An eleven amino acid residue deletion expands the substrate specificity of acetyl xylan esterase II (AXE II) from <Emphasis Type="Italic">Penicillium purpurogenum</Emphasis>

The soft-rot fungus Penicillium purpurogenum secretes to the culture medium a variety of enzymes related to xylan biodegradation, among them three acetyl xylan esterases (AXE I, II and III). AXE II has 207 amino acids; it belongs to family 5 of the carbohydrate esterases and its structure has been determined by X-ray crystallography at 0.9 A resolution (PDB 1G66). The enzyme possesses the alpha/beta hydrolase fold and the catalytic triad typical of serine esterases (Ser90, His187 and Asp175). AXE II can hydrolyze esters of a large variety of alcohols, but it is restricted to short chain fatty acids. An analysis of its three-dimensional structure shows that a loop that covers the active site may be responsible for this strict specificity. Cutinase, an enzyme that hydrolyzes esters of long chain fatty acids and shows a structure similar to AXE II, lacks this loop. In order to generate an AXE II with this broader specificity, the preparation of a mutant lacking residues involving this loop (Gly104 to Ala114) was proposed. A set of molecular simulation experiments based on a comparative model of the mutant enzyme predicted a stable structure. Using site-directed mutagenesis, the loop's residues have been eliminated from the AXE II cDNA. The mutant protein has been expressed in Aspergillus nidulans A722 and Pichia pastoris, and it is active towards a range of fatty acid esters of up to at least 14 carbons. The availability of an esterase with broader specificity may have biotechnological applications for the synthesis of sugar esters.     

On the interaction of metal extractant reagents. Investigation of the aggregation ofdi(2,4,4-trimethylpentyl)phosphinic acid anddi(n-octyl)phosphinic acid in toluene by vapour-pressure osmometry

Summary The aggregation equilibria ofdi(2,4,4-trimethylpentyl)phosphinic acid (HDTMPP) anddi(n-octyl)phosphinic acid (HDOP) dissolved in toluene have been investigated by vapour-pressure osmometry (VPO) at different temperatures. The experimental data have been treated both graphically and numerically, and the average aggregation numberñ as well as the aggregation constant have been determined. The results suggest that dimers are formed and that the dimerization constant decreases with temperature. Finally, the enthalpies for the aggregation ofdi(2,4,4-trimethylpentyl)phosphinic acid anddi(n-octyl)phosphinic acid in toluene have been estimated using thevan't Hoff equation.

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Über die Wechselwirkung metallextrahierender Reagentien. Untersuchung der Aggregation vondi-(2,2,4-Trimethylpentyl)-phosphinsäure unddi-(n-Octyl)-phosphinsäure in Toluol mittels Dampfdruckosmometrie

Zusammenfassung Die Aggregationsgleichgewichte von in Toluol gelösterdi-(2,4,4-Trimethylphentyl)-phosphinsäure (HDTMPP) unddi-(n-Octyl)-phosphinsäure (HDOP) wurden mittels Dampfdruckosmometrie (VPO) bei verschiedenen Temperaturen untersucht. Die experimentellen Daten wurden sowohl graphisch als auch numerisch ausgewertet; es wurden sowohl die mittlere Aggregationszahlñ als auch die Aggregationskonstante bestimmt. Die Resultate lassen vermuten, daß Dimere gebildet werden und die Dimerisationskonstante mit der Temperatur abnimmt. Mit Hilfe dervan't Hoffschen Gleichung wurden die Enthalpien der Aggregation vondi-(2,4,4-Trimethylpentyl)-phosphinsäure unddi-(n-Octyl)-phosphinsäure in Toluol abgeschätzt.

     

Modification of the Peripheral Substituents in Chlorophylls a and b and Their Derivatives (Review)

The extensive published data on methods for the modification of peripheral substituents in chlorophylls a and b and their various semisynthetic derivatives are classified and analyzed. Special attention is paid to specific transformations arising from the presence of the unique macrocyclic tetrapyrrole fragment. Data on certain main pathways to modification of the exocyclic fragment of chlorophylls are also presented.     

