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In an increasingly miniaturised technological world, non-destructive testing (NDT) methodologies able to detect defects at the micro scale are necessary to prevent failures. Although several existing methods allow the detection of defects at that scale, their application may be hindered by the small size of the samples to examine. In this study, the application of bacterial cells to help the detection of fissures, cracks, and voids on the surface of metals is proposed. The application of magnetic and electric fields after deposition of the cells ensured the distribution of the cells over the entire surfaces and helped the penetration of the cells inside the defects. The use of fluorophores to stain the cells allowed their visualisation and the identification of the defects. Furthermore, the size and zeta potential of the cells and their production of siderophores and biosurfactants could be influenced to detect smaller defects. Micro and nano surface defects made in aluminium, steel, and copper alloys could be readily identified by two Staphylococcus strains and Rhodococcus erythropolis cells. [ABSTRACT FROM AUTHOR]

The exposure of bacterial cells to pulsed electric fields (PEF) leads to the reversible formation of pores in the cell membrane if an applied energy is below the critical level. Therefore, the effect of electric field pulses with amplitudes below 14 kV/cm and the applied energy up to 12.2 J/cm³ on the growth of Lactobacillus plantarum 564 cells was investigated. After PEF treatments, the growth of lactobacilli in De Man-Rogosa-Sharpe broth at 37 °C was monitored by isothermal calorimetry, absorbance and plate counts. All the applied treatments resulted in a higher growth rate of PEF-treated cells during early and mid-log phase, especially bacterial samples treated with lower field intensities (1.3-5.5 J/cm³). The transport of ions and molecules through the cell membrane (which facilitates the growth of electroporated lactobacilli) was particularly evident in the mid-exponential growth phase, where the doubling time was reduced more than 3 times after the exposure to electric pulses of 5.5 J/cm³. The heat production rate during the growth of electroporated cells was also higher, indicating the enhanced metabolic activity of PEF-treated cells. Moreover, the electroporated cells had a better acidification ability than the untreated ones. It can be summarized that the applied PEF treatments with an energy input of below 12 J/cm³ potentially induce reversible electroporation of the cell membrane, which has a positive impact on the growth and metabolic activity of the cells of lactobacilli. [ABSTRACT FROM AUTHOR]

Alcanivorax borkumensis strain SK2 is a cosmopolitan oil-degrading oligotrophic marine γ-proteobacterium that exclusively uses petroleum hydrocarbons as sources of carbon and energy. Its ubiquity and unusual physiology suggest its global importance in the removal of hydrocarbons from polluted marine systems. The genome of A. borkumensis SK2 was recently sequenced. Two ferredoxin-nicotinamide adenine dinucleotide phosphate (NADPH) reductase genes ( ABO_0145 and ABO_0203) have been annotated for this bacterium. In the present study, the expression, purification, and kinetic properties of these two genes were explored by constructing the prokaryotic expression vectors (pET21a) for the first time. Isopropyl β- d-thiogalactoside (0.5 mM) was used for induction of exponentially growing cells (30 °C, overnight). Most of the proteins were expressed in inclusion body. Partial purification of recombinant enzymes was performed by ion-exchange chromatography on a DEAE-sepharose column using only one linear gradient of sodium chloride ranging between 0 and 500 mM. The recombinant enzymes displayed reductase activity, which was optimal at pH 6.0 and 45 °C. Ferredoxin-NADPH reductases exhibited several outstanding properties that made them excellent model proteins to address broad biological questions. This study serves as the basis for further investigations of the biotechnological potential of these enzymes. [ABSTRACT FROM AUTHOR]

Oil pollution is an environmental problem of increasing importance. Alcanivorax borkumensis, with a high potential for biotechnological applications, is a key marine hydrocarbonoclastic bacterium and plays a critical role in the bioremediation of oil-polluted marine systems. In oil degrading bacteria, the first step of alkane degradation is catalyzed by a monooxygenase. The reducing electrons are tunneled from NAD(P)H via rubredoxin, one of the most primitive metalloproteins, to the hydroxylase. Rubredoxin reductase is a flavoprotein catalyzing the reduction of rubredoxin. There are two rubredoxin genes, alkG and rubA, in A. borkumensis genome. In this work, the genes encoding rubredoxin reductase (ABO_0162, rubB) and AlkG(ABO_2708, alkG) were cloned and functionally overexpressed in E. coli. Our results demonstrate that RubB could reduce AlkG, therefore compensating for the absence of AlkT, also a rubredoxin reductase, missing in A. borkumensis SK2 genome. These results will increase our knowledge concerning biological alkane degradation and will lead us to design more efficient biotransformation and bioremediation systems. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011 [ABSTRACT FROM AUTHOR]

