Modulation of the catalytic performance of OYE3 by engineering key residues at the entrance of the catalytic pocket.

The amino acid residues at the entrance of the catalytic pocket may impose steric hindrance on the substrate to enter the active center of the enzyme. Based on the analysis of the three-dimensional structure of the Saccharomyces cerevisiae old yellow enzyme 3 (OYE3), four bulky residues were chosen and mutated to small amino acids. The results showed that mutation of the W116 residue had interesting impacts on the catalytic performance. All four variants became inactive for the reduction of (R)-carvone and (S)-carvone, but inverted the stereoselectivity for the reduction of (E/Z)-citral. The mutation of the F250 residue had a more positive effect on the activity and stereoselectivity. Two variants, F250A and F250S, showed excellent diastereoselectivity and activity for the reduction of (R)-carvone (de > 99%, c > 99%) and increased diastereoselectivity and activity for the reduction of (S)-carvone (de > 96%, c > 80%). One variant of the P295 residue, P295G, displayed excellent diastereoselectivity and activity only for the reduction of (R)-carvone (de > 99%, c > 99%). Mutation of the Y375 residue had a negative impact on the activity of the enzyme. These findings provide some solutions for rational enzyme engineering of OYE3.

Combined genomic and transcriptomic analysis of the dibutyl phthalate metabolic pathway in Arthrobacter sp. ZJUTW.

Dibutyl phthalate (DBP) is an environmental pollutant that can threaten human health. The strain Arthrobacter sp. ZJUTW, isolated from the sludge of a river of Hangzhou, can efficiently degrade DBP. Its genomic and transcriptomic differences when cultivated with DBP compared with glucose revealed specific DBP metabolic pathways in the ZJUTW strain. The degrading gene clusters localize separately on a circular chromosome and a plasmid pQL1. Genes related to the initial steps of DBP degradation from DBP to phthalic acid (PA), the pehA gene, and pht gene cluster, are located on the plasmid pQL1. However, the pca gene cluster related to the transforming of intermediate protocatechuic acid (PCA) to acetyl-CoA, is located on the chromosome. After comparative analysis with the reported gene clusters, we found that there were a series of homologous genes in pht and pca gene clusters that contribute to the efficient degradation of DBP by ZJUTW. In addition, transcriptomic analysis suggested a synergistic effect between pht and pca clusters, which also favor ZJUTW allowing it to efficiently degrade DBP. Combined genomic and transcriptomic analyses revealed a complete DBP metabolic pathway in Arthrobacter sp. ZJUTW that is different from that of other reported Arthrobacter strains. After necessary modification based on its metabolic characteristics, Arthrobacter sp. ZJUTW or its derivatives might represent promising candidates for the bioremediation of DBP pollution.

Biodegradation of bis(2-hydroxyethyl) terephthalate by a newly isolated Enterobacter sp. HY1 and characterization of its esterase properties.

Bis(2-hydroxyethyl) terephthalate (BHET) is an important compound produced from poly(ethylene terephthalate) (PET) cleavage. It was selected as the representative substance for the study of PET degradation. A bacterial strain HY1 that could degrade BHET was isolated and identified as Enterobacter sp. The optimal temperature and pH for BHET biodegradation were determined to be 30°C and 8.0, respectively. The half-life of degradation was 70.20 h at an initial BHET concentration of 1,000 mg/L. The results of metabolites' analysis by liquid chromatograph-mass spectrometer revealed that BHET was first converted to mono-(2-hydroxyethyl) terephthalate (MHET) and then to terephthalic acid. Furthermore, an esterase-encoding gene, estB, was cloned from strain HY1, and the expressed enzyme EstB was characterized. The esterase has a molecular mass of approximately 25.13 kDa, with an isoelectric point of 4.68. Its optimal pH and temperature were pH 8.0 and 40°C, respectively. The analysis of the enzymatic products showed that EstB could hydrolyze one ester bond of BHET to MHET. To the best of authors' knowledge, this is the first report on the biodegradation characteristics of BHET by a member of the Enterobacter genus.