Regioselective preparation of (R)-2-(4-hydroxyphenoxy)propionic acid with a fungal peroxygenase

The extracellular heme-thiolate peroxygenase of Agrocybe aegerita catalyzed the H₂O₂-dependent hydroxylation of 2-phenoxypropionic acid (POPA) to give the herbicide precursor 2-(4-hydroxyphenoxy)propionic acid (HPOPA). The reaction proceeded regioselectively with an isomeric purity near 98%, and yielded the desired R-isomer of HPOPA with an enantiomeric excess of 60%. ¹⁸O-labeling experiments showed that the phenolic hydroxyl in HPOPA originated from H₂O₂, which establishes that the reaction is mechanistically a peroxygenation. Our results raise the possibility that fungal peroxygenases may be useful for a variety of organic oxidations.     

Human urine certified reference material CZ 6010: creatinine and toluene metabolites (hippuric acid and o-cresol) and a benzene metabolite (phenol)

A reference material for the biological monitoring of occupational exposure to toluene, benzene and phenol was prepared. O-cresol and hippuric acid (metabolites of toluene) are used for the biological monitoring of occupational exposure to toluene. Phenol, a metabolite of benzene, is used for the biological monitoring of exposure to benzene, but phenol can of course also be used as an indicator of exposure to phenol as well. The reference material (RM) used for the determination of these metabolites was prepared by freeze-drying pooled urine samples obtained from healthy persons occupationally exposed to toluene and those taking part in an inhalation experiment. Tests for homogeneity and stability were performed by determining urine concentrations of o-cresol, hippuric acid, creatinine and phenol. To investigate the stability of the RM, the urinary concentrations of o-cresol and phenol were monitored for eighteen months using GC and HPLC, while those of hippuric acid and creatinine were followed for five and six years, respectively, using HPLC. Analysis of variance showed that the concentrations did not change. The certified concentration values (and their uncertainties) of the substances in this reference material (phenol concentration c=6.46±0.58 mg l⁻¹; o-cresol concentration c=1.17±0.15 mg l⁻¹; hippuric acid concentration c=1328±30 mg l⁻¹; creatinine concentration c=0.82±0.10 g l⁻¹) were evaluated via the interactive statistical programme IPECA.     

Synthesis of 12-methoxyabietic acid methylester,a feeding deterrent of the larch sawflyPristiphora erichsonii (Hartig)

The diastereomeric methylesters of 12-methoxyabietic acid, an antifeedant for the larch sawflyPristiphora erichsonii (Hartig) have been synthesised from abietic acid methylester (2). The unknown configuration at C-12 of the natural product has been determined by comparing the spectral and analytical data of the synthesised compounds with data given in the literature for the natural product as being (S).

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Synthese von 12-Methoxyabietinsäuremethylester

Zusammenfassung 12-Methoxyabietinsäuremethylester spielt eine Rolle als Repellent beim Freßverhalten der Larven vonPristiphora erichsonii (Hartig). Es wird die erstmalige Synthese (ausgehend) von Abietinsäuremethylester (2, in zwei Stufen) beschrieben. Beide Diastereomere wurden erhalten. Aufgrund von Vergleichen der spektroskopischen und analytischen Daten der Produkte mit den Literaturdaten des Naturproduktes konnte die Konfiguration an C-12 für den Naturstoff mit (S) festgelegt werden.

     

Simultaneous determination of iron(III) and cobalt(II) withN-phenylcinnamohydroxamic acid and thiocyanate by extraction and spectrophotometry

Simple, precise, sensitive, and highly selective methods for the separate determination of iron(III) and cobalt(II) and for the simultaneous determination of both metal ions are described. Iron(III) and cobalt(II) react with thiocyanate in the presence ofN-phenylcinnamohydroxamic acid (PCHA) to form pinkish red and blue coloured complexes, respectively. Both the iron(III) and cobalt(II) complexes having stoichiometric composition of 122 (FeSCN^–PCHA) and 14 (CoSCN^–), respectively, are quantitatively extractable into ethylacetate from 0.5–1.5 M hydrochloric acid solutions. The spectra of iron(III) and cobalt(II) complexes in the visible region exhibit absorption maxima at 495 and 625 nm, respectively. The coloured systems obey Beer's law in the concentration range of 0.2–4.0g/ml of iron and 2–40g/ml of cobalt. The effects of foreign ions and of various experimental parameters were studied to establish the optimum conditions for the extraction and determination of iron and cobalt. The methods have been applied successfully to the analysis of blood, vitamin B₁₂, and standard steels for iron and cobalt.     

