Immobilization of Pseudomonas fluorescens Lipase onto Magnetic Nanoparticles for Resolution of 2-Octanol

The lipase from Pseudomonas fluorescens (Lipase AK, AKL) was immobilized onto the magnetic Fe₃O₄ nanoparticles via hydrophobic interaction. Enzyme loading and immobilization yield were determined as 21.4?±?0.5?mg/g and 49.2?±?1.8?%, respectively. The immobilized AKL was successfully used for resolution of 2-octanol with vinyl acetate used as acyl donor. Effects of organic solvent, water activity, substrate ratio, and temperature were investigated. Under the optimum conditions, the preferred isomer for AKL is the (R)-2-octanol and the highest enantioselectivity (E?=?71.5?±?2.2) was obtained with a higher enzyme activity (0.197?±?0.01???mol/mg/min). The results also showed that the immobilized lipase could be easily separated from reaction media by the magnetic steel and remained 89?% of its initial activity as well as the nearly unchanged enantioselectivity after five consecutive cycles, indicating a high stability in practical operation.     

The biosynthesis of the antibiotic obafluorin from p-aminophenylalanine in Pseudomonas fluorescens

The separate units which are used to construct the unique β-lactone antibiotic obafluorin (1) in Pseudomonas fluorescens are defined by the results of [U-¹³C]glucose incorporation. A key intermediate in the biosynthesis of (1) is established to be L-p-aminophenylalanine (7); L-p-nitrophenylalanine (8) is a relatively insignificant precursor. Similar results were obtained for p-nitrophenylacetic acid (9) which is also a metabolite of Ps. fluorescens. L-phenylalanine is an insignificant precursor for obafluorin (1).     

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.     

Characterization of the mupirocin biosynthesis gene cluster from Pseudomonas fluorescens NCIMB 10586

The polyketide antibiotic mupirocin (pseudomonic acid) produced by Pseudomonas fluorescens NCIMB 10586 competitively inhibits bacterial isoleucyl-tRNA synthase and is useful in controlling Staphylococcus aureus, particularly methicillin-resistant Staphylococcus aureus. The 74 kb mupirocin biosynthesis cluster has been sequenced, and putative enzymatic functions of many of the open reading frames (ORFs) have been identified. The mupirocin cluster is a combination of six larger ORFs (mmpA-F), containing several domains resembling the multifunctional proteins of polyketide synthase and fatty acid synthase type I systems, and individual genes (mupA-X and macpA-E), some of which show similarity to type II systems (mupB, mupD, mupG, and mupS). Gene knockout experiments demonstrated the importance of regions in mupirocin production, and complementation of the disrupted gene confirmed that the phenotypes were not due to polar effects. A model for mupirocin biosynthesis is presented based on the sequence and biochemical evidence.     

Cloning and characterization of Pfl_1841, a 2-methylenebornane synthase in Pseudomonas fluorescens PfO-1

The pfl_1841 gene from Pseudomonas fluorescens PfO-1 is the only gene in any of the three sequenced genomes of the Gram-negative bacterium P. fluorescens, that is, annotated as a putative terpene synthase. The predicted Pfl_1841 protein, which harbors the two strictly conserved divalent metal binding domains found in all terpene cyclases, is closely related to several known or presumed 2-methylisoborneol synthases, with the closest match being to the MOL protein of Micromonaspora olivasterospora KY11048 that has been implicated as a 2-methylenebornane synthase. A synthetic gene encoding P. fluorescens Pfl_1841 and optimized for expression in Escherichia coli was expressed and purified as an N-terminal His₆-tagged protein. Incubation of recombinant Pfl_1841 with 2-methylgeranyl diphosphate produced 2-methylenebornane as the major product accompanied by 1-methylcamphene as well as other minor, monomethyl-homomonoterpene hydrocarbons and alcohols. The steady-state kinetic parameters for the Pfl_1841-catalyzed reaction were K_M=110±13 nM and k_cat=2.4±0.1×10⁻² s⁻¹. Attempts to identify the P. fluorescens SAM-dependent 2-methylgeranyl diphosphate synthase have so far been unsuccessful.     