Effects of polystyrene microbeads on cytotoxicity and transcriptomic profiles in human Caco-2 cells.

Microplastics (MPs) pollution is a global paradigm that raises concern in relation to environment and human health. In order to investigate the molecular toxicity mechanisms of MPs, transcriptomic analyses were performed on in vitro Caco-2 cell model. After observing that polystyrene microplastics (PS-MPs) decreased cell viability in a dose-dependent manner, the responsible genes and involved pathways that might make contribution to PS-MBs-induced toxicity to Caco-2 cells were identified with Illumina RNA seq. A total of 442 genes including, 210 up-regulated ones and 232 down-regulated ones, showed differential expression after treatment by PS-MPs with a concentration of 12.5 mg L-1 or 50.0 mg L-1 for 24 hours. Gene Ontology (GO) annotation enriched unigenes can be grouped into three separated clusters: cellular component (CC), biological process (BP), and molecular function (MF). The dominate pathways related to NF-κB, MAPK signaling, cytokine-cytokine receptor interaction, and toll-like receptor were strongly influenced by PS-MBs. These pathways are involved in modulating cell inflammatory and proliferation. The qPCR were applied to investigate the transcriptional level of five proliferation related genes (Ras, ERK, MER, CDK4, Cyclin D1) and four inflammation related genes (TRPV1, iNOS, IL-1ß, IL-8), and the results were consistent with RNA-seq data. This study has provided new insight into the understanding of the toxicity effects of PS-MBs-induced intestinal inflammatory diseases.

Transcriptomic analyses of human bronchial epithelial cells BEAS-2B exposed to brominated flame retardant (tetrabromobisphenol A).

Brominated flame retardants (BFRs) are supposed to act as disruptors of cell signaling, but the underlying mechanisms remain less clear. Human bronchial epithelial cells (BEAS-2B) were used to investigate the toxic effect and gene expression changes induced by tetrabromobisphenol A (TBBPA). By genome-wide approaches with Illumina RNA-seq, 87 genes were identified to exhibit ≥1.5-fold changes in expression after treatment by TBBPA for 48 h, among which, 79 were upregulated and 8 were downregulated. Gene ontology (GO) annotation enriched unigenes were divided into three clusters: biological process (BP), cellular component (CC) and molecular function (MF). Pathway analysis showed that NF-κB, TNF signaling, toll-like receptor, MAPK signaling and B-cell receptor were the most prominent pathways affected by TBBPA, which play key roles in regulating cell proliferation and cell differentiation, inflammatory response. Finally, for verifying the accuracy of microarray analysis, qRT-PCR was used to analyze the transcription level of key genes in the above signaling pathways, and ELISA assay confirmed the effect of TBBPA on the levels of CXCL-2, CCL-3, CCL-4, IL-1ß, TNF-α, and IL-6. These findings provided important information for further exploitation of the mechanisms under-lying BFR-induced adverse health effects.

Effects of novel brominated flame retardant TBBPA on human airway epithelial cell (A549) in vitro and proteome profiling.

The cellular toxicity response of human airway epithelial cells (A549) to tetrabromobisphenol (TBBPA) was assessed in vitro. Cell viability, levels of intracellular reactive oxygen species (ROS), lipid peroxidation (MDA), and caspase-3 activity were determined after A549 treated with varying concentrations of TBBPA. A comparative proteomic analysis was performed in cells treated with different concentrations of TBBPA (0, 10, and 40 µg/mL). Two-way anova analysis showed that cell viability was significantly decreased after treatment by TBBPA with a concentration of 16 µg/mL for 48 hr, however, the caspase-3 activities, ROS generation, and MDA content increased. Ultrastructural observation revealed that the cell was morphological damaged after exposure to 64 µg/mL TBBPA, with mitochondria seriously injured and the smooth endoplasmic reticulum dilated. There was a good correlation between ROS generation and mitochondrial dysfunction. Seventeen differentially expressed proteins involved in various biological processes were identified. These findings provide a basis for understanding the mechanisms of cell dysfunction and perturbation of antioxidant status induced by additive flame retardant on airway epithelial cells.