The basidiomycetous yeast, Xanthophyllomyces dendrorhous, is one of the very few organisms which can be used for biological production of the carotenoid astaxanthin. crtE cDNA has been cloned from this fungus for engineering of the terpenoid pathway. The function of its gene product as a geranylgeranyl pyrophosphate synthase was established. X. dendrorhous was transformed with the crtE cDNA to divert metabolite flow from the sterol pathway towards carotenoid biosynthesis. Transformants were obtained with increased levels of geranylgeranyl pyrophosphate synthase leading to higher carotenoid levels including astaxanthin. Physiological conditions for maximum carotenoid synthesis for wild type and the CrtE transformant were dim light and extra air supply of the shaking culture. These conditions and the transformation with crtE had additive effects and resulted in an 8-fold higher astaxanthin formation as compared to the initial wild type culture without illumination and extra air supply yielding 451 μg/g dry wt within 4 days of growth. [ABSTRACT FROM AUTHOR]

Rhodococcus erythropolis was found to utilize C5 to C16 n-alkane hydrocarbons as sole source of carbon and energy when growing as planktonic or biofilm cells attached to polystyrene surfaces. Growth rates on even numbered were two- to threefold increased relatively to odd-numbered n-alkanes and depended on the chain length of the n-alkanes. C10-, C12-, C14-, and C16- n-alkanes gave rise to two- to fourfold increased maximal growth rates relative to C5- to C9-hydrocarbons. In reaction to the extremely poor water solubility of the n-alkanes, both planktonic and biofilm cells exhibited distinct adaptive changes. These included the production of surface active compounds and substrate-specific modifications of the physicochemical cell surface properties as expressed by the microbial adhesion to hydrocarbon- and contact angle-based hydrophobicity, as well as the zeta potential of the cells. By contrast, n-alkane-specific alterations of the cellular membrane composition were less distinct. The specificity of the observed autecological changes suggest that R. erythropolis cells may be used in the development and application of sound biocatalytic processes using n-alkanes as substrates or substrate reservoirs or for target-specific bioremediation of oils and combustibles, respectively. [ABSTRACT FROM AUTHOR]

The article examines the ability of a highly hydrophobic biocatalyst to improve the bioproduction of carvone in a two-liquid-phase partitioning bioreactor (TPPB) using 1-dodecene and silicone oil. The agitation in the reactor was temporarily stopped after silicone oil was added while 1-dodecene improved the reactor's overall operability. Biotranformation occurred 13.5 hours longer in TPPB with silicone oil compared to single aqueous phase reactor. Another advantages of TPPB over the former are increased productivity and less monitoring and maintenance.

Cells of Rhodococcus erythropolis DCL14 were adapted to increasing toluene concentrations in a mechanically stirred reactor. When the initial non-adapted cells were placed in contact with toluene, only 10.5% of cells remained viable after 1 h in the presence of 20% ( v/ v) toluene, while 8.6% of cells were viable after 28 h in the presence of an organic phase containing 80% ( v/ v) toluene in n-dodecane. Cell adaptation was studied by following the toluene consumption rate, the viability of the cell population, and the composition of the bacteria cellular membrane in the presence of increasing concentrations of toluene in the reactor. A maximum toluene concentration of 4.9 M, which corresponds to 52.4% ( v/ v) toluene in the organic phase, was achieved, toluene being consumed at 10.7 mg/(h mg protein). The adapted cells showed a substantially increased resistance to 50% ethanol and to concentrations of Betadine® and Micropur® tablets currently used in water purification, when compared to non-adapted cells. [ABSTRACT FROM AUTHOR]

The degradation of alkanes and alcohols by Rhodococcus erythropolis DCL14 growing cells was tested at different reactor scales, ranging from a 2-L fermenter to 300 μL 96-well plates. The highest growth rates were attained in the fermenter, where pH, temperature and aeration rate, in particular, are monitored and controlled. However, the results acquired in reactors smaller than 100 mL indicate that these reactors may be successfully used in testing carbon sources, since the normalized results obtained with the different carbons sources were maintained in the 100-0.3 mL range. This study also shows the great potential of R. erythropolis DCL14 cells to degrade a wide range of alcohols and alkanes, which may be used in bioremediation of contaminated sites. [ABSTRACT FROM AUTHOR]

