Insights into the Binding Interaction of Catechol 1,2-Dioxygenase with Catechol in Achromobacter xylosoxidans DN002.

Microbial remediation has become one of the promising ways to eliminate polycyclic aromatic hydrocarbons (PAHs) pollution due to its efficient enzyme metabolism system. Catechol 1,2-dioxygenase (C12O) is a crucial rate-limiting enzyme in the degradation pathway of PAHs in Achromobacter xylosoxidans DN002 that opens the benzene ring through the ortho-cleavage pathway. However, little attention has been given to explore the interaction mechanism of relevant enzyme-substrate. This study aims to investigate the binding interaction between C12O of strain DN002 and catechol by means of a molecular biological approach combined with homology modeling, molecular docking, and multiple spectroscopies. The removal rate of catechol in the mutant strain of cat A deletion was only 12.03%, compared to the wild-type strain (54.21%). A Ramachandran plot of active site regions of the primary amino acid sequences in the native enzyme showed that 93.5% sequences were in the most favored regions on account of the results of homology modeling, while an additional 6.2% amino acid sequences were found in conditionally allowed regions, and 0.4% in generously allowed regions. The binding pocket of C12O with catechol was analyzed to obtain that the catalytic trimeric group of Tyr164-His224-His226 was proven to be great vital for the ring-opening reaction of catechol by molecular docking. In the native enzyme, binding complexes were spontaneously formed by hydrophobic interactions. Binding constants and thermodynamic potentials from fluorescence spectra indicated that catechol effectively quenched the intrinsic fluorescence of C12O in the C12O/catechol complex via conventional static and dynamic quenching mechanisms of C12O. The results of ultraviolet and visible (UV) spectra, synchronous fluorescence, and circular dichroism (CD) spectra revealed conspicuous changes in the local conformation, and site-directed mutagenesis confirmed the role of predicted key residues during catalysis, wherein His226 had a significant effect on catechol utilization by C12O. This is the first report to reveal interactions of C12O with substrate from the molecular docking results, providing the mechanistic understanding of representative dioxygenases involved in aromatic compound degradation, and a solid foundation for further site modifications as well as strategies for the directed evolution of this enzyme.

[Colonization performance and pyrene degradation characteristics of Stenotrophomonas maltophilia PX1]. / å—œéº¦èŠ½å¯¡å »å•èƒžèŒPX1的降解芘特性及定殖效能.

We screened and identified an endophytic bacterium that could efficiently degrade PAHs, which would expand the library of polycyclic aromatic hydrocarbons (PAHs) degrading microorganisms and reduce the pollution risk of crops. Its degradation mechanism and colonization performance were preliminarily examined. The results showed that strain PX1 belonged to Stenotrophomonas maltophilia. The strain had broad spectrum ability to remove PAHs. In PAH mineral salt (MS) media, almost 100% naphthalene was degraded by strain PX1 after 7-d incubation. In a cultivation system solely containing phenanthrene of 50.0 mg·L-1, pyrene of 20.0 mg·L-1, fluoranthene of 20.0 mg·L-1 or benzo[a]pyrene of 10.0 mg·L-1, the degradation efficiency of phenanthrene, pyrene, fluoranthene and benzo[a]pyrene by strain PX1 reached 72.6%, 50.7%, 31.9%, and 12.9%, respectively. Pyrene was selected as PAHs model to study the degradation characteristics of strain PX1. Enzyme activity tests showed that the activities of phthalate dioxygenase, catechol-1,2-dioxygenase, and catechol-2,3-dioxygenase in strain PX1 were induced by pyrene. Some metabolic intermediates such as 4,5-epoxypyrene, 4,5-dihydroxypyrene, gentilic acid/protocatechuic acid, salicylic acid, cis-hexadienedioic acid/2-hydroxymyxofuroic acid semialdehyde, cis-2'-carboxyphenylpyruvic acid, 1-hydroxy-2-naphthoic acid, and salicylaldehyde were detected during the degradation of pyrene by strain PX1. Results of the seed soaking experiment showed that strain PX1 could efficiently colonize in Ipomoea aquatic and Triticum aestivum. After inoculated with strain PX1, the growth of I. aquatic and T. aestivum was significantly increased, and the pyrene concentration in I. aquatic, T. aestivum and MS media was reduced by 29.8%-50.7%, 52.4%-67.1% and 8.0%-15.3%, respectively. Our results suggested that strain PX1 degraded pyrene mainly through 'salicylate pathway' and 'phthalate pathway', and could be colonized into plants and promote plant growth.

