A Novel and Efficient Phthalate Hydrolase from Acinetobacter sp. LUNF3: Molecular Cloning, Characterization and Catalytic Mechanism.

Phthalic acid esters (PAEs), which are widespread environmental contaminants, can be efficiently biodegraded, mediated by enzymes such as hydrolases. Despite great advances in the characterization of PAE hydrolases, which are the most important enzymes in the process of PAE degradation, their molecular catalytic mechanism has rarely been systematically investigated. Acinetobacter sp. LUNF3, which was isolated from contaminated soil in this study, demonstrated excellent PAE degradation at 30 °C and pH 5.0-11.0. After sequencing and annotating the complete genome, the gene dphAN1, encoding a novel putative PAE hydrolase, was identified with the conserved motifs catalytic triad (Ser201-Asp295-His325) and oxyanion hole (H127GGG130). DphAN1 can hydrolyze DEP (diethyl phthalate), DBP (dibutyl phthalate) and BBP (benzyl butyl phthalate). The high activity of DphAN1 was observed under a wide range of temperature (10-40 °C) and pH (6.0-9.0). Moreover, the metal ions (Fe2+, Mn2+, Cr2+ and Fe3+) and surfactant TritonX-100 significantly activated DphAN1, indicating a high adaptability and tolerance of DphAN1 to these chemicals. Molecular docking revealed the catalytic triad, oxyanion hole and other residues involved in binding DBP. The mutation of these residues reduced the activity of DphAN1, confirming their interaction with DBP. These results shed light on the catalytic mechanism of DphAN1 and may contribute to protein structural modification to improve catalytic efficiency in environment remediation.

Composting reduces the risks of antibiotic resistance genes in maize seeds posed by gentamicin fermentation waste.

Using high-throughput quantitative PCR and next generation sequencing, the impact of land application of raw and composted gentamicin fermentation waste (GFW) on antibiotic resistance genes (ARGs) in maize seeds was studied in a three-year field trial. The raw and composted GFW changed both the bacterial community composition and the ARGs diversity in the maize seeds compared to non-amended controls and chemical fertilizer. The abundance of ARGs after raw GFW amendment was significantly higher than other treatments because of a high abundance of aadA1, qacEdeltal and aph(2')-Id-02; probably induced by gentamicin selection pressure in maize tissues. Meanwhile, the potential host of these three ARGs, pathogenic bacteria Tenacibaculum, also increased significantly in maize seeds after the application of raw GFW. But our result proved that composting could weaken the risk posed by GFW. We further reveal that the key biotic driver for shaping the ARG profiles in maize seeds is bacterial community followed by heavy metal resistance genes, and ARGs are more likely located on bacterial chromosomes. Our findings provide new insight into ARGs dispersal mechanism in maize seeds after long-term GFW application, demonstrate the potential benefits of composting the GFW to reduce risks as well as the potential efficient management method to GFW.

Cometabolic biodegradation of quizalofop-p-ethyl by Methylobacterium populi YC-XJ1 and identification of QPEH1 esterase

BACKGROUND: Quizalofop-p-ethyl (QPE), a unitary R configuration aromatic oxyphenoxypropionic acid ester (AOPP) herbicide, was widely used and had led to detrimental environmental effects. For finding the QPEdegrading bacteria and promoting the biodegradation of QPE, a series of studies were carried out. RESULTS: A QPE-degrading bacterial strain YC-XJ1 was isolated from desert soil and identified as Methylobacterium populi, which could degrade QPE with methanol by cometabolism. Ninety-seven percent of QPE (50 mg/L) could be degraded within 72 h under optimum biodegradation condition of 35°C and pH 8.0. The maximum degradation rate of QPE was 1.4 mg/L/h, and the strain YC-XJ1 exhibited some certain salinity tolerance. Two novel metabolites, 2-hydroxy-6-chloroquinoxaline and quinoxaline, were found by high-performance liquid chromatography/mass spectroscopy analysis. The metabolic pathway of QPE was predicted. The catalytic efficiency of strain YC-XJ1 toward different AOPPs herbicides in descending order was as follows: haloxyfop-pmethyl ≈ diclofop-methyl ≈ fluazifop-p-butyl N clodinafop-propargyl N cyhalofop-butyl N quizalofop-p-ethyl N fenoxaprop-p-ethyl N propaquizafop N quizalofop-p-tefuryl. The genome of strain YC-XJ1 was sequenced using a combination of PacBio RS II and Illumina platforms. According to the annotation result, one α/ß hydrolase gene was selected and named qpeh1, for which QPE-degrading function has obtained validation. Based on the phylogenetic analysis and multiple sequence alignment with other QPE-degrading esterases reported previously, the QPEH1 was clustered with esterase family V. CONCLUSION: M. populi YC-XJ1 could degrade QPE with a novel pathway, and the qpeh1 gene was identified as one of QPE-degrading esterase gene.