Carvone has previously been found to highly inhibit its own production at concentrations above 50 mM during conversion of a diastereomeric mixture of (−)-carveol by whole cells of Rhodococcus erythropolis. Adaptation of the cells to the presence of increasing concentrations of carveol and carvone in n-dodecane prior to biotransformation proved successful in overcoming carvone inhibition. By adapting R. erythropolis cells for 197 h, an 8.3-fold increase in carvone production rate compared to non-adapted cells was achieved in an air-driven column reactor. After an incubation period of 268 h, a final carvone concentration of 1.03 M could be attained, together with high productivity [0.19 mg carvone h−1 (ml organic phase)−1] and high yield (0.96 g carvone g carveol−1). [ABSTRACT FROM AUTHOR]

Rhodococcus erythropolisDCL14 has the ability to convert the terpene (-)-carveol to the valuable flavour compound (-)-carvone when growing on a wide range of carbon sources. To study the effect of carbon and energy sources such as alkanes, alkanols and terpenes on the biotechnological process, the cellular adaptation at the level of fatty acid composition of the membrane phospholipids and the (-)-carvone production were examined. All tested carbon sources caused a dose-dependent increase in the degree of saturation of the fatty acids. The exception was observed with short-chain alcohols such as methanol and ethanol, to which the cells adapted with a concentration-dependent decrease in the saturation degree of the membrane phospholipids. This influence of the different carbon sources on the rigidity of the cell membrane also had an impact on the (-)-carvone productivity of the strain. [ABSTRACT FROM AUTHOR]

Abstract: Rhodococcus erythropolis DCL14 cells were able to metabolise C5–C16 hydrocarbons and C1–C12 alcohols as sole carbon and energy sources, both at 15 and 28 °C. Metabolic activity was also observed at 1.00%, 1.95% and 2.50% sodium chloride. Almost complete degradation of n-, iso- and cyclo-alkanes and aromatic compounds present in fuel oil was achieved after 9 months, 60% being consumed in the first three months. The results from the conditions tested here suggest that this type of bacterium could be involved in bioremediation processes in marine environments such as the Atlantic, Pacific and Indian Ocean. [Copyright &y& Elsevier]

The behaviour of cells of Rhodococcus erythropolis DCL14, Xanthobacter Py2, Arthrobacter simplex and Mycobacterium sp. NRRL B-3805, in biphasic systems containing different organic solvents was evaluated and compared. The data, obtained mainly by fluorescence microscopy and image analysis, was interpreted using principal components analysis (PCA). With this technique, the variability of the data could be summarised in 7 components, representing 75.8% of the variance of the data. Over a third of the variance could be explained by the first two principal components which represent solvent toxicity. Apparently this is the major factor influencing cell behaviour in an organic:aqueous system. However, factors such as substrate concentration, cell adaptation ability (resulting in morphological changes and aggregation or separation of cells) and membrane composition (specific to each strain) also play an important role in cell resistance to solvent toxicity. The results regarding cell shape indicate that loss of viability occurs, in the tested bacterial strains, after incorporation of molecules of solvent in the cellular membrane. This should result in an increase in membrane fluidity, and thus, in an alteration of cell shape. The ability to form "self-defence" clusters was observed to be different amongst the four strains. X. Py2 showed, in general, a low tendency to form aggregates under the tested conditions; A. simplex and R. erythropolis aggregated mainly in the presence of low log P solvents; and Mycobacterium. sp. cells showed a high ability to aggregate. [ABSTRACT FROM AUTHOR]

The effect of some solvents, present in different amounts, upon whole cells of Rhodococcus erythropolis DCL14 carrying out the biotransformation of (-)-carveol to (-)-carvone was studied. The solvents tested were ethyl butyrate, n -hexane, cyclohexane, iso -octane, n -dodecane, dimethyl sulfoxide, bis(2-ethylhexyl) phthalate and FC-70. The volumes of each solvent corresponded to organic:aqueous phase ratios of 0.0005, 0.0025, 0.005, 0.025 and 0.2. To assess any potential solvent protection towards substrate toxicity, assays were carried out at two initial carveol concentrations (15 and 50 mM). Carvone accumulation was followed by gas chromatography. Cell viability, several aspects of cell morphology and the ability to form clusters were monitored by fluorescence microscopy. Principal components analysis (PCA) was used as a tool to explain the differences in the observations of the multidimensional data set obtained from the multiple conditions. PCA using the different volumes of each solvent as variable suggests that the variability of the observations can be summarised in six components which represent 79.4% of the variance of the data. Conversely, using cell and solvent data to perform the PCA, 97.1% of the variance of the data can be summarised in three components, the first two capturing 91.0% of the information. These components seem to represent solvent toxicity and a protective effect of the solvent from carveol toxicity. [ABSTRACT FROM AUTHOR]