Shifts of microbial community structure in soils of a photovoltaic plant observed using tag-encoded pyrosequencing of 16S rRNA.

The environmental risk of fluoride and chloride pollution is pronounced in soils adjacent to solar photovoltaic sites. The elevated levels of fluoride and chloride in these soils have had significant impacts on the population size and overall biological activity of the soil microbial communities. The microbial community also plays an essential role in remediation of these soils. Questions remain as to how the fluoride and chloride contamination and subsequent remediation at these sites have impacted the population structure of the soil microbial communities. We analyzed the microbial communities in soils collected from close to a solar photovoltaic enterprise by pyrosequencing of the 16S rRNA tag. In addition, we used multivariate statistics to identity the relationships shared between sequence diversity and heterogeneity in the soil environment. The overall microbial communities were surprisingly diverse, harboring a wide variety of taxa and sharing significant correlations with different degrees of fluoride and chloride contamination. The contaminated soils harbored abundant bacteria that were probably resistant to the high acidity, high fluoride and chloride concentration, and high osmotic pressure environment. The dominant genera were Sphingomonas, Subgroup_6_norank, Clostridium sensu stricto, Nitrospira, Rhizomicrobium, and Acidithiobacillus. The results of this study provide new information regarding a previously uncharacterized ecosystem and show the value of high-throughput sequencing in the study of complex ecosystems.

Toxicological Responses of Chlorella vulgaris to Dichloromethane and Dichloroethane.

The aim of this study was to evaluate the acute toxicity effects of dichloromethane and dichloroethane on Chlorella vulgaris at the physiological and molecular level. Data showed that the cell number, chlorophyll a, and total protein content gradually decreased with increasing dichloromethane and dichloroethane concentrations over a 96-h exposure. Lower doses of two organic solvents had stimulatory effects on catalase and superoxide dismutase activity. Malondialdehyde showed a concentration-dependent increase in response to dichloromethane and dichloroethane exposure. Electron microscopy also showed that there were some chloroplast abnormalities in response to different concentrations of dichloromethane and dichloroethane exposure. Real-time polymerase chain reaction assay demonstrated that dichloromethane and dichloroethane reduced the transcript abundance of psaB, whereas that of psbC changed depending on the toxicant after 24 h of exposure. Dichloromethane and dichloroethane affected the activity of antioxidant enzymes, disrupted the chloroplast ultrastructure, and reduced transcription of photosynthesis-related genes in C. vulgaris, leading to metabolic disruption and cell death.

Peculiarities of metabolism of anthracene and pyrene by laccase-producing fungus Pycnoporus sanguineus H1.

The metabolic peculiarities of anthracene and pyrene by Pycnoporus sanguineus H1 were investigated. The fungus H1 could grow on potato dextrose agar plates with anthracene and anthraquinone as carbon sources. In liquid medium, the strain degraded 8.5% of anthracene as the sole carbon source, with no ligninolytic enzymes detected, indicating that intracellular catabolic enzymes might be responsible for the initial oxidation of anthracene. When bran was added to the medium, the degradation rate of anthracene and pyrene increased to 71.3% and 30.2%, respectively, and the laccase activities increased to a maximal value of 501.2 and 587.6 U/L, respectively. By gas chromatography-mass spectrometry analysis, anthraquinone was detected as the unique intermediate product of anthracene oxidation, with a yield molar ratio of 0.3. In vitro experiments showed that the extracellular culture fluid containing laccase transformed anthracene to anthraquinone with a yield molar ratio of 1.0, which was less than that of the in vivo experiment, indicating that anthraquinone could be further metabolized by the strain. Pyrene could not be oxidized by culture fluid. These results showed that both extracellular laccase and intracellular catabolic enzymes might play an important role in the initial oxidation of anthracene, whereas pyrene could be only oxidized by intracellular catabolic enzymes through cometabolism.