[Characterization and 16S rRNA gene-based metagenomic analysis of the organophosphorous flame retardants degrading consortium YC-BJ1].

As flame retardants, organophosphate is recognized as a global environmental contaminant because of its wide application. This contaminant is hardly degradable by hydrolysis in the environment due to its special physicochemical properties. Therefore, it is of urgent needs to study the microbial degradation of organophosphate. Through continuous enrichment, we isolated one bacterial consortium, named YC-BJ1, from leachate of waste treatment plant in Beijing. The bacterial consortium YC-BJ1 could efficiently degrade 99.8% of triphenyl phosphate (TPhP) and 91.9% of tricresyl phosphate (TCrP) with the concentration of 100 mg/L within 4 days. Besides aryl phosphates, it could degrade chloro-phosphates, tris(1,3-dichloroisopropyl) phosphate (TDCPP) and tris(2-chloroethyl) phosphate (TCEP) by 16.5% and 22.0% respectively. The degradation of the consortium on TPhP was optimized through a broad range of temperature (15-40 ℃), pH (5.0-12.0) and salinity (0%-4%). 16S rRNA gene-based metagenomic analysis revealed that Hyphomicrobium (38.80%), Chryseobacterium (17.57%) and Sphingopyxis (17.46%) were the dominant genera of the consortium YC-BJ1. Compared with the reported organophosphorus flame retardants (OPFRs) degrading bacteria and microflora, the mixed microflora YC-BJ1 exhibited great advantages in degradation efficiency and environmental adaptability, demonstrating its wide application potential. The enrichment and isolation of highly efficient degrading flora can provide abundant microbial resources for the degradation of OPFRs and the bioremediation towards OPFRs-contaminated environments, and also lay a solid foundation for the exploration of its degradation mechanism.

Characterization and 16S metagenomic analysis of organophosphorus flame retardants degrading consortia.

Three bacterial consortia, named YC-SY1, YC-BJ1 and YC-GZ1, were enriched from different areas of China. Bacterial consortia YC-SY1, YC-BJ1 and YC-GZ1 could efficiently degrade triphenyl phosphate (TPhP) (100 mg/L) by approximately 79.4%, 99.8% and 99.6%, tricresyl phosphate (TCrP) by 90.6%, 91.9% and 96.3%, respectively, within 4 days. And they could retain high degrading efficiency under a broad range of temperature (15-40 ℃), pH (6.0-10.0) and salinity (0-4%). A total of 10 bacterial isolates were selected and investigated their degradation capacity. Among these isolates, two were significantly superior to the others. Strain Rhodococcus sp. YC-JH2 could utilize TPhP (50 mg/L) as sole carbon source for growth with 37.36% degradation within 7 days. Strain Sphingopyxis sp. YC-JH3 could efficiently degrade 96.2% of TPhP (50 mg/L) within 7 days, except that no cell growth was observed. Combined with 16S diversity analysis, our results suggest that the effective components of three bacterial consortia responsible for TPhP and TCrP degradation were almost the same, that is, bacteria capable of degrading TPhP and TCrP are limited, in this study, the most efficient component is Sphingopyxis. This study provides abundant microorganism sources for research on organophosphorus flame retardants (OPFRs) metabolism and bioremediation towards OPFRs-contaminated environments.

Identification and characterization of a meta-cleavage product hydrolase involved in biphenyl degradation from Arthrobacter sp. YC-RL1.