Abstract Aims: The partial characterization of bacteriocins produced by an environmental strain Enterococcus faecium EK13, isolated from cattle dung water. Methods and Results: A bacteriocin was partially purified by ammonium sulphate precipitation, followed by a SP-Sepharose column, reverse-phase chromatography and N-terminal region sequenced. The anti-microbial substance produced was found to be a heat-stable polypeptide with molecular mass 4·83 kDa, which was determined by N-terminal amino acid sequencing to be enterocin A. A second substance was specified by PCR as enterocin P. Bacteriocins were stable at 4 and -20°C for long storage periods. The optimum of bacteriocin production was observed in the range of pH 5·0–6·5 at 30 and 37°C. The most active substances are produced by strain EK13 in logarithmic growth phase and bacteriocins are produced after 1 h of fermentation. The highest activity detected in fermentation experiments was 51 200 AU ml-1 and the most sensitive indicator strain was found to be Listeria innocua LMG 13568. Differences in bacteriocin activity against two indicators could be explained by more than one type of enterocin production by strain EK13, or with different mode of action or in different sensitivity of strains. Conclusion: Enterococcus faecium strain EK13 isolated from cattle dung water produces two bacteriocins, enterocin A and P, with an inhibitory effect against the strain of the genera Enterococcus , Leuconostoc , Lactobacillus , Streptococcus , Staphylococcus , Bacillus and Listeria (in different origin). Significance and Impact of the Study: Enterococcus faecium EK13 environmental strain is a new producer of enterocin A and P. The E. faecium EK13, isolated from cattle dung water, is presented with the further aim to utilize it for waste treatment by biotechnological processes. [ABSTRACT FROM AUTHOR]

Develops a genetically engineered Escherichia coli strain for the epoxide hydrolase-catalysed kinetic resolution of epoxides. Evidence of a 138-fold increase in Eph1 activity in cell extracts of the recombinant E. coli; Results of optimization of cultivation conditions and co-expression of molecular chaperones.

A novel lyase, combining hydratase and aldolase activity, that converts citral into methylheptenone and acetaldehyde, was purified from spores of Penicillium digitatum . Remarkably, citral lyase activity was induced 118-fold by incubating nongerminating spores with the substrate, citral. This cofactor independent hydratase/aldolase, was purified and found to be a monomeric enzyme of 31 kDa. Citral lyase has a K m of 0.058 mm and a V max of 52.6 U·mg-1 . Enzyme activity was optimal at 20 °C and pH 7.6. The enzyme has a strong preference for the trans isomer of citral (geranial). Citral lyase also converts other α,β-unsaturated aldehydes (farnesal, methyl-crotonaldehyde, decenal and cinnemaldehyde). [ABSTRACT FROM AUTHOR]

A selection of different glycosidases was screened for the glycosylation of 1-propanethiol. The β-glucosidases from almond, Aspergillus niger and Caldocellum saccharolyticum were capable of 1-propanethioglucoside (1-PTG) formation. The almond β-glucosidase showed the highest activity in this reversed hydrolysis type of reaction using glucose as glucosyl donor. Besides 1-propanethiol, also thioglucosides of 2-propanethiol and furfuryl mercaptan were formed by the almond β-glucosidase. The substrate specificity of the almond β-glucosidase with respect to thioglucosylation is restricted to primary and secondary aliphatic thiols. Once the thioglucosides are formed, they are not hydrolyzed at a significant rate by almond β-glucosidase. As a consequence the synthesis of 1-PTG could be observed at very low aglycone concentrations (0.5% v/v based on the reaction solution) and high yields (68% based on 1-PT and 41% based on glucose) were obtained. An excess of aglycone, otherwise frequently applied in reversed hydrolysis glycosylation, is therefore not necessary in the glucosylation of 1-PT. [ABSTRACT FROM AUTHOR]