Effects of operating conditions on the adhesive strength of Pseudomonas fluorescens biofilms in tubes

Understanding the mechanical properties of biofilms, especially the force required to disrupt them and remove them from substrata is very important to development of antibiofouling strategies. In this work, a novel micromanipulation technique with a specially designed T-shaped probe has been developed to serve as an experimental means to measure directly the adhesive strength of biofouling deposits on the surface of a glass test stud. The basic principle of this novel technique is to pull away a whole biofilm accumulated on the surface of a glass test stud with T-shaped probe, and to measure simultaneously the force imposed on the biofilm. The adhesive strength between the biofilms and the surface to which they are attached, is defined as the work per unit area required to remove the biofilms from the surface. The biofouling experiments were performed on an elaborate design of a simulated heat exchanger system. A monoculture of Pseudomonas fluorescens was chosen as the fouling microorganism for the laboratory studies. Results indicate that the adhesive strength of the biofilm was affected by the conditions of operation, such as biofilm age, nutrient concentration, suspended cell concentration, pH, surface roughness of the substratum and fluid velocity. As noted, the effect of fluid velocity on the biofilm adhesive strength seemed to overwhelm other factors. At the same operating conditions, the biofilm adhesive strength increased as the fluid velocity increased within the range of 0.6-1.6m/s. In addition, the flow-related biofilm structures were observed that biofilms generally grew as a more compact pattern at the higher fluid velocity. Apparently, the fluid velocity can affect the biofilm structure, which in turn determines the biofilm adhesive strength. The knowledge of the biofilm adhesive strength with associated influences of the operating conditions may be used to define better cleaning procedures.     

Aldol Reactions of the trans-o-Hydroxybenzylidenepyruvate Hydratase-Aldolase (tHBP-HA) from Pseudomonas fluorescens N3

In this paper, a recombinant trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (tHBP-HA) of Pseudomonas fluorescens N3 was used as a new catalyst for aldol condensation reactions. The reaction of some aldehydes with a different electronic activation catalyzed by tHBP-HA is presented and discussed together with some hints on the product structure. The enzyme is strictly pyruvate-dependent but uses different aldehydes as acceptors. The structure of the products is highly dependent on the electronic characteristics of the aldehyde. The results are interesting for both their synthetic importance and the mechanism of the formation of the products. Not only the products obtained and the recognition power are reported, but also some characteristics of its mechanism are analyzed. The results clearly show that the enzyme is efficiently prepared, purified, and stored, that it recognizes many different substrates, and that the products depend on the substrate electronic nature.     

Oscillatory regime during catalytic formation and consumption of oxygen in the system H2O2-ascorbic acid-CuTAAB2+

Using potentiometric, amperometric, and specific methods we studied oscillations in the oxygen concentration in the catalytic system ascorbic acid-O₂-H₂O₂-CuTAAB²⁺ (TAAB = tetrabenzo-[b, f, j, n][5, 9, 13]-tetraazacycIoheoadecine). We determine the conditions for existence of the oscillatory regime and we discuss the nature of the oscillations. We plot attractors for this system, confirming the presence of a chaotic oscillation regime in the course of this reaction.L. V. Pisarzhevskii Institute of Physical Chemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from hTeoreticheskaya i Éksperimental'naya Khimii. Vol. 27, No, l, pp. 39–45, January–February 1991. Original article submitted March 14, 1989.     

Simulation of growth and corrosion during the formation of a passive layer

From the analysis of experiments relative to the formation of a passive layer at the Li-solvent interface a model is proposed in order to describe the gross features concerning the growth of this layer. In this model the growth is initiated by chemical reactions partially counterbalanced by a poisoning process. The model which works at a mesoscopic scale is simulated on a two-dimensional lattice. A quantitative analysis of the results shows that this model appears as the superposition of the Eden model which describes the structure of the front and a similar percolation process which determines the properties of the bulk part of the layer as its porosity. A second model in which the corrosion is taken into account is introduced. It is analyzed in a very simple case in which there is no poisoning. The corrosion induces a restructuring in the layer and this gives rise to a porosity in the bulk part of the layer. Some unexpected results are interpreted.     

Effect of surface melting on the formation and growth of nanocrystals in the Bi2O3-Fe2O3 system

Formation of nanocrystals in the Bi₂O₃-Fe₂O₃ system prepared by the co-precipitation of bismuth and iron hydroxides has been studied. The temperature of onset of the BiFeO₃ and Bi₂Fe₄O₉ nanocrystals formation is correlated with the melting point of the non-autonomous phases. The optimal temperature of BiFeO₃ and Bi₂Fe₄O₉ nanoparticles synthesis is 460–520 and 500–550°C, respectively.     