Polychlorinated biphenyls (PCBs) are a group of persistent organic pollutants (POPs) widely existing in the environment. Arthrobacter sp. YC-RL1 is a biphenyl-degrading bacterium that shows metabolic versatility towards aromatic compounds. A 2-hydroxy-6-oxo-6-phenylhexa-2, 4-dienoate (HOPDA) hydrolase (BphD) gene involved in the biodegradation of biphenyl was cloned from strain YC-RL1 and heterologously expressed in Escherichia coli BL21 (DE3). The recombinant BphDYC-RL1 was purified and characterized. BphDYC-RL1 showed the highest activity at 45 °C and pH 7. It was stable under a wide range of temperature (20-50 °C). The enzyme had a Km value of 0.14 mM, Kcat of 11.61 s-1, and Vmax of 0.027 U/mg. Temperature dependence catalysis exhibited a biphasic Arrhenius Plot with a transition at 20 °C. BphDYC-RL1 was inactivated by SDS, Tween 20, Tween 80, Trition X-100, DTT, CHAPS, NBS, PMSF, and DEPC, but insensitive to EDTA. Site-directed mutagenesis of the active-site residues revealed that the catalytic triad residues (Ser115, His275, and Asp247) of BphDYC-RL1 were necessary for its activity. The investigation of BphDYC-RL1 not only provides new potential enzyme resource for the biodegradation of biphenyl but also helps deepen our understanding on the catalytic process and mechanism.

In silico genome analysis reveals the metabolic versatility and biotechnology potential of a halotorelant phthalic acid esters degrading Gordonia alkanivorans strain YC-RL2.

Members of genus Gordonia are known to degrade various xenobitics and produce secondary metabolites. The genome of a halotorelant phthalic acid ester (PAEs) degrading actinobacterium Gordonia alkanivorans strain YC-RL2 was sequenced using Biosciences RS II platform and Single Molecular Real-Time (SMRT) technology. The reads were assembled de novo by hierarchical genome assembly process (HGAP) algorithm version 2. Genes were annotated by NCBI Prokaryotic Genome Annotation Pipeline. The generated genome sequence was 4,979,656 bp with an average G+C content of 67.45%. Calculation of ANI confirmed previous classification that strain YC-RL2 is G. alkanivorans. The sequences were searched against KEGG and COG databases; 3132 CDSs were assigned to COG families and 1808 CDSs were predicted to be involved in 111 pathways. 95 of the KEGG annotated genes were predicted to be involved in the degradation of xenobiotics. A phthalate degradation operon could not be identified in the genome indicating that strain YC-RL2 possesses a novel way of phthalate degradation. A total of 203 and 22 CDSs were annotated as esterase/hydrolase and dioxygenase genes respectively. A total of 53 biosynthetic gene clusters (BGCs) were predicted by antiSMASH (antibiotics & Secondary Metabolite Analysis Shell) bacterial version 4.0. The genome also contained putative genes for heavy metal metabolism. The strain could tolerate 1 mM of Cd2+, Co2+, Cu2+, Ni2+, Zn2+, Mn2+ and Pb2+ ions. These results show that strain YC-RL2 has a great potential to degrade various xenobiotics in different environments and will provide a rich genetic resource for further biotechnological and remediation studies.

Excellent Degradation Performance of a Versatile Phthalic Acid Esters-Degrading Bacterium and Catalytic Mechanism of Monoalkyl Phthalate Hydrolase.

Despites lots of characterized microorganisms that are capable of degrading phthalic acid esters (PAEs), there are few isolated strains with high activity towards PAEs under a broad range of environmental conditions. In this study, Gordonia sp. YC-JH1 had advantages over its counterparts in terms of di(2-ethylhexyl) phthalate (DEHP) degradation performance. It possessed an excellent degradation ability in the range of 20⁻50 °C, pH 5.0⁻12.0, or 0⁻8% NaCl with the optimal degradation condition 40 °C and pH 10.0. Therefore, strain YC-JH1 appeared suitable for bioremediation application at various conditions. Metabolites analysis revealed that DEHP was sequentially hydrolyzed by strain YC-JH1 to mono(2-ethylhexyl) phthalate (MEHP) and phthalic acid (PA). The hydrolase MphG1 from strain YC-JH1 hydrolyzed monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP), mono-n-hexyl phthalate (MHP), and MEHP to PA. According to molecular docking and molecular dynamics simulation between MphG1 and monoalkyl phthalates (MAPs), some key residues were detected, including the catalytic triad (S125-H291-D259) and the residues R126 and F54 potentially binding substrates. The mutation of these residues accounted for the reduced activity. Together, the mechanism of MphG1 catalyzing MAPs was elucidated, and would shed insights into catalytic mechanism of more hydrolases.