Spores of Penicillium digitatum ATCC 201167 transform geraniol, nerol, citral, and geranic acid into methylheptenone. Spore extracts of P. digitatum convert geraniol and nerol NAD+-dependently into citral. Spore extract also converts citral NAD+-dependently into geranic acid. Furthermore, a novel enzymatic activity, citral lyase, which cofactor-independently converts citral into methylheptenone and acetaldehyde, was detected. These result show that spores of P. digitatum convert geraniol via a novel biotransformation pathway. This is the first time a biotransformation pathway in fungal spores has been substantiated by biochemical studies. Geraniol and nerol are converted into citral by citrol dehydrogenase activity. The citral formed is subsequently deacetylated by citral lyase activity, forming methylheptenone. Moreover, citral is converted reversibly into geranic acid by citral dehydrogenase activity. [ABSTRACT FROM AUTHOR]

A recently isolated white-rot fungal strain, Merulius tremellosus ono991, displays high stereoselectivity during the reduction of arylketones. In order to increase the productivity and specific yield of the optically active alcohols, the culture conditions for the reduction of the model ketone compound 1′-acetonaphtone to α-methyl-1-naphtalenemethanol were optimized with respect to oxygen supply, choice of primary substrate and arylketone concentration. Alternative electron acceptors were also used to elucidate the role of reduction equivalents in the reduction process. The optimal yields of α-methyl-1-naphtalenemethanol were obtained in N2-flushed incubations with glycerol as primary substrate. The specific yield was increased from 57% to 98% compared to incubations under air with glucose. Most of the yield increase was due to N2-flushing and could be attributed to two factors. First, an increased stability of the product, α-methyl-1-naphtalenemethanol, in anaerobic compared to aerobic atmosphere was demonstrated. Second, fermentative metabolism increased reduced enzyme cofactors available for the reduction. Diverting reducing equivalents away from fermentation with alternative electron acceptors correlated with a decreased yield of α-methyl-1-naphtalenemethanol. Furthermore, the dependency of ketone reductase for common occurring metabolic reducing equivalents, NAD(P)H, was demonstrated by the reduction of 1′-acetonaphtone in cell extracts of M. tremellosus ono991. [ABSTRACT FROM AUTHOR]

Pseudomonas sp. strain DCA1, which is capable of utilizing 1,2-dichloroethane (DCA) as sole carbon and energy source, was used to oxidize chlorinated methanes, ethanes, propanes, and ethenes. Chloroacetic acid, an intermediate in the DCA degradation pathway of strain DCA1, was used as a co-substrate since it was readily oxidized by DCA-grown cells of strain DCA1 and did not compete for the monooxygenase. All of the tested compounds except tetrachloroethylene (PER) were oxidized by cells expressing DCA monooxygenase. Strain DCA1 could not utilize any of these compounds as a growth substrate. Co-metabolic oxidation during growth on DCA was studied with 1,2-dichloropropane. Although growth on this mixture occurred, 1,2-dichloropropane strongly inhibited growth of strain DCA1. This inhibition was not caused by competition for the monooxygenase. It was shown that the oxidation of 1,2-dichloropropane resulted in the accumulation of 2,3-dichloro-1-propanol and 2-chloroethanol. [ABSTRACT FROM AUTHOR]

The solvent-tolerant bacterium Pseudomonas putida S12, which adapts its membrane lipids to the presence of toxic solvents by a cis to trans isomerization of unsaturated fatty acids, was used to study possible in vivo regiospecificity of the isomerase. Cells were supplemented with linoleic acid (C18:2Δ9-cis,Δ12-cis), a fatty acid that cannot be synthesized by this bacterium, but which was incorporated into membrane lipids up to an amount of 15% of total fatty acids. After addition of 1-octanol, which was used as an activator of the cis-trans isomerase, the linoleic acid was converted into the Δ9-trans,Δ12-cis isomer, while the Δ9-cis,Δ12-trans and Δ9-trans,Δ12-trans isomers were not synthesized. Thus, for the first time, regiospecific in vivo formation of novel, mixed cis/trans isomers of dienoic fatty acid chains was observed. The maximal conversion (27–36% of the chains) was obtained at 0.03–0.04% (v/v) octanol, after 2 h. The observed regiospecificity of the enzyme, which is located in the periplasmic space, could be due to penetration of the enzyme to a specific depth in the membrane as well as to specific molecular recognition of the substrate molecules. [ABSTRACT FROM AUTHOR]