Biodegradation kinetics and metabolism of Benzo(a)fluorene by Pseudomonas strains isolated from refinery effluent.

The final sinkers of polyaromatic hydrocarbons are water sources, where they undergo bioaccumulation and biomagnification, leading to adverse mutagenic, carcinogenic, and teratogenic effects on exposure in flora, fauna, and humans. Two indigenous strains, Pseudomonas sp. WDE11 and Pseudomonas sp. WD23, isolated from refinery effluent, degraded over 97.5% of benzo(a)fluorene (10 mg/L) in 7 days. On growth at concentration dependent amounts (50 mg/L and 100 mg/L), the degradation reduced to approximately 90% and 80% respectively in 56 days. Degradation kinetics was concentration dependent, as degradation followed first-order and second-order kinetics for 50 mg/L and 100 mg/L respectively. The half-life for degradation of benzo(a)fluorene ranged between 11.64 - 12.26 days and 13.11-14.5 days for strains WDE11 and WD23 respectively. The values of Andrew-Haldane kinetic parameters i.e. µmax, Ks, and Ki were 0.306 day-1, 11.11 mg/L, and 120.41 mg/L for strain WDE11 respectively, while for strain WD23, the respective values were 0.312 day-1, 9.97 mg/L, and 152 mg/L. Degradation metabolites were identified by their MS patterns as 3,4-dihydroxy fluorene, 2-(1-oxo-2,3-dihydro-1H-inden-2-yl) acetic acid, 3,4-dihydrocoumarin, salicylic acid, catechol, and oxalic acid. Metabolic pathway of degradation constructed, revealed that benzo(a)fluorene was metabolized via the formation of fluorene, further metabolized by salicylate pathway forming catechol. The catechol formed was degraded into simpler metabolites by meta-cleavage pathway, which was validated by catechol 2,3 dioxygenase enzyme activity.

Discovery by metagenomics of a functional tandem repeat sequence that controls gene expression in bacteria.

The ability to degrade exogenous compounds is acquired by adaptive processes of microorganisms when they are exposed to compounds that are foreign to their existing enzyme systems. Previously, we reported that simultaneous point mutations and mobile genetic elements cause the evolution and optimization of the degradation systems for aromatic compounds. In the present study, we propose another element with this role-tandem repeats. The novel metagenomic tandem repeat (MTR) sequence T(G/A)ACATG(A/C)T was identified in the 5'-untranslated regions of catechol 2,3-dioxygenase (C23O)-encoding genes by metagenomic analysis. Recombinant Escherichia coli carrying a C23O gene with various numbers of MTRs exhibited increased C23O protein expression and enzyme activity compared with cells expressing the C23O gene without MTRs. Real-time reverse transcription PCR showed that changes in the numbers of MTRs affected the levels of detectable C23O mRNA in the E. coli host. Furthermore, the mRNAs transcribed from C23O genes containing various numbers of MTRs had longer half-lives than those transcribed from a C23O gene without MTRs. Thus, MTRs would affect the translation efficiency of the gene expression system. MTRs may change the expression levels of their downstream genes for adaptation to a fluctuating environment.

Shape-function of a novel metapyrocatechase, RW4-MPC: Metagenomics to SAXS data based insight into deciphering regulators of function.

The oxygenases have attracted considerable attention in enzyme-mediated bioremediation of xenobiotic compounds due to their high specificity, cost-effectiveness, and targeted field applications. Here, we performed a functional metagenomics approach to cope with culturability limitations to isolate a novel extradiol dioxygenase. Fosmid clone harboring dioxygenase gene was sequenced and analyzed by bioinformatics tools. One ring-cleaving dioxygenase RW4-MPC (metapyrocatechase) was purified and characterized to examine its degradation efficiency. The RW4-MPC was significantly active in the temperature and pH range of 5 to 40 °C, and 7-10, respectively, with an optimum temperature of 25 °C and pH 8. To gain insight into observed differential activity, Small-Angle X-ray Scattering (SAXS) data of the protein samples were analyzed, which brought forth that the RW4-MPC molecules form tight globular tetramers in solution. This native association was stable till 35 °C, and protein started to associate at higher temperatures, explaining heat-induced loss of function. Similarly, RW4-MPC aggregated or lost globular profile below pH 7 or at pH 10, respectively. The kinetic parameters showed the six folds high catalytic efficiency of RW4-MPC towards 2,3-dihydroxy biphenyl than catechol and its derivatives. RW4-MPC molecules showed remarkable retention of functionality in hypersaline conditions with more than 70% activity in a buffer having 3 M NaCl concentration. In concordance, SAXS data analysis showed retention of functional shape profile in hypersaline conditions. The halotolerant and oxygen insensitive nature of this enzyme makes it a potential candidate for bioremediation.