Biodegradation of Di-(2-ethylhexyl) Phthalate by Rhodococcus ruber YC-YT1 in Contaminated Water and Soil.

Di-(2-ethylehxyl) phthalate (DEHP) is one of the most broadly representative phthalic acid esters (PAEs) used as a plasticizer in polyvinyl chloride (PVC) production, and is considered to be an endocrine-disrupting chemical. DEHP and its monoester metabolites are responsible for adverse effects on human health. An efficient DEHP-degrading bacterial strain Rhodococcus ruber YC-YT1, with super salt tolerance (0⁻12% NaCl), is the first DEHP-degrader isolated from marine plastic debris found in coastal saline seawater. Strain YC-YT1 completely degraded 100 mg/L DEHP within three days (pH 7.0, 30 °C). According to high-performance liquid chromatography⁻mass spectrometry (HPLC-MS) analysis, DEHP was transformed by strain YC-YT1 into phthalate (PA) via mono (2-ethylehxyl) phthalate (MEHP), then PA was used for cell growth. Furthermore, YC-YT1 metabolized initial concentrations of DEHP ranging from 0.5 to 1000 mg/L. Especially, YC-YT1 degraded up to 60% of the 0.5 mg/L initial DEHP concentration. Moreover, compared with previous reports, strain YC-YT1 had the largest substrate spectrum, degrading up to 13 kinds of PAEs as well as diphenyl, p-nitrophenol, PA, benzoic acid, phenol, protocatechuic acid, salicylic acid, catechol, and 1,2,3,3-tetrachlorobenzene. The excellent environmental adaptability of strain YC-YT1 contributed to its ability to adjust its cell surface hydrophobicity (CSH) so that 79.7⁻95.9% of DEHP-contaminated agricultural soil, river water, coastal sediment, and coastal seawater were remedied. These results demonstrate that R. ruber YC-YT1 has vast potential to bioremediate various DEHP-contaminated environments, especially in saline environments.

Complete genome sequence of Gordonia sp. YC-JH1, a bacterium efficiently degrading a wide range of phthalic acid esters.

Phthalic acid esters (PAEs) are a family of recalcitrant pollutants mainly used as plasticizer. The strain Gordonia sp.YC-JH1, isolated from petroleum-contaminated soil, is capable of efficiently degrading a wide range of PAEs. In order to pertinently investigate the genetic mechanism of PAEs catabolism by strain YC-JH1, its complete genome sequencing has been performed by SMRT sequencing technology. The genome comprises a circular chromosome and a plasmid with a size of 4,101,557 bp and 91,767 bp respectively. Based on the genome sequence, 3563 protein-coding genes are predicted, of which the genes responsible for PAEs degradation are identified, including the two genes of PAEs hydrolase and the gene clusters for phthalic acid and protocatechuic acid degradation. The genome information provides genomic basis of PAEs degradation to allow the complete metabolism of PAEs. The wide substrate spectrum and its genetic basis of this strain should expand its application potential for environments bioremediation, provide novel gene resources involved in PAEs degradation for biotechnology and gene engineering, and contribute to shed light on the mechanism of PAEs metabolism.