Screening for novel thioglucoside hydrolase activity resulted in the isolation of Sphingobacterium sp. strain OTG1 from enrichment cultures containing octylthioglucoside (OTG). OTG was hydrolysed into octanethiol and glucose by cell free extracts. Besides thioglucoside hydrolysis, several other glucoside hydrolase activities were detected in the Sphingobacterium sp. strain OTG1 cell free extract. By adding β-glucosidase inhibitors it was possible to discriminate between these different activities. Ascorbic acid and D-gluconic acid lactone inhibited the hydrolysis of p-nitrophenyl β-glucoside, but did not affect octyl- and octylthioglucoside hydrolase activity. Besides OTG, various other thioglucosides were hydrolysed by the novel thioglucosidase, with almost the same activities regardless of the nature of the aglycone, including the myrosinase model substrate sinigrin (a glucosinolate). Sinigrin could also be used as a growth substrate by Sphingobacterium sp. strain OTG1, although at concentrations exceeding 0.15 mM degradation was not complete. [ABSTRACT FROM AUTHOR]

The bioconversion of toluene into 3-methylcatechol was studied as a model system for the production of valuable 3-substituted catechols in general. For this purpose, an improved microbial system for the production of 3-methylcatechol was obtained. Pseudomonas putida strains containing the todC1C2BAD genes involved in the conversion of toluene into 3-methylcatechol were used as hosts for introducing extra copies of these genes by means of a novel integrative expression system. A construct was made containing an expression cassette with the todC1C2BAD genes cloned under the control of the inducible regulatory control region for naphthalene and phenanthrene degradation, nagR. Introducing this construct into wild-type P. putida F1, which degrades toluene via 3-methylcatechol, or into mutant P. putida F107, which accumulates 3-methylcatechol, yielded biocatalysts carrying multiple copies of the expression cassette. As a result, up to 14 mM (1.74 g l–1) of 3-methylcatechol was accumulated and the specific production rate reached a level of 105 µmol min–1 g–1cell dry weight, which is four times higher than other catechol production systems. It was shown that these properties were kept stable in the biocatalysts without the need for antibiotics in the production process. This is an important step for obtaining designer biocatalysts. [ABSTRACT FROM AUTHOR]

Twenty-one microorganisms were screened for their ability to convert nitroaromatics into 3-nitrocatechol as a result of the action of an oxygenase. Cultures containing toluene dioxygenases and phenol monooxygenases accumulated 3-nitrocatechol during incubation with nitrobenzene and nitrophenol, respectively. Nocardia S3 was selected and studied in more detail. Toluene-pregrown cultures were able to degrade nitrobenzene with a concomitant formation of 3-nitrocatechol. The rates of nitrobenzene utilization decreased throughout the biotransformation period and finally the accumulation ceased. The gradual deterioration of the biotransformation rates was not a consequence of depletion of the NADH pool, but was due to the accumulation of 3-nitrocatechol. The inhibition of nitrobenzene biotransformation by 3-nitrocatechol greatly impacts 3-nitrocatechol production processes . [ABSTRACT FROM AUTHOR]

A two-phase membrane bioreactor was developed to continuously produce enantiopure epoxides using the epoxide hydrolase activity of Rhodotorula glutinis. An aqueous/organic cascade, hydrophilic, hollow-fiber membrane bioreactor was used: (1) to carry out large-scale resolution of epoxides, (2) to continuously extract residual enantiopure epoxides from the aqueous phase, and (3) to separate inhibitory formed diol from the yeast cells contained in the aqueous phase. Dodecane was employed to dissolve-feed epoxide as well as to extract residual epoxide. 1,2-Epoxyhexane was used as a model substrate. By use of this membrane bioreactor, enantiopure (S)-1,2-epoxyhexane (>98% enantiomeric excess) was obtained with a volumetric productivity of 3.8 g l-1 h-1. The continuous-production system was operated for 12 days and resulted in 38 g enantiopure (S)-1,2-epoxyhexane. [ABSTRACT FROM AUTHOR]

The active efflux system contributing to the solvent tolerance of Pseudomonas putida S12 was characterized physiologically. The mutant P. putida JK1, which lacks the active efflux system, was compared with the wild-type organism. None of 20 known substrates of common multi-drug-resistant pumps had a stronger growth-inhibiting effect on the mutant than on the wild type. The amount of [14C]toluene accumulating in P. putida S12 increased in the presence of the solvent xylene and in the presence of uncouplers. The effect of uncouplers confirms the proton dependency of the efflux system in P. putida S12. Other compounds, potential substrates for the solvent pump, did not affect the accumulation of [14C]toluene. These results show that the efflux system in P. putida S12 is specific for organic solvents and does not export antibiotics or other known substrates of multi-drug-resistant pumps. [ABSTRACT FROM AUTHOR]