Rhizosphere assisted biodegradation of benzo(a)pyrene by cadmium resistant plant-probiotic Serratia marcescens S2I7, and its genomic traits.

Melia azedarach-rhizosphere mediated degradation of benzo(a)pyrene (BaP), in the presence of cadmium (Cd) was studied, using efficient rhizobacterial isolate. Serratia marcescens S2I7, isolated from the petroleum-contaminated site, was able to tolerate up to 3.25 mM Cd. In the presence of Cd, the isolate S2I7 exhibited an increase in the activity of stress-responsive enzyme, glutathione-S-transferase. Gas Chromatography-Mass spectroscopy analysis revealed up to 59% in -vitro degradation of BaP after 21 days, while in the presence of Cd, the degradation was decreased by 14%. The bacterial isolate showed excellent plant growth-promoting attributes and could enhance the growth of host plant in Cd contaminated soil. The 52,41,555 bp genome of isolate S. marcescens S2I7 was sequenced, assembled and annotated into 4694 genes. Among these, 89 genes were identified for the metabolism of aromatic compounds and 172 genes for metal resistance, including the efflux pump system. A 2 MB segment of the genome was identified to contain operons for protocatechuate degradation, catechol degradation, benzoate degradation, and an IclR type regulatory protein pcaR, reported to be involved in the regulation of protocatechuate degradation. A pot trial was performed to validate the ability of S2I7 for rhizodegradation of BaP when applied through Melia azedarach rhizosphere. The rhizodegradation of BaP was significantly higher when augmented with S2I7 (85%) than degradation in bulk soil (68%), but decreased in the presence of Cd (71%).

Biodecomposition of Phenanthrene and Pyrene by a Genetically Engineered Escherichia coli.

BACKGROUND: Genetically engineered microorganisms (GEMs) can be used for bioremediation of the biological pollutants into nonhazardous or less-hazardous substances, at lower cost. Polycyclic aromatic hydrocarbons (PAHs) are one of these contaminants that associated with a risk of human cancer development. Genetically engineered E. coli that encoded catechol 2,3- dioxygenase (C230) was created and investigated its ability to biodecomposition of phenanthrene and pyrene in spiked soil using high-performance liquid chromatography (HPLC) measurement. We revised patents documents relating to the use of GEMs for bioremediation. This approach have already been done in others studies although using other genes codifying for same catechol degradation approach. OBJECTIVE: In this study, we investigated biodecomposition of phenanthrene and pyrene by a genetically engineered Escherichia coli. METHODS: Briefly, following the cloning of C230 gene (nahH) into pUC18 vector and transformation into E. coli Top10F, the complementary tests, including catalase, oxidase and PCR were used as on isolated bacteria from spiked soil. RESULTS: The results of HPLC measurement showed that in spiked soil containing engineered E. coli, biodegradation of phenanthrene and pyrene comparing to autoclaved soil that inoculated by wild type of E. coli and normal soil group with natural microbial flora, were statistically significant (p<0.05). Moreover, catalase test was positive while the oxidase tests were negative. CONCLUSION: These findings indicated that genetically manipulated E. coli can provide an effective clean-up process on PAH compounds and it is useful for bioremediation of environmental pollution with petrochemical products.

Mechanistic investigations in ultrasound-assisted biodegradation of phenanthrene.

