Production of recombinant bleaching enzymes from thermophilic microorganisms in fungal hosts

Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host-vector expression system critical. We have developed two systems for the bulk production of bleaching enzymes from thermophiles. Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2μ-like plasmid pKD1 of Kluyveromyces drosophilarium. Our second system involves the filamentous fungus Trichoderma reesei. Fusion and nonfusion vectors have been constructed using the strong cellobiohydrolase 1 (cbh1) promoter. The KEX2 protease cleavage site and a 6 × HIS-tag have been incorporated to facilitate both cleavage and purification of the mature foreign proteins.     

Palladium-arene interactions in catalytic intermediates: an experimental and theoretical investigation of the soft rearrangement between eta1 and eta2 coordination modes

A series of dichloro-bridged arylbicycloheptylpalladium complexes have been synthesized and characterized by means of NMR spectroscopy. The compound [(C16H19)PdCl]2*CH2Cl2 with ortho and para methyl substituents at the arene has been characterized by means of X-ray diffraction techniques. The C(ipso) atom of the arene lies almost at the fourth planar coordination site of the metal [Pd-C(ipso) = 2.22(1) A (average)], and due to the arene's tilting, the substituted C(ortho) atom is relatively close to the metal atom [2.54(1) A (average)]. The coordinated C(ipso)-C(ortho) linkage, in a seemingly dihapto coordination, is anti with respect to the CH2 bridge of the bicycloheptyl unit. Variable-temperature NMR experiments for the para-substituted dimer 9 reveal restricted rotation of the two aryl groups about the corresponding C-C(ipso) bonds (DeltaE < or =17 kcal x mol(-1)). DFT-B3LYP calculations have been carried out on the known and similar monomer (phenylbicycloheptenyl)Pd(PPh3)I (4) and its related substituted derivatives. The essential results are as follows: (i) The potential energy surface for twisting the phenyl ring away from the symmetric eta(1) coordination in 4 is very flat (DeltaE < or = 1 kcal x mol(-1)) whereas an Atoms in Molecules analysis excludes the existence of an actual Pd-C(ortho) bond in the seemingly eta2-type conformer. (ii) Complete rotation of the unsubstituted phenyl ring is not facile but feasible. A significant strain affects the transition-state structure featuring a Pd-HC(aryl) agostic-type bond. The calculated destabilization of 10.3 kcal x mol(-1), with respect to the ground state, can be compared to the experimental barrier of the dimer 9. (iii) Various methyl-substituted derivatives of 4 have been optimized, and their structural and energetic trends are discussed. An almost ideal eta1 coordination is shown by the anti conformer of the C(ortho)-substituted complex due steric effects. For all of the other cases, a slipped eta2 coordination may be described. As a general conclusion, the unsaturated metal center receives pi electron density of the arene mainly through its C(ipso) atom. The effect may be slightly improved if the C(ortho) atom also gets closer to the metal, but in no case, does the slipped eta2 coordination seem to be crucial for the stability of the system.     

Interpretation of the Metabolic Enthalpy Change, ΔHmet,Calculated for Microbial Growth Reactions in Soils

The microcalorimetric method was used to calculate the metabolic enthalpy change per mol of glucose degraded by soil microorganisms, ΔH _met. This parameter has been calculated by microcalorimetry for many organic, inorganic and biochemical reactions, but there is only some information about its quantification for microbial growth reactions in soils. Values of ΔH _met were calculated for different soil samples collected in Galicia (Spain) and Campinas (Săo Paolo, Brazil). Exponential microbial growth was stimulated in all soil samples by the addition of glucose and power-time curves were recorded. Results showed changes in the values of ΔH _met calculated for all the soil samples, suggesting a dependence of this value with the microbial growth rate constant, with the percentage of growth, with the initial number of microorganisms of soil samples, with the quantity of glucose added and with the strain of bacteria growing in soil. The interpretation of variations of ΔH _met provides important qualitative and quantitative information. It reports data that allow to interpret from a qualitative point of view, the increase in biomass as a consequence of the degradation of the organic matter in soil, to understand changes in the percentages of soil organic matter and to know if the microbial population growing in differential soil samples is homogeneous. Therefore, to report that value would be very important in ecological studies, but beforehand, it is necessary to solve some problems that can appear in the experiments done to make the quantification . This revised version was published online in August 2006 with corrections to the Cover Date.     

Carbon fiber cylindrical microelectrode-based detector for the determination of antithyroid drugs

An amperometric flow-injection method for the determination of antithyroid drugs such as 6-methyl-2-thiouracil (MTU) using a carbon fiber cylindrical microelectrode-based detector is reported. A home-made flow-cell specially adapted for working with cylindrical microelectrodes ranging between 4 and 10 mm in length was used for this purpose. Methanol containing 0.05 mol l(-1) tetrabutylammonium perchlorate as the supporting electrolyte was employed, and a potential of +1.6 V was selected for the amperometric detection of MTU. No cleaning or electrode surface regeneration of the fiber was necessary during the whole working day. A limit of detection of 2.6x10(-7) mol l(-1) (37 mug MTU l(-1)) was achieved. The method was applied to the determination of MTU in spiked feed samples (peas and corn flour), at the 142 mug g(-1) level. The procedure involved the extraction of the drug in methanol, a clean-up step using Florisil cartridges and the use of the standard additions method. Recoveries of 81+/-6 and 85+/-8% were obtained for peas and corn flour, respectively.     