Properties of the Macrophomina phaseolina endoglucanase (EGL 1) gene product in bacterial and yeast expression systems

Functional expression of a β-d-1,4 glucanase-encoding gene (egl1) from a filamentous fungus was achieved in both Escherichia coli and Saccharomyces cerevisiae using a modified version of pRS413. Optimal activity of the E. coli-expressed enzyme was found at incubation temperatures of 60°C, whereas the enzyme activity was optimal at 40°C when expressed by S. cerevisiae. Enzyme activity at different pH levels was similar for both bacteria and yeast, being highest at 5.0. Yeast expression resulted in a highly glycosylated protein of approx 60 kDa, compared to bacterial expression, which resulted in a protein of 30 kDa. The hyperglycosylated protein had reduced enzyme activity, indicating that E. coli is a preferred vehicle for production scale-up.     

Properties of mixed monomolecular films of poly(benzyl-methacrylate) and arachidic acid

Binary mixed monomolecular films of poly(benzyl-methacrylate) and arachidic acid at the water/air interface have been studied with respect to compatibility and stability. The surface pressure — area isotherms indicate compatibility of the two components. However, the miscible state is unstable at high surface pressures. This is demonstrated by the constant pressure relaxation of the mixtures. There is a mechanism of separation and nucleation of the arachidic acid from the film for mixtures with high polymer content at a surface pressure of 30 mN/m. For lower concentrations of polymer in the mixtures and at a surface pressure of 20 mN/m the fatty acid is stabilized by the polymer.     

Unexpected transformation of 1,1"-methylenebis(2-naphthol) into 1,2,1",2"-tetrahydrospiro(naphthalene-1,3"-naphtho[2,1-b]pyran)-2-one, a spirodimer of 1,2-naphthoquinone 1-methide, in the presence of boric acid and paraformaldehyde

When heated in benzene with paraformaldehyde and boric acid, 1,1"-methylenebis(2-naphthol) easily decomposes to 1,2-naphthoquinone 1-methide, which dimerizes into a spirodimer. The by-product of the reaction is 14Í-dibenzo[a,j]xanthene.     

Potential of ethylenediaminedi(o-hydroxyphenylacetic acid) and N,N′-bis(hydroxybenzyl)ethylenediamine-N,N′-diacetic acid for the determination of metal ions by capillary electrophoresis

Two aromatic polyaminocarboxylate ligands, ethylenediaminedi(o-hydroxyphenylacetic acid) (EDDHA) and N,N′-bis(hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED), were applied for the separation of transition and heavy metal ions by the ion-exchange variant of electrokinetic chromatography. EDDHA structure contains two chiral carbon centers. It makes it impossible to use the commercially available ligand. All the studied metal ions showed two peaks, which correspond to meso and rac forms of the ligand. The separation of metal–HBED chelates was performed using poly(diallyldimethylammonium) polycations in mixed acetate–hydroxide form. Simultaneous separation of nine single- and nine double-charged HBED chelates, including In(III), Ga(III), Co(II)–(III) and Mn(II)–(III) pairs demonstrated the efficiency of 40 000–400 000 theoretical plates. The separation of Co(III), Fe(III) complexes with different arrangements of donor groups and oxidation of Co(II), Mn(II), Fe(II) ions in reaction with HBED have been discussed.     

On the inverse miniemulsion copolymerization and terpolymerization of acrylamide, <Emphasis Type="Italic">N,N′</Emphasis>-methylenebis(acrylamide) and methacrylic acid

The kinetics of free-radical copolymerization and terpolymerization of acrylamide (AAm), N, N′-methylenebis(acrylamide) (MBA) and methacrylic acid (MA) in the inverse water/monomer/cyclohexane/Tween 85 miniemulsion was investigated. Polymerizable sterically-stable miniemulsions were formulated in cyclohexane as a continuous medium. Polymerizations are very fast and reach the final conversion within several minutes. The dependence of the polymerization rate vs. conversion is described by a curve with two nonstationary rate intervals. The maximum rate of polymerization slightly increases with increasing concentration of crosslinking monomer (MBA) and strongly decreases by the addition of MA. The rate of polymerization is inversely proportional to the 0.9 ^th and 1.8 ^th power of the particle concentration without and with MA, respectively. The number of polymer particles is inversely proportional to the 0.18 ^th and 0.13 ^th power of MBA concentration. The kinetic and colloidal parameters of the miniemulsion polymerization are discussed in terms of microemulsion polymerization model.     

