Reduction of nitroaromatic compounds mediated by Streptomyces sp. exudates.

Exudates from Streptomyces griseoflavus Tü 2484 effectively mediated electron transfer between hydrogen sulfide and various nitrobenzenes. In general, pseudo-first-order kinetics were observed, except for the initial phase of the reaction at higher pH values. Under fixed pH and Dh conditions, linear free energy relationships were found between the logarithms of the reaction rate constants and the one-electron reduction potentials of the nitroaromatic compounds. No competition was observed between various compounds. Comparison of the results of this study with the results of experiments conducted with model quinones and an iron porphyrin suggest that the secondary metabolites cinnaquinone and dicinnaquinone, excreted by strain Tü 2484 on the order of 100 mg/liter, are responsible for the catalytic activity of the exudate. Further support for this hypothesis comes from the facts that the catalytic activity of the exudate became prominent only after the growth phase of the microorganisms and that the mediating substances have a molecular weight of less than 3,000.     

Stereo- and Regioselective Hydroxylation of α-Ionone by Streptomyces Strains

A total of 215 Streptomyces strains were screened for their capacity to regio- and stereoselectively hydroxylate β- and/or α-ionone to the respective 3-hydroxy derivatives. With β-ionone as the substrate, 15 strains showed little conversion to 4-hydroxy- and none showed conversion to the 3-hydroxy product as desired. Among these 15 Streptomyces strains, S. fradiae Tü 27, S. arenae Tü 495, S. griseus ATCC 13273, S. violaceoniger Tü 38, and S. antibioticus Tü 4 and Tü 46 converted α-ionone to 3-hydroxy-α-ionone with significantly higher hydroxylation activity compared to that of β-ionone. Hydroxylation of racemic α-ionone [(6R)-(−)/(6S)-(+)] resulted in the exclusive formation of only the two enantiomers (3R,6R)- and (3S,6S)-hydroxy-α-ionone. Thus, the enzymatic hydroxylation of α-ionone by the Streptomyces strains tested proceeds with both high regio- and stereoselectivity.     

Circumventing the Effect of Product Toxicity: Development of a Novel Two-Stage Production Process for the Lantibiotic Gallidermin

Lantibiotics such as gallidermin are lanthionine-containing polypeptide antibiotics produced by gram-positive bacteria that might become relevant for the treatment of various infectious diseases. So far, self-toxicity has prevented the isolation of efficient overproducing strains, thus hampering their thorough investigation and preventing their exploitation in fields other than the food area. We wanted to investigate the effect of lantibiotic precursor peptides on the producing strains in order to evaluate novel strategies for the overproduction of these promising peptides. In this study, gallidermin was chosen as a representative example of the type A lantibiotics. A Staphylococcus gallinarum Tü3928 mutant, whose gene for the extracellular pregallidermin protease GdmP was replaced by a kanamycin-resistance gene, was constructed. Mass spectrometry (MS) analysis indicated that this mutant produced fully posttranslationally modified gallidermin precursors with truncated versions of the leader peptide, but not the entire leader as predicted from the gdmA sequence. In filter-on-plate assays, these truncated pregallidermins showed no toxicity against Staphylococcus gallinarum Tü3928 up to a concentration of 8 g/liter (corresponding to approximately 2.35 mM), while gallidermin produced clear inhibitory zones at concentrations as low as 0.25 g/liter (0.12 mM). We showed that the lack of toxicity is due entirely to the presence of the truncated leader, since MS as well as bioassay analysis showed that the peptides resulting from tryptic cleavage of pregallidermins and gallidermin produced by S. gallinarum Tü3928 had identical masses and approximately the same specific activity. This demonstrates that even a shortened leader sequence is sufficient to prevent the toxicity of mature gallidermin. In nonoptimized fermentations, the gdmP mutant produced pregallidermin to a 50%-higher molar titer, suggesting that the absence of self-toxicity has a beneficial effect on gallidermin production and giving a first confirmation of the suitability of the overproduction strategy.     

