonia with a rate constant of 1.5 × 10?? s?1. In the presence of hydroxide, the evolution of ammonia was also the rate-limiting step with a rate constant of 1.4 × 10?2? s?1. In addition, desorption of the cyanate ion presented an energy barrier of 6190 kJ mol?1 suggesting that the cyanate ion cannot be separated from NiOOH unless further reactions occurred. Finally, elementary dissociation reactions with hydroxide ions deprotonating urea to produce nitrogen and carbon dioxide were analyzed. These elementary reactions were investigated along three paths differing in the order that protons were removed and the nitrogen atoms were rotated. The rate-limiting step was found to be the removal of carbon dioxide with a rate constant of 4.3 × 10??? s?1. Therefore, the catalyst could be deactivated by the surface blockage caused by carbon dioxide adsorption.     

Determination of beta 2-receptor agonists in bovine urine and liver by gas chromatography-tandem mass spectrometry.

A highly specific and sensitive method for the simultaneous detection of seven beta 2-receptor agonists in bovine liver homogenates and urine was developed. A 10-g amount of liver was homogenized and treated with Subtilisin A. The resulting enzymatic digest was extracted with tert.-butanol-ethyl acetate (3:7) and the crude extract was purified on a 6-ml Bakerbond alumina neutral disposable extraction column. Subsequently, the hydrous eluate from the alumina column was buffered at pH 6 and loaded on top of a preconditioned 3-ml Bond-Elut Certify column. Urine was buffered and loaded onto a 3-ml Certify column without pretreatment. The analytes were eluted with dichloromethane-isopropanol (8:2) containing 2% ammonia. The extract obtained was trimethylsilylated and analysed by gas chromatography-tandem mass spectrometry using multiple selected reaction monitoring. The limits of detection for the beta 2-receptor agonists evaluated were between 0.5 and 5 ppb.     

Designed chimaeric galactosyl-mimodye ligands for the purification of Pseudomonas fluorescens beta-galactose dehydrogenase

Two chimaeric galactosyl-mimodye ligands were designed and applied to the purification of Pseudomonas fluorescens galactose dehydrogenase (GaDH). The chimaeric affinity ligands comprised a triazine ring on which were anchored: (i) an anthraquinone moiety that pseudomimics the adenine part of NAD+, (ii) a galactosyl-mimetic moiety (D-galactosamine for ligand BM1 or shikimate for ligand BM2), bearing an aliphatic 'linker', that mimics the natural substrate galactose, and (iii) a long hydrophilic 'spacer'. The mimodye-ligands were immobilised to 1,1-carbonyldiimidazole-activated agarose chromatography support, via the spacer's terminal amino-group, to produce the respective mimodye adsorbents. Both immobilized mimodyes successfully bound P. fluorescens GaDH but failed to bind the enzyme from rabbit muscle. Adsorbent BM1 bound GaDH from green peas and Baker's yeast, but adsorbent BM2 failed to do so. The mimodye-ligand comprising D(+)-galactosamine (BM1), compared to BM2, exhibited higher purifying ability and enzyme recovery for P. fluorescens GaDH. The dissociation constants (KD) of BM1 and BM2 for P. fluorescens GaDH were determined by analytical affinity chromatography to be 5.9 microM and 15.4 microM, respectively. The binding capacities of adsorbents BM1 and BM2 were 18 U/mg adsorbent and 6 U/mg adsorbent, respectively. Adsorbents BM1 and BM2 were integrated in two different protocols for the purification P. fluorescens GaDH. Both protocols comprised as a common first step DEAE anion-exchange chromatography, with a second step of affinity chromatography on BM1 or BM2, respectively. The purified GaDH obtained from the protocols using BM1 and BM2 showed specific activities equal to 1077 and 854 U/mg, respectively. The former is the highest reported so far and the enzyme appeared as a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.     

NMR investigations of the possible cross reactions between cyanate and epoxy resins

The possible cross reactions indicated by solid-state NMR between cyanate functionalized resin and epoxy functionalized resin have been investigated by using both natural abundance and labeled monofunctional model compounds. These soluble products were isolated and purified by silica gel adsorption chromatography and gel permeation chromatography. They were fully characterized by high resolution ¹H-, ¹³C-, ¹⁵N-NMR spectroscopy and by mass spectrometry. The major cross-reaction product is a racemic mixture of enantiomers, which contain an oxazolidinone ring formed by one cyanate molecule and two epoxy molecules. However, epoxy consumption lags cyanate consumption in the overall reaction as triazine formation from the cyanate is much faster than the two competing reactions, the cross reaction between cyanate and epoxy, and the self-polymerization of epoxy, under the conditions investigated. The cross reaction between cyanate and epoxy is limited. Approximately 12% of cross reaction between cyanate and epoxy was found in the overall reaction. In addition to the cross reactions of epoxy and cyanate, the reactions of epoxy and the carbamate, which is the major side product for the curing reaction of cyanate resin in solution, have also been investigated, and the mechanism of these reactions discussed. From the reactions of epoxy and carbamate, several products related to cross reaction between epoxy and cyanate have been isolated and identified. It is suggested that the reaction of epoxy and carbamate is one of the pathways in the overall cross reaction between epoxy and cranate resins. Finally, the mechanism of the overall cross-curing reaction between the diepoxy and dicyanate mixed resins is discussed. © 1994 John Wiley & Sons, Inc.