This study has addressed the biodegradation of polycyclic aromatic hydrocarbon, phenanthrene using Candida tropicalis. Optimization using central composite statistical design yielded optimum experimental parameters as: pH = 6.2, temperature = 33.4 °C, mechanical shaking = 190 rpm and % inoculum = 9.26% v/v. Sonication of biodegradation mixture at 33 kHz and 10% duty cycle in log phase (12 h per day for 4 days) resulted in a 25% enhancement in phenanthrene removal. Profiles of specific growth rate (µ) and specific degradation rate (q) versus initial substrate concentration were fitted to Haldane substrate inhibition model. Both µ and q showed maxima for initial concentration of 100 mg L-1. Kinetic analysis of degradation profiles showed higher biomass yield coefficient and smaller decay coefficient in presence of sonication. Expression of total intracellular proteins in control and test experiments were analyzed using SDS-PAGE. This analysis revealed overexpression of enzyme catechol 2,3-dioxygenase (in meta route metabolism) during sonication which is involved in ring cleavage of phenanthrene. Evaluation of cell viability after sonication by flow cytometry analysis revealed > 80% live cells. These effects are attributed to enhanced cellular transport induced by intense microturbulence generated by sonication.

Novel PCB-degrading Rhodococcus strains able to promote plant growth for assisted rhizoremediation of historically polluted soils.

Extended soil contamination by polychlorinated biphenyls (PCBs) represents a global environmental issue that can hardly be addressed with the conventional remediation treatments. Rhizoremediation is a sustainable alternative, exploiting plants to stimulate in situ the degradative bacterial communities naturally occurring in historically polluted areas. This approach can be enhanced by the use of bacterial strains that combine PCB degradation potential with the ability to promote plant and root development. With this aim, we established a collection of aerobic bacteria isolated from the soil of the highly PCB-polluted site "SIN Brescia-Caffaro" (Italy) biostimulated by the plant Phalaris arundinacea. The strains, selected on biphenyl and plant secondary metabolites provided as unique carbon source, were largely dominated by Actinobacteria and a significant number showed traits of interest for remediation, harbouring genes homologous to bphA, involved in the PCB oxidation pathway, and displaying 2,3-catechol dioxygenase activity and emulsification properties. Several strains also showed the potential to alleviate plant stress through 1-aminocyclopropane-1-carboxylate deaminase activity. In particular, we identified three Rhodococcus strains able to degrade in vitro several PCB congeners and to promote lateral root emergence in the model plant Arabidopsis thaliana in vivo. In addition, these strains showed the capacity to colonize the root system and to increase the plant biomass in PCB contaminated soil, making them ideal candidates to sustain microbial-assisted PCB rhizoremediation through a bioaugmentation approach.

Highly sensitive microbial biosensor based on recombinant Escherichia coli overexpressing catechol 2,3-dioxygenase for reliable detection of catechol.

A highly sensitive whole cell based electrochemical biosensor was developed for catechol detection in this study. The carE gene of Sphingobium yanoikuyae XLDN2-5 encoding catechol 2,3-dioxygenase (C23O), a key enzyme in the biodegradation of aromatic compound, was cloned and over-expressed in Escherichia coli BL21 (E. coli BL21). Compared to Sphingobium yanoikuyae XLDN2-5, the recombinant E. coli BL21 over-expressed C23O exhibited higher catalytic activity towards catechol. Moreover, the whole cells provided a better environment for C23O to maintain its catalytic activity and stability compared with crude enzyme. The distinctive features of the recombinant E. coli BL21 over-expressed C23O made it an ideal bio-recognition element for the fabrication of a microbial biosensor. Additionally, nanoporous gold (NPG) with unique properties of structure and function was selected as a support to immobilized the recombinant E. coli BL21 over-expressed C23O. Based on the synergistic effect of C23O and NPG, the E. coli BL21-C23O/NPG/GCE bioelectrode showed a good linear response for catechol detection ranging from 1 µM to 500 µM with a high sensitivity of 332.24 µA mM-1 cm-2 and a low detection limit of 0.24 µM. Besides, the E. coli BL21-C23O/NPG/GCE bioelectrode exhibited strong anti-interference and good stability. For the detection of catechol in wastewater samples, the concentrations detected by the E. coli BL21-C23O/NPG/GCE bioelectrode were in good agreement with the standard concentrations that added in the wastewater samples, which make the E. coli BL21-C23O/NPG/GCE bioelectrode an ideal tool for reliable catechol detection.

Versatile catechol dioxygenases in Sphingobium scionense WP01T.