FURTHER OBSERVATIONS ON THE MECHANISM OF PHAGE ACTION

1. The reaction between an antistaphlycoccal phage and the homologous bacterium has been studied, applying the following experimental technics not used in earlier work reported from this laboratory: (a) Both the activity assay and the plaque count were utilized for determining [phage]. (b) Sampling was done at short intervals; i.e., every 0.1 hour. (c) Extracellular phage was separated from the cell-bound fraction by a filtration procedure permitting passage of < 95 per cent of free phage. 2. Using these technics, the reaction was followed: (a) with pH maintained at 6.10 and temperature at 28°C. to slow the process; (b) with pH maintained at 7.2 and temperature at 36°C. 3. In addition separate experiments were performed on the sorption of phage by bacteria at 30°, 23°, and 0°C. 4. At pH 6.10 and 28°C. the phage-bacterium reaction proceeds in the following sequence: (a) There is an initial phase of rapid logarithmic sorption of phage to susceptible cells, during which the total phage activity and the plaque numbers in the mixtures remain constant. (b) When 90 per cent of the phage has been bound, there is a sudden very rapid increase in phage activity not paralleled by an increase in plaques; i.e., phage is formed intracellularly, but is retained within cellular confines. (c) After a further drop in the extracellular phage fraction there occurs a pronounced increase in the total phage plaque count not accompanied by any increase in total activity. This indicates a redistribution of phage formed intracellularly. At the same time there is a rise in the extracellular phage curves (both activity and plaque). (d) With the concentrations of phage and bacteria used in the experiment carried out at pH 6.1 and 28°C. there are two further increments in [phage]_act. before massive lysis begins. (e) During terminal lysis there are sharp rises in the curves for [total phage]_plaq., [extracellular phage]_act., and [extracellular phage]_plaq.. (f) Immediately after the completion of lysis there is a considerable disparity between measurements of total phage and extracellular phage, probably occasioned by the association of phage molecules with cellular debris, the latter being of sufficient size to be removed by the super-cel filters. 5. At pH 7.2 and 36°C. the steps in the phage production curve as determined by activity assay and plaque count are much less prominent than those observed at pH 6.1 and 28°C. However, the plateaus described by Ellis and Delbrück (10) for B. coli and coli phage can be detected also in the present case if frequent samples are taken. 6. The sorption experiments show a significant rise in the rate of phage uptake with increase in temperature, again supporting the view that the reaction involves more than a purely physical adsorption. 7. Delbrück''s objections to: (a) the use of the activity assay for determining [total phage] in mixtures of phage and susceptible cells, and (b), to the demonstration of phage precursor in "activated" bacteria have been analyzed. 8. The activity assay has been demonstrated to be an accurate procedure for determining either phage free in solution or phage bound to living susceptible cells, under the conditions of the experiments reported here and in earlier work. 9. The titration values obtained in the experiments designed to exhibit intracellular phage precursor are not the result of artifacts as Delbrück has inferred. The data can be interpreted in terms of the precursor theory, although other explanations are not ruled out.     

Analysis of the Staphylococcus epidermidis genes epiF, -E, and -G involved in epidermin immunity.

The lantibiotic epidermin is produced by Staphylococcus epidermidis Tü3298. The known genes involved in epidermin biosynthesis and regulation are organized as operons (epiABCD and epiQP) that are encoded on the 54-kb plasmid pTü32. Here we describe the characterization of a DNA region that mediates immunity and increased epidermin production, located upstream of the structural gene epiA. The sequence of a 2.6-kb DNA fragment revealed three open reading frames, epiF, -E, and -G, which may form an operon. In the cloning host Staphylococcus carnosus, the three genes mediated an increased tolerance to epidermin, and the highest level of immunity (sevenfold) was achieved with S. carnosus carrying epiFEG and epiQ. The promoter of the first gene, epiF, responded to the activator protein EpiQ and contained a palindromic sequence similar to the EpiQ binding site of the epiA promoter, which is also activated by EpiQ. Inactivation of epiF, -E, or -G resulted in the complete loss of the immunity phenotype. An epidermin-sensitive S. epidermidis Tü3298 mutant was complemented by a DNA fragment containing all three genes. When the epiFEG genes were cloned together with plasmid pTepi14, containing the biosynthetic genes epiABCDQP, the level of epidermin production was approximately fivefold higher. The proteins EpiF, -E, and -G are similar in deduced sequence and proposed structure to the components of various ABC transporter systems. EpiF is a hydrophilic protein with conserved ATP-binding sites, while EpiE and -G have six alternating hydrophobic regions and very likely constitute the integral membrane domains. When EpiF was overproduced in S. carnosus, it was at least partially associated with the cytoplasmic membrane. A potential mechanism for how EpiFEG mediates immunity is discussed.     