The aim of the study was to investigate whether toxic fine chemical production can be improved using the solvent-tolerant Pseudomonas putida S12 in a two-liquid-phase system consisting of aqueous media and a water-immiscible octanol phase with production of 3-methylcatechol from toluene as the model conversion. For this purpose the genes involved in this conversion, todC1C2BAD from P. putida F1, were introduced into P. putida S12 with high stable expression. Production of 3-methylcatechol was monitored in batch incubations with different media using a single medium and a two-liquid medium–octanol system. The maximum concentration of 3-methylcatechol increased two-fold using the two-liquid medium–octanol system, irrespective of the selected medium. [ABSTRACT FROM AUTHOR]

We cloned and characterized the epoxide hydrolase gene, EPH1, from Rhodotorula glutinis. The EPH1 open reading frame of 1230 bp was interrupted by nine introns and encoded a polypeptide of 409 amino acids with a calculated molecular mass of 46.3 kDa. The amino acid sequence was similar to that of microsomal epoxide hydrolase, which suggests that the epoxide hydrolase of R. glutinis also belongs to the α/β hydrolase fold family. EPH1 cDNA was expressed in Escherichia coli and resting cells showed a specific activity of 200 nmol min-1 (mg protein)-1 towards 1,2-epoxyhexane. [ABSTRACT FROM AUTHOR]

The epoxide hydrolase from Rhodotorula glutinis was isolated and initially characterized. The enzyme was membrane associated and could be solubilized by Triton X-100. Purification yielded an enzyme with sp. act. of 66 μmol 1,2-epoxyhexane hydrolyzed min−1 mg−1 protein. The enzyme was not completely purified to homogeneity but, nevertheless, a major protein was isolated by SDS-PAGE for subsequential amino acid determination of peptide fragments. From sequence alignments to related enzymes, a high homology towards the active site sequences of other microsomal epoxide hydrolases was found. Molecular mass determinations indicated that the native enzyme exists as a homodimer, with a subunit molecular mass of about 45 kDa. Based upon these, this epoxide hydrolase is structurally related to other microsomal epoxide hydrolases. [ABSTRACT FROM AUTHOR]

Ligninolytic basidiomycetes were screened for their ability to reduce aryl acids to the corresponding aldehydes and alcohols. Seven fungal strains converted p-anisic acid in high molar yields to the reduced products. The white-rot fungus Bjerkandera sp. strain BOS55 was one of the best reducing strains and was highly tolerant towards high concentrations of different aromatic acids. It was tested for the reduction of p-anisic, veratric, 3-chloro-4-methoxybenzoic, 3,5-dichloro-4-methoxybenzoic, 3,4-dichlorobenzoic, 4-fluorobenzoic, and 3-nitrobenzoic acids. All of these compounds were reduced to their corresponding aldehydes and alcohols. [ABSTRACT FROM AUTHOR]

Limonene-1,2-epoxide hydrolase (LEH) from Rhodococcus erythropolis DCL14, an enzyme involved in the limonene degradation pathway of this microlorganism, has a narrow substrate specificity. Of the compounds tested, the natural substrate, limonene-1,2-epoxide, and several alicyclic and 2-methyl-1,2-epoxides (e.g. 1-methylcyclohexene oxide and indene oxide), were substrates for the enzyme. When LEH was incubated with a diastereomeric mixture of limonene-1,2-epoxide, the sequential hydrolysis of first the (1 R,2 S)- and then the (1 S,2 R)-isomer was observed. The hydrolysis of (4 R)- and (4 S)-limonene-1,2-epoxide resulted in, respectively, (1 S,2 S,4 R)- and (1 R,2 R,4 S)-limonene-1,2-diol as the sole product with a diastereomeric excess of over 98%. With all other substrates, LEH showed moderate to low enantioselectivities ( E ratios between 34 and 3). [ABSTRACT FROM AUTHOR]

A gene library of Cellulomonas pachnodae was constructed in Escherichia coli and was screened for endoglucanase activity. Five endoglucanase-positive clones were isolated that carried identical DNA fragments. The gene, designated cel6A, encoding an endoglucanase enzyme, belongs to the glycosyl hydrolase family 6 (cellulase family B). The recombinant Cel6A had a molecular mass of 53 kDa, a pH optimum of 5.5, and a temperature optimum of 50–55 °C. The recombinant endoglucanase Cel6A bound to crystalline cellulose and beech litter. Based on amino acid sequence similarity, a clear cellulose-binding domain was not distinguished. However, the regions in the Cel6A amino acid sequence at the positions 262–319 and 448–473, which did not show similarity to any of the known family-6 glycosyl hydrolases, may be involved in substrate binding. [ABSTRACT FROM AUTHOR]

Examines the mechanism of veratryl alcohol(VA)-mediated oxidation of isoeugenyl acetate (IEA) by lignin peroxidase. Formation of the predominant VA products on IEA; Function of VA during IEA oxidation; Enhancement of lignin peroxidase during oxidation of substrates.