The objective was to understand the roles of multiple catechol dioxygenases in the type strain Sphingobium scionense WP01T (Liang and Lloyd-Jones in Int J Syst Evol Microbiol 60:413-416, 2010a) that was isolated from severely contaminated sawmill soil. The dioxygenases were identified by sequencing, examined by determining the substrate specificities of the recombinant enzymes, and by quantifying gene expression following exposure to model priority pollutants. Catechol dioxygenase genes encoding an extradiol xylE and two intradiol dioxygenases catA and clcA that are highly similar to sequences described in other sphingomonads are described in S. scionense WP01T. The distinct substrate specificities determined for the recombinant enzymes confirm the annotated gene functions and suggest different catabolic roles for each enzyme. The role of the three enzymes was evaluated by analysis of enzyme activity in crude cell extracts from cells grown on meta-toluate, benzoate, biphenyl, naphthalene and phenanthrene which revealed the co-induction of each enzyme by different substrates. This was corroborated by quantifying gene expression when cells were induced by biphenyl, naphthalene and pentachlorophenol. It is concluded that the ClcA and XylE enzymes are recruited in pathways that are involved in the degradation of chlorinated aromatic compounds such as pentachlorophenol, the XylE and ClcA enzymes will also play a role in degradation pathways that produce alkylcatechols, while the three enzymes ClcA, XylE and CatA will be simultaneously involved in pathways that generate catechol as a degradation pathway intermediate.

Biotransformation of benzo[a]pyrene by the thermophilic bacterium Bacillus licheniformis M2-7.

Benzo[a]pyrene (BaP) is recognized as a potentially carcinogenic and mutagenic hydrocarbon, and thus, its removal from the environment is a priority. The use of thermophilic bacteria capable of biodegrading or biotransforming this compound to less toxic forms has been explored in recent decades, since it provides advantages compared to mesophilic organisms. This study assessed the biotransformation of BaP by the thermophilic bacterium Bacillus licheniformis M2-7. Our analysis of the biotransformation process mediated by strain M2-7 on BaP shows that it begins during the first 3 h of culture. The gas chromatogram of the compound produced shows a peak with a retention time of 17.38 min, and the mass spectra shows an approximate molecular ion of m/z 167, which coincides with the molecular weight of the chemical formula C6H4(COOH)2, confirming a chemical structure corresponding to phthalic acid. Catechol 2,3-dioxygenase (C23O) enzyme activity was detected in minimal saline medium supplemented with BaP (0.33 U mg-1 of protein). This finding suggests that B. licheniformis M2-7 uses the meta pathway for biodegrading BaP using the enzyme C23O, thereby generating phthalic acid as an intermediate.

Catechol 2,3-dioxygenase from a new phenolic compound degrader Thauera sp. K11: purification and biochemical characterization.

Catechol 2,3-dioxygenase (C23O) from a new phenolic compound degrader Thauera sp. K11 was purified and characterized. The native form of the enzyme was determined as a homotetramer with a molecular weight of 140 kDa, and its isoelectric point was close to 6.4. One iron per enzyme subunit was detected using atom absorption spectroscopy, and the effective size of C23O in its dilute solution (0.2 g L-1 , pH 8.0) was 14.5 nm. The optimal pH and temperature were 8.4 and 45 °C, respectively. The addition of Mg2+ , Cu2+ , Fe2+ , and Mn2+ could improve the enzyme activity, while Ag+ was found to be a strong inhibitor. C23O was stable in alkali conditions (pH 7.6-11.0) and thermostable below 50 °C. The final purified C23O had a sheet content of 53%, consistent with the theoretical value. This showed that the purified catechol 2,3-dioxygenase folded with a reasonable secondary structure.

Assessment of active bacteria metabolizing phenolic acids in the peanut (Arachis hypogaea L.) rhizosphere.

Phenolic acids can enhance the mycotoxin production and activities of hydrolytic enzymes related to pathogenicity of soilborne fungus Fusarium oxysporum. However, characteristics of phenolic acid-degrading bacteria have not been investigated. The objectives of this study were to isolate and characterize bacteria capable of growth on benzoic and vanillic acids as the sole carbon source in the peanut rhizosphere. Twenty-four bacteria were isolated, and the identification based on 16S rRNA gene sequencing revealed that pre-exposure to phenolic acids before sowing shifted the dominant culturable bacterial degraders from Arthrobacter to Burkholderia stabilis-like isolates. Both Arthrobacter and B. stabilis-like isolates catalysed the aromatic ring cleavage via the ortho pathway, and Arthrobacter isolates did not exhibit higher C12O enzyme activity than B. stabilis-like isolates. The culture filtrate of Fusarium sp. ACCC36194 caused a strong inhibition of Arthrobacter growth but not B. stabilis-like isolates. Additionally, Arthrobacter isolates responded differently to the culture filtrates of B. stabilis-like isolates. The Arthrobacter isolates produced higher indole acetic acid (IAA) levels than B. stabilis-like isolates, but B. stabilis-like isolates were also able to produce siderophores, solubilize mineral phosphate, and exert an antagonistic activity against peanut root rot pathogen Fusarium sp. ACCC36194. Results indicate that phenolic acids can shift their dominant culturable bacterial degraders from Arthrobacter to Burkholderia species in the peanut rhizosphere, and microbial interactions might lead to the reduction of culturable Arthrobacter. Furthermore, increasing bacterial populations metabolizing phenolic acids in monoculture fields might be a control strategy for soilborne diseases caused by Fusarium spp.