THE KINETICS OF TRYPSIN DIGESTION : V. SCHUTZ'S RULE.

The rate of digestion of concentrated casein solutions by low concentrations of trypsin at 0° has been followed. Under these conditions the enzyme is inhibited by the product of the reaction and under certain conditions this effect should lead to Schütz''s rule, i.e. the amount of hydrolysis should be proportional to the square root of the product of the time into the enzyme concentration. This is the result obtained. Both Schütz''s rule and Arrhenius'' equation fail to hold accurately owing to the incorrect relation assumed to hold between the rate of hydrolysis and the substrate concentration.     

Proliferation Potential of Müller Glia after Retinal Damage Varies between Mouse Strains

Retinal Müller glia can serve as a source for regeneration of damaged retinal neurons in fish, birds and mammals. However, the proliferation rate of Müller glia has been reported to be low in the mammalian retina. To overcome this problem, growth factors and morphogens have been studied as potent promoters of Müller glial proliferation, but the molecular mechanisms that limit the proliferation of Müller glia in the mammalian retina remain unknown. In the present study, we found that the degree of damage-induced Müller glia proliferation varies across mouse strains. In mouse line 129×1/SvJ (129), there was a significantly larger proliferative response compared with that observed in C57BL/6 (B6) after photoreceptor cell death. Treatment with a Glycogen synthase kinase 3 (GSK3) inhibitor enhanced the proliferation of Müller glia in 129 but not in B6 mouse retinas. We therefore focused on the different gene expression patterns during retinal degeneration between B6 and 129. Expression levels of Cyclin D1 and Nestin correlated with the degree of Müller glial proliferation. A comparison of genome-wide gene expression between B6 and 129 showed that distinct sets of genes were upregulated in the retinas after damage, including immune response genes and chromatin remodeling factors.     

Development of Models for Predicting the Predominant Taste and Odor Compounds in Taihu Lake,China

Taste and odor (T&O) problems, which have adversely affected the quality of water supplied to millions of residents, have repeatedly occurred in Taihu Lake (e.g., a serious odor accident occurred in 2007). Because these accidents are difficult for water resource managers to forecast in a timely manner, there is an urgent need to develop optimum models to predict these T&O problems. For this purpose, various biotic and abiotic environmental parameters were monitored monthly for one year at 30 sites across Taihu Lake. This is the first investigation of this huge lake to sample T&O compounds at the whole-lake level. Certain phytoplankton taxa were important variables in the models; for instance, the concentrations of the particle-bound 2-methylisoborneol (p-MIB) were correlated with the presence of Oscillatoria, whereas those of the p-β-cyclocitral and p-β-ionone were correlated with Microcystis levels. Abiotic factors such as nitrogen (TN, TDN, NO₃-N, and NO₂-N), pH, DO, COND, COD and Chl-a also contributed significantly to the T&O predictive models. The dissolved (d) T&O compounds were related to both the algal biomass and to certain abiotic environmental factors, whereas the particle-bound (p) T&O compounds were more strongly related to the algal presence. We also tested the validity of these models using an independent data set that was previously collected from Taihu Lake in 2008. In comparing the concentrations of the T&O compounds observed in 2008 with those concentrations predicted from our models, we found that most of the predicted data points fell within the 90% confidence intervals of the observed values. This result supported the validity of these models in the studied system. These models, basing on easily collected environmental data, will be of practical value to the water resource managers of Taihu Lake for evaluating the probability of T&O accidents.     