Biodegradation of aniline in an alkaline environment by a novel strain of the halophilic bacterium, Dietzia natronolimnaea JQ-AN.

Dietzia natronolimnaea JQ-AN was isolated from industrial wastewater containing aniline. Under aerobic conditions, the JQ-AN strain degraded 87% of the aniline in a 300 mg L(-1) aniline solution after 120 h of shake flask incubation in a medium containing sodium acetate. This strain had an unusually high salinity tolerance in minimal medium (0-6% NaCl, w/v). The optimal pH for microbial growth and aniline biodegradation was pH 8.0. Two liters of simulated aniline wastewater was created in a reactor at pH 8.0 and 3% NaCl (w/v), and biodegradation of aniline was tested over 7 days at 30 °C. For the initial concentrations of 100, 300, and 500 mg L(-1), 100%, 80.5% and 72% of the aniline was degraded, respectively. Strain JQ-AN may use an ortho-cleavage pathway for dissimilation of the catechol intermediate.

Construction of a native promoter-containing transposon vector for the stable monitoring of the denitrifying bacterium Pseudomonas stutzeri LYS-86 by chromosomal-integrated gfp.

Green fluorescent protein (GFP) is the most potential useful marker for the in situ monitoring of biofilm microbes. The objective of this study was to construct and compare the efficacy of transposon vectors containing native and foreign promoters in monitoring the denitrifying bacterium Pseudomonas stutzeri LYS-86 by chromosomal-integrated gfp. The promoter of nitrite reductase (Pnir) was cloned from LYS-86 and utilized to construct the transposon vector pUT/mini-Tn5-km2-Pnir-gfp. Another transposon vector, pUT/mini-Tn5-km2-Plac-gfp, containing the lactose promoter Plac was also constructed. These two transposon vectors and pUT-luxAB-gfp containing the promoter PpsbA were individually inserted into the chromosome of P. stutzeri LYS-86 by conjugation. Three GFP-tagged recombinant strains, LYS-Plac-gfp, LYS-Pnir-gfp, and LYS-PpsbA-gfp, were selected from the conjugants. Green fluorescence was observed only in LYS-Pnir-gfp, suggesting that the native promoter Pnir may be more suitable for GFP expression in P. stutzeri than the foreign promoters Plac and PpsbA. Indeed, LYS-Pnir-gfp maintained stable GFP fluorescence over 16 subcultures without significant changes in the denitrifying capacity.

Coenzyme Q(10) production by immobilized Sphingomonas sp. ZUTE03 via a conversion-extraction coupled process in a three-phase fluidized bed reactor.

A three-phase fluidized bed reactor (TPFBR) was designed to evaluate the potential of CoQ(10) production by gel-entrapped Sphingomonas sp. ZUTE03 via a conversion-extract coupled process. In the reactor, the CoQ(10) yield reached 46.99 mg/L after 8 h of conversion; a high-level yield of about 45 mg/L was maintained even after 15 repetitions (8 h/batch). To fully utilize the residual precursor (para-hydroxybenzoic acid, PHB) in the aqueous phase, the organic phase was replaced with new solution containing 70 mg/L solanesol for each 8 h batch. The CoQ(10) yield of each batch was maintained at a level of about 43 mg/L until the PHB ran out. When solid solanesol was fed to the organic phase for every 8 h batch, CoQ(10) could accumulate and reach a yield of 171.52 mg/L. When solid solanesol and PHB were fed to the conversion system after every 8 h batch, the CoQ(10) yield reached 441.65 mg/L in the organic phase after 20 repetitions, suggesting that the conversion-extract coupled process could enhance CoQ(10) production in the TPFBR.