Biodegradation of phenol via meta cleavage pathway triggers de novo TAG biosynthesis pathway in oleaginous yeast.

Phenol is reported to be one of the most toxic environmental pollutants present in the discharge of various industrial effluents causing a serious threat to the existing biome. Biodegradation of phenol by oleaginous yeast Rhodosporidium kratochvilovae HIMPA1 was found to degrade 1000mg/l phenol. The pathways for phenol degradation by both ortho and meta-cleavage were proposed by the identification of metabolites and enzymatic assays of ring cleavage enzymes in the cell extracts. Results suggest that this oleaginous yeast degrade phenol via meta-cleavage pathway and accumulates a high quantity of lipid content (64.92%; wt/wt) as compared to control glucose synthetic medium (GSM). Meta-cleavage pathway of phenol degradation leads to formation of pyruvate and acetaldehyde. Both these end products feed as precursors for de novo triacylglycerols (TAG) biosynthesis pathway which causes accumulation of TAG in the lipid droplets (LD) of 6.12±0.78µm grown on phenol while 2.38±0.52µm observed on GSM. This was confirmed by fluorescence microscopic images of BODIPY505-515nm stained live yeast cells. GC-MS analysis of extracted total lipid showed enhanced amount of monounsaturated fatty acid (MUFA) which was as 51.87%, 58.33% and 62.98% in presence of 0.5, 0.75 and 1g/l of phenol.

Microaerobic degradation of 2-Mercaptobenzothiazole present in industrial wastewater.

Microaerobic degradation of 2-Mercaptobenzothiazole (2-MBT) was investigated using an isolated bacterial strain CSMB1. It was identified as Alcaligenes sp. MH146 by genomic analysis. The isolate degraded 50mg/L concentration of 2-MBT which was measured in terms of Total organic carbon (TOC) (700mg/L). A maximum degradation of 86% with a residual TOC concentration of 101mg/L was obtained after 72h, with the biomass growth of 290mg/L. The presence of specific activity of catechol 2, 3 oxygenase was observed in all the tested derivatives of benzothiazoles and the benzene ring opening was observed through meta cleavage. By analyzing the 72h incubated culture supernatant, 2-MBT, and all its biotransformed products were degraded into polar compounds. With the analytical results obtained, a possible microaerobic degradative pathway was proposed and illustrated for 2-MBT. It is concluded that microaerophilic isolate CSMB1 was able to degrade 2-MBT and its intermediates by utilizing them as sole carbon and energy.

Development of nitrilase promoter-derived inducible vectors for Streptomyces.

An inducible expression vector, pSH19, which harbors regulatory expression system PnitA-NitR, for streptomycetes was constructed previously. Here, we have modified pSH19 to obtain shuttle vectors for Streptomyces-E. coli by introducing the replication origin of a plasmid for E. coli (ColE1) and an antibiotic-resistant gene. Six inducible shuttle vectors, pESH19cF, pESH19cR, pESH19kF, pESH19kR, pESH19aF, and pESH19aR, for Streptomyces-E. coli, were successfully developed. The stability of these vectors was examined in five different E. coli strains and Streptomyces lividans TK24. The stability test showed that the pSH19-derived shuttle vectors were stable in E. coli Stbl2 and S. lividans TK24. Heterologous expression experiments involving each of the catechol 2,3-dioxygenase, nitrilase, and N-substituted formamide deformylase genes as a reporter gene showed that pESH19cF, pESH19kF, and pESH19aF possess inducible expression ability in S. lividans TK24. Thus, these vectors were found to be useful expression tools for experiments on both Gram-negative and Gram-positive bacterial genes.