Studies on sex-organ development. Oestrogenic effect on ornithine decarboxylase activity in the differentiating Müllerian ducts and other organs of the chick embryo.

The activity of ornithine decarboxylase in the differentiating left and right Müllerian ducts was assayed and compared with that in other embryonic organs, i.e. the liver and the brain throughout the stages of development. In general the enzyme activity was high in the early stages and decreased extensively in the late stages of development. Specifically, in the left and righ Müllerian ducts, the enzyme activity was high from day 8 to day 9 of incubation. In the right duct the enzyme activity started to decline on day 9 and then continuously decreased to an almost undetectable value on day 18 of incubation. In the left duct the enzyme activity also decreased slightly from day 9 to day 12; however, it increased from day 13 to day 15 and finally decreased to a constant value from day 18 until hatching. The alteration in enzyme activity in the Müllerian duct as assayed in vitro during development is not due to the effect of the size of the endogenous ornithine pool. When the enzyme activity was subjected to oestrogen stimulation, an increase of 5--10-fold for the left duct and of 5--3-fold for the right duct was observed during the course of development. No such stimulation was observed with the treatment of progesterone. Testosterone consistently caused a 25--30% inhibition of the enzyme activity in the Müllerian duct. Oestrogen slightly stimulated the enzyme activity in the developing liver but inhibited that of the brain. The concentration of the three polyamines measured in the Müllerian duct corresponds to the activity of the enzyme determined.     

Lactococcus lactis subsp. cremoris of Plant Origin Produces Antifungal Cyclo-(Leu-Pro) and Tetradecanoic Acid

The antifungal cyclo-depeptide and the fatty acid were isolated and purified from an indigenous strain of Lactococcus lactis subsp. cremoris. Maximal activity was observed at pH 5.5 and 6.5, and at 30 °C under stationary conditions, which was detected in the culture supernatant 8 h post-inoculation in MRS broth until 22 h. The activity of antifungal compounds in the culture supernatant was sensitive to pH and temperature; and was protease-resistant. The antifungal compounds were concentrated by freeze-drying and ultrafiltration with activity retained in 1 kDa filtrates indicating low molecular weight metabolites. The compounds were further extracted by using different solvents amongst which, ethyl acetate provided the highest recovery. Antifungal compounds were separated on a silica gel column into two active fractions that were revealed to be tetradecanoic acid and cyclo-(Leu-Pro), a cyclic dipeptide, by GC–MS. Herein, we describe and attribute the biocontrol potential of L. lactis subsp. cremoris to the low molecular weight antifungal compounds isolated, which is the first report of their isolation from this strain. The broad antifungal spectrum of this candidate advocates further exploration of its biocontrol potential in managing fungal infections in different food and feed systems.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12088-020-00917-z.     

Electron paramagnetic resonance spectroscopic and electrochemical characterization of the partially purified N5-methyltetrahydromethanopterin:coenzyme M methyltransferase from Methanosarcina mazei Gö1.

The N5-methyltetrahydromethanopterin:coenzyme M methyltransferase is a membrane-bound cobalamin-containing protein of Methanosarcina mazei Gö1 that couples the methylation of coenzyme M by methyltetra-hydrosarcinopterin to the translocation of Na+ across the cell membrane (B. Becher, V. Müller, and G. Gottschalk, J. Bacteriol. 174:7656-7660, 1992). We have partially purified this enzyme and shown that, in addition to the cobamide, at least one iron-sulfur cluster is essential for the transmethylation reaction. The membrane fraction or the partly purified protein contains a "base-on" cobamide with a standard reduction potential (Eo') for the Co2+/1+ couple of -426 mV. The iron-sulfur cluster appears to be a [4Fe-4S]2+/1+ type with an Eo' value of -215 mV. We have determined the methyltransferase activity at various controlled redox potentials and demonstrated that the enzyme activity is activated by a one-electron reduction with half-maximum activity occurring at -235 mV in the presence of ATP and -450 mV in its absence. No activation was observed when ATP was replaced by other nucleoside triphosphates or nonhydrolyzable ATP analogs.     