Glass formation in the ZnCl2-H2O system

A scheme of structural groups responsible for glass formation in the ZnCl₂-H₂O system was proposed. Possible glass formation mechanisms in the ZnCl₂-H₂O system and in glassy ZnCl₂ were compared.     

Crystal formation and growth during the hydrothermal synthesis of beta-Ni(OH)2 in one-dimensional nano space

Hydrothermal synthesis of beta-Ni(OH)(2) was performed inside uniform carbon-coated nanochannels of an anodic aluminium oxide film. The time course of crystal formation and growth of Ni(OH)(2) in such one-dimensional nano space was observed using transmission electron microscopy (TEM), and the changes in the number and size of crystals with the hydrothermal reaction period were quantitatively analyzed using the TEM images. Moreover, the effect of the channel size (25, 100 and 300 nm in diameter) on the crystal growth was examined. In the early stage of the reaction, the crystal formation and growth of beta-Ni(OH)(2) in the one-dimensional channels took place in the same manner as in conventional hydrothermal synthesis. However, except for the 300 nm-channels, further crystal growth was hampered by the spatial restriction, and it allowed only the growth toward the channel axis. In the case of the 25 nm-channels, many Ni(OH)(2) crystals of less than 40 nm formed initially, but slowly disappeared except for a few that grew larger at the expense of the small crystals. This finding clearly indicates that the crystal growth of Ni(OH)(2) during the whole hydrothermal process was governed by the Ostwald ripening. With this mechanism and the spatial restriction, single crystals of beta-Ni(OH)(2) nanorods with a length of over 150 nm were finally formed.     

Study of the interaction and activation of lipase from Pseudomonas fluorescens in surfactant monolayers and precipitates

The activation of lipase from Pseudomonas fluorescens (PFL) upon its immobilization in surfactant coprecipitates (hexadecane-1,2-diol (HDD), cetyl alcohol (CetOH), N-cetylacetamide (CetAA), and cetylamine (CetNH₂)) organized in monolayers at the interface were studied by the Langmuir—Blodgett monolayer technique. Incorporation of the enzyme into surfactant monolayers at the surface pressure = 10 mN m^–1 results in an apparent increase in the area per molecule. In the series of noncharged surfactants CetOH—HDD—CetAA, this effect increases in proportion to the amount of the enzyme incorporated in the monolayer. The catalytic activity of the lipase—surfactant coprecipitates in an organic solvent as regards esterification increases in the same sequence, indicating similarity of the interaction of lipase with surfactant monolayers and coprecipitates. For = 10 mN m^–1, the CetNH₂ monolayer with liquid-expanded state incorporates the largest amount of the enzyme (PFL : CetNH₂ = 1 : 290); the CetOH monolayer, which exists in the condensed state under the same conditions, incorporates the smallest amount (PFL : CetOH = 1 : 1700). The hydrolytic activity of PFL in mixed monolayers with surfactants increases 1.5—11-fold; the esterification activity in surfactant coprecipitates, 1.6—9-fold. The lipase activation effects are explained by facilitated transport of substrates into mixed monolayers and surfactant—enzyme precipitates in aqueous and organic media, respectively.     

Mutations in distant residues moderately increase the enantioselectivity of Pseudomonas fluorescens esterase towards methyl 3bromo-2-methylpropanoate and ethyl 3phenylbutyrate

Directed evolution combined with saturation mutagenesis identified six different point mutations that each moderately increases the enantioselectivity of an esterase from Pseudomonas fluorescens (PFE) towards either of two chiral synthons. Directed evolution identified a Thr230Ile mutation that increased the enantioselectivity from 12 to 19 towards methyl (S)-3-bromo-2-methylpropanoate. Saturation mutagenesis at Thr230 identified another mutant, Thr230Pro, with higher-than-wild-type enantioselectivity (E=17). Previous directed evolution identified mutants Asp158Asn and Leu181Gln that increased the enantioselectivity from 3.5 to 5.8 and 6.6, respectively, towards ethyl (R)-3-phenylbutyrate. In this work, saturation mutagenesis identified other mutations that further increase the enantioselectivity to 12 (Asp158Leu) and 10 (Leu181Ser). A homology model of PFE indicates that all mutations lie outside the active site, 12-14 A from the substrate and suggests how the distant mutations might indirectly change the substrate-binding site. Since proteins contain many more residues far from the active site than close to the active site, random mutagenesis is strongly biased in favor of distant mutations. Directed evolution rarely screens all mutations, so it usually finds the distant mutations because they are more common, but probably not the most effective.