The red heterobasidiomycetous yeast Xanthophyllomyces dendrorhous (perfect state of Phaffia rhodozyma) contains a novel type of carotenoid biosynthetic enzyme. Its structural gene, designated crtYB, was isolated by functional complementation in a genetically modified, carotenogenic Escherichia coli strain. Expression studies in different carotenogenic E. coli strains demonstrated that the crtYB gene encodes a bifunctional protein involved both in synthesis of phytoene from geranylgeranyl diphosphate and in cyclisation of lycopene to β-carotene. By sequence comparison with other phytoene synthases and complementation studies in E. coli with various deletion derivatives of the crtYB gene, the regions responsible for phytoene synthesis and lycopene cyclisation were localised within the protein. [ABSTRACT FROM AUTHOR]

This is the first report describing the complete oxidation of dimethyl sulfide (DMS) to sulfate by an anoxygenic, phototrophic purple sulfur bacterium. Complete DMS oxidation was observed in cultures of Thiocapsa roseopersicina M11 incubated under oxic/light conditions, resulting in a yield of 30.1 mg protein mmol–1. No oxidation of DMS occurred under anoxic/light conditions. Chloroform, methyl butyl ether, and 3-amino-1,2,4-triazole, which are specific inhibitors of aerobic DMS oxidation in thiobacilli and hyphomicrobia, did not affect DMS oxidation in strain M11. This could be due to limited transport of the inhibitors through the cell membrane. The growth yield on sulfide as sole electron donor was 22.2 mg protein mmol–1 under anoxic/light conditions. Since aerobic respiration of sulfide would have resulted in yields lower than 22 mg protein mmol–1, the higher yield on DMS under oxic/light conditions suggests that the methyl groups of DMS have served as an additional carbon source or as an electron donor in addition to the sulfide moiety. The kinetic parameters V max and K m for DMS oxidation under oxic/light conditions were 12.4 ± 1.3 nmol (mg protein)–1 min–1 and 2 μM, respectively. T. roseopersicina M11 also produced DMS by cleavage of dimethylsulfoniopropionate (DMSP). Specific DMSP cleavage rates increased with increasing initial substrate concentrations, suggesting that DMSP lyase was only partly induced at lower initial DMSP concentrations. A comparison of T. roseopersicina strains revealed that only strain M11 was able to oxidize DMS and cleave DMSP. Both strain M11 and strain 5811 accumulated DMSP intracellularly during growth, while strain 1711 showed neither of these characteristics. Phylogenetic comparison based on 16S rRNA gene sequence revealed a similarity of 99.0% between strain M11 and strain 5811, and 97.6% between strain M11 and strain 1711. DMS and DMSP utilization thus appear to be strain-specific. [ABSTRACT FROM AUTHOR]

This paper follows the development in the activity of the key enzymes of glycolysis and dehydro- genases of the pentose phosphate shunt throughout the in vitro growth and metacyclogenesis of two human strains of Leishmania infantum– one visceral (VL) and the other cutaneous (CL) – together with changes in the glucose, ammonium, and proton concentrations in the culture medium. In the first stage, ammonium was generated and no glucose was consumed. Later on, all the glucose was consumed and, finally, ammonium was generated again. The ammonium concentration increased 16- and 21-fold in cultures of VL and CL strains, respectively. The activities of the glycosomal enzymes hexokinase and phosphofructokinase differed in each strain, always being higher in CL than in VL and increasing throughout the culture period in CL while decreasing in VL. This probably indicates a different capability to adapt to the culture medium conditions. The activities of the pentose phosphate shunt enzymes examined indicate that 6-phosphogluconate dehydrogenase is possibly a rate-limiting enzyme for this pathway. Pyruvate kinase is a cytosolic control enzyme of glycolysis in trypanosomatids, and its activity decreased throughout the growth and differentiation of both strains of L. infantum, as occurs in other trypanosomatids. It was also observed that glucose catabolism was more active in the cutaneous strain than in the visceral one. [ABSTRACT FROM AUTHOR]