Thermodynamic and kinetic stability of intermolecular triple helices containing different proportions of C+*GC and T*AT triplets

We have used oligonucleotides containing appropriately placed fluorophores and quenchers to measure the stability of 15mer intermolecular triplexes with third strands consisting of repeats of TTT, TTC, TCC and TCTC. In the presence of 200 mM sodium (pH 5.0) triplexes that contain only T·AT triplets are unstable and melt below 30°C. In contrast, triplets with repeats of TTC, TCC and CTCT melt at 67, 72 and 76°C, respectively. The most stable complex is generated by the sequence containing alternating C⁺·GC and T·AT triplets. All four triplexes are stabilised by increasing the ionic strength or by the addition of magnesium, although triplexes with a higher proportion of C⁺·GC triplets are much less sensitive to changes in the ionic conditions. The enthalpies of formation of these triplexes were estimated by examining the concentration dependence of the melting profiles and show that, in the presence of 200 mM sodium at pH 5.0, each C⁺·GC triplet contributes about 30 kJ mol^–1, while each T·AT contributes only 11 kJ mol^–1. Kinetic experiments with these oligonucleotides show that in 200 mM sodium (pH 5.0) repeats of TCC and TTC have half-lives of ~20 min, while the triplex with alternating C⁺·GC and T·AT triplets has a half-life of ~3 days. In contrast, the dissociation kinetics of the triplex containing only T·AT are too fast to measure.     

Towards the mechanism and comparative effect of diphenyl diselenide, diphenyl ditelluride and ebselen under various pathophysiological conditions in rat's kidney preparation

As an extension of our previous work we not only evaluated the relationship between acidosis and lipid peroxidation in rat's kidney homogenate, but also determined for the first time the potential anti-oxidant activity of diphenyl diselenide, diphenyl ditelluride and ebselen at a range of pH values (7.4–5.4). Because of the pH dependency of iron redox cycling, pH and iron need to be well controlled and for the reason we tested a number of pH values (from 7.4 to 5.4) to get a closer idea about the role of iron under various pathological conditions. Acidosis increased rate of lipid peroxidation in the absence Fe (II) in kidney homogenates especially at pH 5.4. This higher extent of lipid peroxidation can be explained by; the mobilized iron which may come from reserves where it is weakly bound. Addition of iron (Fe) chelator desferoxamine (DFO) to reaction medium completely inhibited the peroxidation processes at all studied pH values including acidic values (5.8–5.4). In the presence of Fe (II) acidosis also enhanced detrimental effect of Fe (II) especially at pH (6.4–5.4). Diphenyl diselenide significantly protected lipid peroxidation at all studied pH values, while ebselen offered only a small statistically non-significant protection. The highest anti-oxidant potency was observed for diphenyl ditelluride. These differences in potencies were explained by the mode of action of these compounds using their catalytic anti-oxidant cycles. However, changing the pH of the reaction medium did not alter the anti-oxidant activity of the tested compounds. This study provides evidence for acidosis catalyzed oxidative stress in kidney homogenate and for the first time anti-oxidant potential of diphenyl diselenide and diphenyl ditelluride not only at physiological pH but also at a range of acidic values.     

Effect of a molecular dipole on the ionic strength dependence of a biomolecular rate constant. Identification of the site of reaction.

A theory is proposed for determining the location of a reaction site on a protein of known tertiary structure with an asymmetric charge distribution by an analysis of the effect of ionic strength on the rate of reaction of the protein with a small ion, using equations of Brønsted (J. N. Brønsted, 1922, Z. Phys, Chem. 102:169-207), Debye and Hückel (P. Debye and E. Hückel, 1923, Phys. Z. 24:185-206), and Kirkwood (J. G. Kirkwood, 1934, J. Chem. Phys. 2:351-361). The theory is based on the fact that the dipole moment of the transition complex differs from that of the protein, which will be reflected in the ionic strength dependence of the reaction. The location of the small ion with respect to the dipole axis of the protein can be calculated from this difference. For protein-protein reactions, an a priori assumption has to be made about the orientation of one of the reaction partners, since many different orientations of the reactants with respect to each other result in dipole moments of the same magnitude.     