Production of lactic acid from pulp mill solid waste and xylose using Lactobacillus delbrueckii (NRRL B445)

Using the simultaneoussaccharification and fermentation (SSF) technique, pulp mill solid waste cellulose was converted into glucose using cellulase enzyme and glucose into lacticacid using NRRL B445. SSF experiments were conducted at various pH levels, temperatures, and nutrient concentrations, and the lactic acid yield ranged from 86 to 97%. The depletion of xylose in SSF was further investigated by inoculating NRRL B445 into a xylose-only medium. On prolonged incubation, depletion of xylose with lactic acid production was observed. An experimental procedure with a nonglucose medium was developed to eliminate the lag phase. From xylose fermentation, Lactobacillus delbrueckii yielded 88–92% lactic acid and 2–12% acetic acid.     

Study of Y(III), La(III) and Ce(III) solid compounds of 1-phenylazo-2-naphthol-3, 6-disulphonic acid and its p-nitroderivative

The solid compounds of Y(III), La(III) and Ce(III) with 1-phenylazo-2-naphthol-3, 6-disulphonic acid (disodium salt) and itsp-nitroderivative were synthesized. The nature, composition and structure of the isolated solids have been elucidated by elemental analysis, conductometric titrations, UV-Vis and IR. It may be assumed that in both reagents the bond is only due to the sulphonic groups.     

Reaction of 6-methoxybenzo[b]furan-3(2H)-one and benzo[b]thiophen-3(2H)-one with 2-aryl-1,1-dicyanoethylenes as a convenient synthetic route to substituted 2-amino-4-aryl-1,3-dicyano-7-methoxydibenzo[b,d]furans and 2-amino-4-aryl-3-cyano-4H-benzothieno[3,2-b]pyrans

The reactions of 6-methoxybenzo[b]furan-3(2H)-one with 2-aryl-1,1-dicyanoethylenes and malononitrile or with aromatic aldehyde and two moles of malononitrile afford 2-amino-4-aryl-1,3-dicyano-7-methoxydibenzo[b,d]furans. The reactions of benzo[b]thiophen-3(2H)-one with 2-aryl-1,1-dicyanoethylenes or with aromatic aldehyde and one mole of malononitrile produce 2-amino-4-aryl-3-cyano-4H-benzothieno[3,2-b]pyrans.     

Molecular characterization of a gene for aldose reductase (CbXYL1) from Candida boidinii and its expression in Saccharomyces cerevisiae

Candida boidinii produces significant amounts of xylitol from xylose, and assays of crude homogenates for aldose (xylose) reductase (XYL1p) have been reported to show relatively high activity with NADH as a cofactor even though XYL1p purified from this yeast does not have such activity. A gene coding for XYL1p from C. boidinii (CbXYL1) was isolated by amplifying the central region using primers to conserved domains and by genome walking. CbXYL1 has an open reading frame of 966 bp encoding 321 amino acids. The C. boidinii XYL1p is highly similar to other known yeast aldose reductases and is most closely related to the NAD(P)H-linked XYL1p of Kluyveromyces lactis. Cell homogenates from C. boidinii and recombinant Saccharomyces cerevisiae were tested for XYL1p activity to confirm the previously reported high ratio of NADH:NADPH linked activity. C. boidinii grown under fully aerobic conditions showed an NADH:NADPH activity ratio of 0.76, which was similar to that observed with the XYL1p from Pichia stipitis XYL1, but which is much lower than what was previously reported. Cells grown under low aeration showed an NADH:NADPH activity ratio of 2.13. Recombinant S. cerevisiae expressing CbXYL1 showed only NADH-linked activity in cell homogenates. Southern hybridization did not reveal additional bands. These results imply that a second, unrelated gene for XYL1p is present in C. boidinii.     

Kinetic and mechanistic studies of the iridium(III) catalyzed oxidation of sulphanilic acid by diperiodatocuprate(III) in aqueous alkaline medium

The effect of La₂O₃, K₂O and Li₂O on the properties and catalytic performance of silica-supported nickel catalysts for the hydrogenation of m-dinitrobenzene was investigated. The catalysts promoted with La₂O₃, Li₂O and K₂O showed better catalytic performance than the catalyst without promotion, especially the ones co-promoted with La₂O₃ and K₂O or Li₂O.     