Positive and negative ion chemistry of the anesthetic halothane (1-bromo-1-chloro-2,2,2-trifluoroethane) in air plasma at atmospheric pressure

The ion chemistry of 1-bromo-1-chloro-2,2,2-trifluoroethane (the common anesthetic halothane) in air plasma at atmospheric pressure was investigated by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). The major positive ion observed at low declustering (API interface) energies is the ionized dimer, M(+.)M, an unexpectedly abundant species which possibly is stabilized by two H-bonding interactions. At higher energies [M--HF](+.) and [M--Br](+) prevail; the former, corresponding to ionized olefin [ClBrC=CF(2)](+.), appears to originate from M(+.)M and is quite stable towards fragmentation. The latter fragment ion ([M--Br](+)) and its analogue, [M--Cl](+), which is also observed though at much lower abundance, are originally ethyl cations (+)CHX--CF(3) (X = Br, Cl) which, upon collisional activation, rearrange and fragment to CHFX(+) via elimination of CF(2). All of the above described ions are also observed in humid air: in addition, the oxygenated ion [ClBrC=CFOH](+.) also forms in humid air via water addition to [ClBrC=CF(2)](+.) and HF elimination, as observed earlier for ionized trichloroethene. In contrast with similar chloro- and fluoro-substituted ethanes, halothane does not react with H(3)O(+) in the APCI plasma, a result confirmed by selected ion APCI triple-quadrupole (TQ) experiments. Major negative ions formed from halothane in the air plasma are Br(-) and, to a lesser extent, Cl(-), and their complexes with neutral halothane. APCI-TQ experiments indicated that Br(-) and Cl(-) are formed via reaction of halothane with O(2) (-.), O(2) (-.)(H(2)O) and O(3) (-.), possibly via dissociative electron transfer or nucleophilic substitution. Competing proton transfer was also observed in the reaction with O(2) (-.) and, at high halothane pressure, also with O(2) (-.)(H(2)O); at lower pressures the molecular anion M(-.) was observed instead. The other minor anions of the air plasma, NO(2) (-), N(2)O(2) (-.) and NO(3) (-), were found to be unreactive towards halothane.     

Influence of the pyridine methyl substituent position on the chemical changes of the NCS groups in the thermal decomposition of coordination compounds of the type Cu(NCS)2L2

The influence of the position of the CH₃ group in picoline and lutidine ligands on the degree of chemical change of the NCS groups in coordination compounds of the type Cu(NCS)₂L₂ (whereL=2-, 3- and 4-picoline, and 2,3-, 2,4-, 2,5-, 2,6-, 3,4- and 3,5-lutidine) is dealt with. The most marked effect of the CH₃ group is found to be exerted in position 4. This effect of the methyl group on the degree of chemical change points to the mutual influence of the ligands in coordination compounds of Cu(II).

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Zusammenfassung Der Artikel befaßt sich mit dem Einfluß der Lage der CH₃ Gruppe in Pikolinen und Lutidinen als Liganden auf den Grad der chemischen Änderungen der Gruppen NSC in Koordinationsverbindungen des Typs Cu(NCS)₂L₂ (L=2-, 3- und 4-Pikoline, 2,3-, 2,4-, 2,5-, 2,6-, 3,4- und 3,5-Lutidine). Der ausgeprägteste Effekt der CH₃ Gruppe wurde in der Position 4 beobachtet. Dieser Einfluß der Methylgruppe auf das Ausmaß der chemischen Änderungen deutet auch auf die gegenseitige Wirkung der Liganden in Koordinationsverbindungen von Cu(II).

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Résumé L'article a trait à l'influence de la position du groupe CH₂ dans les picolines et lutidines, en tant que ligands, sur le degré des changements chimiques des groupes SCN dans les composés de coordination du type Cu(SCN)₂L₂ (L=2-, 3 et 4-picoline, 2,3-, 2,4-,2,5-, 2,6-, 3,4- et 3,6-lutidine). L'effet le plus prononcé du groupe CH₃ s'observe en position 4. Cette influence du groupe méthyle sur le degré des changements chimiques indique aussi l'influence mutuelle des ligands dans les composés de coordination du Cu(II).

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- , , NCS Cu(NCS)₂,L₂, L=2-, 3- 4- , 2.3-, 2.4-, 2.5-, 2.6-, 3.4- 3.5-. , 4. Cu(II).