A STUDY OF THE BACTERICIDAL ACTION OF ULTRA VIOLET LIGHT : II. THE EFFECT OF VARIOUS ENVIRONMENTAL FACTORS AND CONDITIONS

1. Wide differences in the intensity of incident ultra violet energy are not accurately compensated by corresponding changes in the exposure time, so that the Bunsen-Roscoe reciprocity law does not hold, strictly, especially for bactericidal action on young, metabolically and genetically active bacteria. In the present series of experiments, however, the energies used at various wave lengths did not differ by so much as to cause a significant error in the reported reactions. 2. The longer wave length limit of a direct bactericidal action on S. aureus was found to be between 302 and 313 mµ. The shorter limit was not determined because the long exposures required vitiate quantitative results. Bactericidal action was observed at λ225 mµ. 3. The temperature coefficient of the bactericidal reaction approaches 1 and thus furnishes empirical evidence that the direct action of ultra violet light on bacteria is essentially physical or photochemical in character. 4. The hydrogen ion concentration of the environment has no appreciable effect upon the bactericidal reaction between the limits of pH 4.5 and 7.5. At pH 9 and 10 evidence of a slight but definite increase in bacterial susceptibility was noted, but this difference may have been due to a less favorable environment for subsequent recovery and multiplication of injured organisms. 5. Plane polarization of incident ultra violet radiation has no demonstrable effect upon its bactericidal action. In a third paper of this group the ratios of incident to absorbed ultra violet energy at various wave lengths and the significance of these relations in an analysis of the bactericidal reaction will be discussed.     

Potential Role of Cyr61 Induced Degeneration of Human Müller Cells in Diabetic Retinopathy

The degeneration of Müller cells has been recognized to involve in the pathogenesis of diabetic retinopathy. However, the mechanism is not yet clear. This study is to explore the potential role of Cyr61, a secreted signaling protein in extracellular matrix, in inducing human Müller cell degeneration in diabetic retinopathy (DR). Twenty patients with proliferative diabetic retinopathy (PDR) and twelve non-diabetic patients were recruited for this study. Vitreous fluid was collected during vitrectomy surgery for Cyr61 ELISA. Human Müller cell line MIO-M1 were cultured to be subconfluent, and then treated with glucose (0–20 mM) or Cyr61 (0–300 ng/ml). Cyr61 expression induced by increasing concentrations of glucose was evaluated by RT-qPCR and Western blot. Effects of Cyr61 on Müller cells viability, migration and apoptosis were observed by MTT assay, Transwell assay, and TUNEL assay. Vitreous Cyr61 levels were observed to be 8-fold higher in patients with PDR (3576.92±1574.58 pg/mL), compared with non-diabetic controls (436.14±130.69 pg/mL). Interestingly, the active PDR group was significantly higher than the quiescent PDR group (P<0.01). In retinal Müller cells culture, high glucose significantly and dose-dependently elevated Cyr61 expression at both mRNA and protein levels. Cyr61 at high concentrations dose-dependently inhibited the viability and migration of Müller cells. TUNEL assay further revealed that high concentration of Cyr61 significantly promoted the cell apoptosis. In conclusion, these findings demonstrated for the first time that the expression of Cyr61 was elevated by high glucose in Müller cells, and Cyr61 inhibited cell viability and migration while induced apoptosis, suggesting the potential role of Cyr61 in Müller cell degeneration. The elevated Cyr61 levels in vitreous fluid of PDR patients further support its role in diabetic retinopathy (DR).     