Coenzyme Q10 production by Sphingomonas sp. ZUTE03 with novel precursors isolated from tobacco waste in a two-phase conversion system.

Coenzyme Q10 (CoQ10) is a widely used supplement in heart diseases treatment or antioxidative dietary. The microbial production of CoQ10 was enhanced by addition of solanesol and novel precursors recovered from waste tobacco. The novel precursors were separated by silica gel and identified as alpha-linolenic acid (LNA) and butylated hydroxytoluene (BHT) based on the effect on CoQ10 production and GC-MS. The effects of novel precursors on CoQ10 production by Sphingomonas sp. ZUTE03 were further evaluated in a two-phase conversion system. The precursor's combination of solanesol (70 mg/l) with BHT (30 mg/l) showed the best effect on the improvement of CoQ10 yield. A maximal CoQ10 productivity (9.5 mg l-1 h-1) was achieved after 8 h conversion, with a molar conversion rate of 92.6% and 92.4% on BHT and solanesol, respectively. The novel precursors, BHT and LNA in crude extracts from waste tobacco leaves, might become potential candidates for application in the industrial production of CoQ10 by microbes.

Effects of phenanthrene on the mortality, growth, and anti-oxidant system of earthworms (Eisenia fetida) under laboratory conditions.

To assess the toxic effects of phenanthrene on earthworms, we exposed Eisenia fetida to artificial soils supplemented with different concentrations (0.5, 2.5, 12.5, mgkg(-1) soil) of phenanthrene. The residual phenanthrene in the soil, the bioaccumulation of phenanthrene in earthworms, and the subsequent effects of phenanthrene on growth, anti-oxidant enzyme activities, and lipid peroxidation (LPO) were determined. The degradation rate of low concentrations of phenanthrene was faster than it was for higher concentrations, and the degradation half-life was 7.3d (0.5 mgkg(-1)). Bioaccumulation of phenanthrene in the earthworms decreased the phenanthrene concentration in soils, and phenanthrene content in the earthworms significantly increased with increasing initial soil concentrations. Phenanthrene had a significant effect on E. fetida growth, and the 14-d LC(50) was calculated as 40.67 mgkg(-1). Statistical analysis of the growth inhibition rate showed that the concentration and duration of exposure had significant effects on growth inhibition (p<0.001). Superoxide dismutase (SOD) activity increased at the beginning (2 and 7d) and decreased in the end (14 and 28 d). Catalase (CAT) activity in all treatments was inhibited from 1 to 14 d of exposure. However, no significant perturbations in malondialdehyde (MDA) content were noted between control and phenanthrene-treated earthworms except after 2d of exposure. These results revealed that bioaccumulation of phenanthrene in E. fetida caused concentration-dependent, sub-lethal toxicity. Growth and superoxide dismutase activity can be regarded as sensitive parameters for evaluating the toxicity of phenanthrene to earthworms.

[Isolation, identification and conditions of bacterial strain capable to metabolize high concentrations of formaldehyde].

One bacterial strain capable to degrade and metabolize formaldehyde as a sole carbon source was isolated from soil. Based on the results of standard morphological identification, physiological and biochemical characters, and 16S rDNA sequence analysis, the strain was identified as Pseudomonas putida. After single factor test and orthogonal test, the optimal condition for formaldehyde degradation was determined as the follows: peptone 1.2 g/L, KH2PO4 4 g/L, K2HPO4 3 g/L, MgSO4 x 7H2O 0.2 g/L, trace elements solution 0.1 mL/L, temperature 30 degrees C, pH 8. Under the optimal conditions, the strain tolerance of original formaldehyde concentration was up to 6 g/L and 86% of formaldehyde was consumed after 54 h. It completely consumed 5 g/L formaldehyde after 46 h and degraded 100% of 4 g/L formaldehyde after 35 h.