A novel chlorpyrifos hydrolase CPD from Paracoccus sp. TRP: molecular cloning, characterization and catalytic mechanism

Background: Biodegradation is a reliable approach for efficiently eliminating persistent pollutants such as chlorpyrifos. Despite many bacteria or fungi isolated from contaminated environment and capable of degrading chlorpyrifos, limited enzymes responsible for its degradation have been identified, let alone the catalytic mechanism of the enzymes. Results: In present study, the gene cpd encoding a chlorpyrifos hydrolase was cloned by analysis of genomic sequence of Paracoccus sp. TRP. Phylogenetic analysis and BLAST indicated that CPD was a novel member of organophosphate hydrolases. The purified CPD enzyme, with conserved catalytic triad (Ser155-Asp251-His281) and motif Gly-Asp-Ser-Ala-Gly, was significantly inhibited by PMSF, a serine modifier. Molecular docking between CPD and chlorpyrifos showed that Ser155 was adjacent to chlorpyrifos, which indicated that Ser155 may be the active amino acid involved in chlorpyrifos degradation. This speculation was confirmed by site-directed mutagenesis of Ser155Ala accounting for the decreased activity of CPD towards chlorpyrifos. According to the key role of Ser155 in chlorpyrifos degradation and molecular docking conformation, the nucleophilic catalytic mechanism for chlorpyrifos degradation by CPD was proposed. Conclusion: The novel enzyme CPD was capable of hydrolyze chlorpyrifos and Ser155 played key role during degradation of chlorpyrifos.

Complete genome sequence of a phthalic acid esters degrading Mycobacterium sp. YC-RL4

ABSTRACT Mycobacterium sp. YC-RL4 is capable of utilizing a broad range of phthalic acid esters (PAEs) as sole source of carbon and energy for growth. The preliminary studies demonstrated its high degrading efficiency and good performance during the bioprocess with environmental samples. Here, we present the complete genome of Mycobacterium sp. YC-RL4, which consists of one circular chromosome (5,801,417 bp) and one plasmid (252,568 bp). The genomic analysis and gene annotation were performed and many potential genes responsible for the biodegradation of PAEs were identified from the genome. These results may advance the investigation of bioremediation of PAEs-contaminated environments by strain YC-RL4.

Complete genome sequence of a phthalic acid esters degrading Mycobacterium sp. YC-RL4.

Mycobacterium sp. YC-RL4 is capable of utilizing a broad range of phthalic acid esters (PAEs) as sole source of carbon and energy for growth. The preliminary studies demonstrated its high degrading efficiency and good performance during the bioprocess with environmental samples. Here, we present the complete genome of Mycobacterium sp. YC-RL4, which consists of one circular chromosome (5,801,417bp) and one plasmid (252,568bp). The genomic analysis and gene annotation were performed and many potential genes responsible for the biodegradation of PAEs were identified from the genome. These results may advance the investigation of bioremediation of PAEs-contaminated environments by strain YC-RL4.

Degradation of Di(2-Ethylhexyl) Phthalate by a Novel Gordonia alkanivorans Strain YC-RL2.

One bacterial strain, YC-RL2, isolated from petroleum-contaminated soil, could utilize environmental hormone Di(2-Ethylhexyl) phthalate (DEHP) as a sole carbon source for growth. Strain YC-RL2 was identified as Gordonia alkanivorans by 16S rRNA gene analysis and Biolog tests. The effects of environmental factors which might affect the degrading process were optimized at 30 °C and pH 8.0. Strain YC-RL2 showed superior halotolerance and could tolerate up to 0-5% NaCl in trace element medium supplemented with DEHP, although the DEHP degradation rates slowed as NaCl concentration increased. It also showed an outstanding performance in a wide range of pH (6.0-11.0). Meanwhile, strain YC-RL2 was able to withstand high concentrations of DEHP (from 100 to 800 mg/L), and the degradation rates were all above 94%. The DEHP intermediates were detected by HPLC-MS, and the degradation pathway was deduced tentatively. DEHP was transformed into phthalic acid (PA) via mono (2-ethylhexyl) phthalate (MEHP), and PA was further utilized for growth via benzoic acid (BA). The enzyme expected to catalyze the hydrolysis of MEHP to PA was identified from strain YC-RL2. Further investigation found that the enzyme could catalyze the transformation of a wide range of monoalkyl phthalates to PA. This study is the first report about species G. alkanivorans which could degrade several kinds of phthalic acid esters (PAEs), and indicates its application potential for bioremediation of PAE-polluted sites.