Matrix Fixed Charge Density Modulates Exudate Concentration during Cartilage Compression

Electrolyte filtration arises due to the presence of fixed charges in cartilage extracellular matrix glycosaminoglycans (GAGs). Commonly assumed negligible, it can be important for design and interpretation of streaming potential measurements and modeling assumptions. To quantify the scale of this phenomenon, chloride ion concentration in exudate of compressed cartilage was measured by Mohr’s titration and explant GAG content was colorimetrically assayed. Pilot studies indicated that an appropriate strain rate for experiments was 8 × 10⁻³ s⁻¹ to eliminate concerns of exudate evaporation and explant damage (at low and high strain rates, respectively). Exudate chloride concentration of explants equilibrated in 1× PBS was significantly (p < 0.05) lower than the bath chloride concentration at strains of 37.5, 50, and 62.5%, with clear dependence on strain magnitude. Exudate chloride concentration was also significantly lower than that of the bath when 50% strain was applied after equilibration in 0.5, 1, and 2× PBS, with a trend for an increase in this relative difference with decreasing bath concentration (p = 0.065 between 0.5 and 2× PBS). Decreasing exudate chloride concentration correlated negatively with increasing postcompression GAG concentration. No difference between exudate chloride concentration and bath chloride concentration was ever observed for compression of uncharged agarose gel controls. Findings show that exudate from compressed cartilage is dilute relative to the bath due to the presence of matrix fixed charges, and this difference can generate diffusion potentials external to the explant, which may affect streaming potential measurements particularly under conditions of low strain rates and high strains.     

Chloroperoxidase from Streptomyces lividans: isolation and characterization of the enzyme and the corresponding gene.

For the first time, a halogenating enzyme which is not known to produce halogenated metabolites has been isolated from a bacterial strain. The gene encoding the nonheme chloroperoxidase (CPO-L) from Streptomyces lividans TK64 was cloned, and its gene product was characterized. S. lividans TK64 produced only very small amounts of the enzyme. After cloning of the gene into Streptomyces aureofaciens Tü24-88, the enzyme was overexpressed up to 3,000-fold. Based on the overexpression, a simple purification procedure using acid precipitation and hydrophobic interaction chromatography was developed. Thus, 54 mg of homogeneous CPO-L could be obtained from 27 g (wet weight) of mycelium. The native enzyme has a molecular weight of 64,000 and consists of two identical subunits. The enzyme does not exhibit an absorption peak in the Soret region of the optical spectrum. X-ray fluorescence spectroscopy revealed that the enzyme does not contain any metal ions in equimolar amounts. CPO-L showed cross-reaction with antibodies raised against the nonheme chloroperoxidase from Pseudomonas pyrrocinia but not with antibodies raised against CPO-T from S. aureofaciens Tü24. CPO-L exhibits substrate specificity only for chlorination, not for bromination. Therefore, monochlorodimedone is only brominated by CPO-L, whereas indole is brominated and chlorinated. The functional chloroperoxidase gene was located on a 1.9-kb SalI DNA fragment. DNA sequence analysis revealed an open reading frame encoding a predicted polypeptide of 276 amino acids. The overall identity of the amino acid sequence to that of chloroperoxidase from P. pyrrocinia was 71%, whereas that to bromoperoxidase BPO-A2 from S. aureofaciens ATCC 10762 was only 42%.     

Inhibition of soluble epoxide hydrolase by phytochemical constituents of the root bark of Ulmus davidiana var. japonica

A novel compound 1 and nine known compounds (2–10) were isolated by open column chromatography analysis of the root bark of Ulmus davidiana. Pure compounds (1–10) were tested in vitro to determine the inhibitory activity of the catalytic reaction of soluble epoxide hydrolase (sEH). Compounds 1, 2, 4, 6–8, and 10 had IC₅₀ values ranging from 11.4 ± 2.3 to 36.9 ± 2.6 μM. We used molecular docking to simulate inhibitor binding of each compound and estimated the binding pose of the catalytic site of sEH. From this analysis, the compound 2 was revealed to be a potential inhibitor of sEH in vitro and in silico. Additionally, molecular dynamics (MD) study was performed to find detailed interaction signals of inhibitor 2 with enzyme. Finally, compound 2 is promising candidates for the development of a new sEH inhibitor from natural plants.