     

Synthesis, structure and conformation of partially-modified retro- and retro-inverso psi[NHCH(CF3)]Gly peptides

Partially modified retro- (PMR) and retro-inverso (PMRI) psi[NHCH(CF(3))]Gly peptides, a conceptually new class of peptidomimetics, have been synthesized in wide structural diversity and variable length by aza-Michael reaction of enantiomerically pure alpha-amino esters and peptides with enantiomerically and geometrically pure N-4,4,4-trifluorocrotonoyl-oxazolidin-2-ones. The factors underlying the observed moderate to good diastereocontrol have been investigated. The conformations of model PMR-psi[NHCH(CF(3))]Gly tripeptides have been studied in solution by (1)H NMR spectroscopy supported by MD calculations, as well as in the solid-state by X-ray diffraction. Remarkable stability of turn-like conformations, comparable to that of parent malonyl-based retropeptides, was evidenced, as a likely consequence of two main factors: 1) severe torsional restrictions about sp(3) bonds in the [CO-CH(2)-CH(CF(3))-NH-CH(R)-CO] module, which is biased by the stereoelectronically demanding CF(3) group and the R side chain; 2) formation of nine-membered intramolecularly hydrogen-bonded rings, which have been clearly detected both in CHCl(3) solution and in some crystal structures. The former factor seems to be more important, as turn-like conformations were found in the solid-state even in the absence of intramolecular hydrogen bonding. The relative configuration of the -C*H(CF(3))NHC*H(R)- stereogenic centers has a major effect on the stability of the turn-like conformation, which seems to require a syn stereochemistry. X-ray diffraction and ab initio computational studies showed that the [-CH(CF(3))NH-] group can be seen as a sort of hybrid between a peptide bond mimic and a proteolytic transition state analogue, as it combines some of the properties of a peptidyl -CONH- group (low NH basicity, CH(CF(3))-NH-CH backbone angle close to 120 degrees, C-CF(3) bond substantially isopolar with the C=O) with some others of the tetrahedral intermediate [-C(OX)(O(-))NH-] involved in the protease-mediated hydrolysis reaction of a peptide bond (high electron density on the CF(3) group, tetrahedral backbone carbon).     

Bicyclic γ-butyrolactones. Relation between conformation of the lactone ring and chiroptical properties

The CD curves of a set of condensed γ-butyrolactones have been investigated. A simple correlation between the sign of the Cotton effect (CE) and the configuration of C can be deduced from the resulting data.     

X-ray diffraction and solid-state NMR studies of a germanium binuclear complex

A compound formulated as (C4H12N2)[Ge2(pmida)2(OH)2] x 4 H2O (where pmida(4-) = N-(phosphonomethyl)iminodiacetate and C4H12N2(2+) = piperazinedium cation), containing the anionic [Ge2(pmida)2(OH)2]2- complex, has been synthesised by the hydrothermal approach and its structure determined by single-crystal X-ray diffraction analysis. Several high-resolution solid-state magic-angle spinning (MAS) NMR techniques, in particular two-dimensional 1H-X(13C,31P) heteronuclear correlation (HETCOR) and 1H-1H homonuclear correlation (HOMCOR) experiments incorporating a frequency-switched Lee-Goldburg (FS-LG) decoupling scheme, have been employed for the first time in such a material. Using these tools in tandem affords an excellent general approach to study the structure of other inorganic-organic hybrids. We assigned the NMR resonances with the help of C...H and P...H internuclear distances obtained through systematic statistical analyses of the crystallographic data. The compound was further characterised by powder X-ray diffraction techniques, IR and Raman spectroscopy, and by elemental and thermal analyses (thermogravimetric analysis and differential scanning calorimetry).     

Heterodinuclear Ln[bond]Na complexes with an asymmetrical macrocyclic compartmental Schiff base

Heterodinuclear lanthanide(III)-sodium(I) complexes [LnNa(L)(Cl)(2)(CH(3)OH)] (Ln=La[bond]Nd, Sm[bond]Lu), where H(2)L is a [1+1] asymmetric compartmental macrocyclic ligand containing a N(3)O(2) Schiff base and a O(3)O(2) crown-ether-like coordination site, have been prepared and characterized by IR, (1)H, (13)C, and (23)Na NMR spectroscopy, mass spectrometry, and electron microscopy. In the solid state, the lanthanide(III) ions coordinate the Schiff-base N(3)O(2) site, and the sodium ion occupies the O(3)O(2) crownlike cavity, as shown by the X-ray crystal structures of the Nd, Eu, Gd, and Yb derivatives. In these complexes, the lanthanide(III) ion is coordinated by two chlorine atoms in the trans position and by three nitrogen and two negatively charged phenol oxygen atoms of the Schiff base, and the ion is heptacoordinated with a pentagonal bipyramidal geometry. The sodium ion is coordinated by three etheric oxygen atoms and the two phenolic oxygens that act as a bridge. A methanol molecule is also coordinated in the apical position of the resulting pentagonal pyramidal polyhedron. A detailed (1)H and (13)C NMR study was carried out in CD(3)OD for both diamagnetic and paramagnetic heterodinuclear complexes [LnNa(L)(Cl)(2)(CH(3)OH)]. The complexes are also isostructural in solution, and their structures parallel those found in the solid state. Moreover, some significative distances determined in the solid state and in solution are comparable. Finally, the potential use of these complexes as molecular probes for the selective recognition of specific metal ions has been tested. In particular, their ability to act as shift reagents and the selectivity of the O(3)O(2) site towards Li(+), Ca(2+), and K(+) were investigated by (23)Na NMR spectroscopy.