Antioxidant enzymes are induced by phenol in the marine microalga Lingulodinium polyedrum.

Knowing the impacts of different anthropogenic activities on ecosystems promotes preservation of aquatic organisms. Aiming to facilitate the identification of polluted or contaminated areas, the study of microalga Lingulodinium polyedrum in phenol-containing medium comprises the determination of toxic and metabolic phenol effects, featuring a possible use of this microorganism as bioindicator for this pollutant. Marine microalga L. polyedrum exposure to phenol increases superoxide dismutase (SOD) and catalase (CAT) activities. The 20% and 50% inhibitory concentrations (IC20 and IC50) of cells exposed to phenol were 40 µmol L(-1) and 120 µmol L(-1), respectively. Phenol biodegradation by L. polyedrum was 0.02 µmol h(-1)cell(-1), and its biotransformation was catalyzed by glutathione S-transferase (GST), phenol hydroxylase and catechol 2,3-dihydroxygenase metabolic pathways. Phenol exposure produced the metabolites 2-hydroxymuconic semialdehyde acid, 1,2-dihydroxybenzene (catechol), and 2-oxo-4-pentenoic acid; also, it induced the activity of key antioxidant biomarker enzymes SOD and CAT by three folds compared to that in the controls. Further, phenol decreased the glutathione/oxidized glutathione ratio (GSH/GSSG), highlighting the effective glutathione oxidation in L. polyedrum. Overall, our results suggest that phenol alters microalga growth conditions and microalgae are sensitive bioindicators to pollution by phenol in marine environments.

Effects of LB broth, naphthalene concentration, and acetone on the naphthalene degradation activities by Pseudomonas putida G7.

Luria-Bertani broth and acetone were usually used in naphthalene degradation experiments as nutrient and solvent. However, their effect on the degradation was seldom mentioned. In this work, we investigated the effect of LB, naphthalene concentration, and acetone on the degradation of naphthalene by Pseudomonas putida G7, which is useful for the degradation of naphthalene on future field remediation. By adding LB, the naphthalene degradation efficiencies and naphthalene dioxygenase were both decreased by 98%, while the catechol dioxygenase was decreased by 90%. Degradation of naphthalene was also inhibited when naphthalene concentration was 56 ppm and higher, which was accompanied with the accumulation of orange-colored metabolism products. However, acetone can stimulate the degradation of naphthalene, and the stimulation was more obvious when naphthalene concentration was lower than 2000 ppm. By assaying the enzyme activities of naphthalene dioxygenase and catechol dioxygenase, it was thought that the degradation efficiency was depending on the more sensitive enzymes on the complicated conditions.

Bioinspired copper(I) complexes that exhibit monooxygenase and catechol dioxygenase activity.

New tripodal ligand L2 featuring three different pyridyl/imidazolyl-based N-donor units at a bridgehead C atom, from which one of the imidazolyl units is separated by a phenylene linker, was synthesized and investigated with regards to copper(I) complexation. The resulting complex [(L2)Cu]OTf (2(OTf)), the known complex [(L1)Cu]OTf (1(OTf); L1 differs from L2 in that it lacks the phenylene spacer) and [(L3)Cu]OTf (3(OTf)), prepared from a known chiral, tripodal, N-donor ligand featuring pyridyl, pyrazolyl, and imidazolyl donors, were tested as catalysts for the oxidation of sodium 2,4-di-tert-butylphenolate (NaDTBP) with O2. Indeed, they mediated NaDTBP oxidation to give mainly the corresponding catecholate and quinone (Q). None of the complexes 1(OTf), 2(OTf), and 3(OTf) is superior to the others, as yields were comparable and, if the presence of protons is guaranteed by concomitant addition of the phenol DTBP, the oxidation can also be performed catalytically. For all complexes stoichiometric oxidations under certain conditions (concentrated solutions, high NaDTBP content) were found to also generate products typical for metal-mediated intradiol cleavage of the catecholate with O2. As shown representatively for 1(OTf) this dioxygenation sets in at a later stage of the reaction. Initially a copper species responsible for the monooxygenation must form from 1(OTf)/NaDTBP/O2, and only thereafter is the copper species responsible for dioxygenation formed and consumes Q as substrate. Hence, under these circumstances complexes 1(OTf)-3(OTf) show both monooxygenase and catechol dioxygenase activity.