Mechanisms of oxidation of K4Fe(CN)6 by hypochlorous acid and catalytic activation by azide, bromide, and iodide

Summary HOCl reacts with Fe(CN) ₆ ^4– to generate an intermediate, presumably FeCl(CN) ₆ ^3– , which exhibits a weak absorption around 282 nm and decays simultaneously with the formation of Fe(CN) ₆ ^3– . When decreasing the HOCl/Fe(CN) ₆ ^4– concentration ratio fromR>1 toR<1, a drastic change in the kinetics of the oxidation is observed. Depending onR, the intermediate obviously oxidizes either Fe(CN) ₆ ^4– or HOCl. AtR1, a further intermediate appears which also precedes the oxidation and absorbs strongly around 360 nm. The intermediates detected may represent reactive high oxidation states of iron (Fe(IV) and Fe(VI)). HOCl induced oxidation of Fe(CN) ₆ ^4– is activated catalytically by Br^–, I^–, and N ₃ ^– , obviously due to generation of stronger oxidants (HOBr, HOI, and ClN₃), but oxidation is efficiently inhibited by CN^– and NCS^–.

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Mechanismen der Oxidation von K₄Fe(CN)₆ durch Hypochlorsäure und katalytische Aktivierung durch Azid, Bromid und Iodid

Zusammenfassung HOCl reagiert mit Fe(CN) ₆ ^4– unter Bildung eines Intermediats, vermutlich FeCl(CN) ₆ ^3– , das bei 282 nm eine schwache Absorption aufweist und parallel zum Erscheinen von Fe(CN) ₆ ^3– verschwindet. Man beobachtet eine drastische Änderung in der Oxidationskinetik, wenn das HOCl/Fe(CN) ₆ ^4– Konzentrationsverhältnis vonR>1 zuR<1 verändert wird. In Abhängigkeit vonR wird offenbar entweder Fe(CN) ₆ ^4– oder HOCl durch das Intermediat oxidiert. FürR1 erscheint ein weiteres Intermediat mit einer starken Absorptionsbande bei 360 nm, das ebenfalls der Oxidation vorangeht. Bei den beobachteten Intermediaten handelt es sich vermutlich um reaktive höhere Oxidationsstufen des Eisens (Fe(IV) und Fe(VI)). Die HOCl-induzierte Oxidation von Fe(CN) ₆ ^4– wird einerseits durch Br^–, I^– und N ₃ ^– katalytish aktiviert (offenbar infolge der Bildung stärkerer Oxidantien wie HOBr, HOI und ClN₃), andererseits durch CN^– und NCS^– effektiv inhibiert.

     

Hydroacridines XXV [1]. First Synthesis of (4aα,8aα,9aβ,10aβ)-Tetradecahydroacridine and New Syntheses of (4aα,8aα,9aα,10aβ)- and (4aα,8aβ,9aα,10aβ)-Tetradecahydroacridine

Summary. On heating in dry DMSO, in the presence of potassium t-butoxide, the N-nitrosamine of (4aα,8aβ,9aβ,10aα)-tetradecahydroacridine is completely converted into the N-nitrosamine of (4aα,8aα,9aα,10aβ)-tetradecahydroacridine. Under similar conditions, the N-nitrosamine of (4aα,8aα,9aβ,10aα)-tetradecahydroacridine yields a ternary equilibrium mixture containing itself (19%), and the N-nitrosamines of (4aα,8aβ,9aα,10aβ)-tetradecahydroacridine (46%) and the so far unknown (4aα,8aα,9aβ,10aβ)-tetradecahydroacridine (35%). The resulting N-nitrosamines can be smoothly denitrosated to the corresponding secondary amines.     

Molecular and crystal structure of 2-cyano-(2E)-pentadien-2,4-oic acid and ethyl-2-cyano-(2E)-pentadien-2,4-oate

X-ray study of 2-cyano-(2E)-pentadien-2,4-oic acid (1) and its ethyl ester (2) showed that the molecules of1 and2 in the crystalline phase form stacks by translating along the shortest crystallographic axis. The nature of the intermolecular interactions favoring the formation of such β-structures was analyzed within the framework of the Bader topological theory. Possible routes of topochemical reactions of compounds1 and2 are considered. Dedicated to the memory of Academician M. I. Kabachnik on his 90th birthday. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 10, pp. 1991–1995, October, 1998.