[Determination of the species origin and thrombin-like enzyme content of Bothrops atrox venom by ultra-high performance liquid chromatography-tandem mass spectrometry based on marker peptide].

Snake venom thrombin drugs are hemostatic drugs prepared from Agkistrodon halys venom, and the main active ingredients are snake venom thrombin-like enzymes (svTLEs). The svTLEs derived from different snake species differ in their structures, hemostatic mechanisms, and pharmacological effects. Therefore, accurate identification of the source of snake venom species and determination of the svTLE content are essential to ensure the quality of these products. Based on proteomics technology, the marker peptides of svTLEs from Bothrops atrox were screened with species specificity for the first time in this study, and an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for species identification and determination of the svTLE content of Bothrops atrox was established. After reductive alkylation and trypsin enzymolysis of the purified svTLE from Bothrops atrox, enzymatic peptide fragments were obtained and determined by easy-nano liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry (Nano LC-Q-Exactive-MS). The mass spectrum data were analyzed by Proteome Discoverer 2.2 software. The maker peptide "EAYNGLPAK", which characterized the svTLE from Bothrops atrox, was finally screened and validated by comparison of the basic local alignment search tool (BLAST) with the NCBI and UniProt databases. For the marker peptide, the enzymolysis temperature, enzymolysis time and amount of enzyme for the sample preparation were optimized. The optimized enzymolysis conditions were as follows: enzymolysis temperature, 37 ℃; enzymolysis time, 4 h; and amount of enzyme, 10 µL. A qualitative and quantitative detection method based on UHPLC-MS/MS was established by optimizing the chromatographic and mass spectrometric conditions. Accordingly, 20 mg of the evenly mixed sample was weighed and placed in a 100 mL volumetric flask. Then, 25 mmol/L ammonium bicarbonate solution was added to dissolve the sample, and the solution was diluted to the scale. Precisely 1.00 mL of the solution was extracted; subsequently, addition of 10 µL trypsin solution was added, followed by shaking, and the mixture was placed in an incubator for 4 h to induce enzymolysis at a constant temperature of 37 ℃. The mixture was subsequently removed from the incubator, cooled to ambient temperature, centrifuged at 12000 r/min for 10 min, and analyzed by LC-MS. Separation was performed on the UPLC system with a Thermo Hypersil GOLD C18 column (100 mm×2.1 mm, 3.0 µm) under the gradient elution of acetonitrile containing 0.1% (v/v) acetic acid and water containing 0.1%(v/v) acetic acid, at a flow rate of 0.3 mL/min, column temperature of 30 ℃, and injection volume of 2 µL. The maker peptides were determined in the electrospray positive ionization (ESI+) and multiple reaction monitoring (MRM) modes using the external standard curve method. The detection ions were m/z 481.9> 315.2 and 481.9> 485.2. There was a good linear relationship between the mass concentration of the marker peptide and the chromatographic peak area in the range of 2.5-30 ng/mL, and the correlation coefficient (r) was 0.9996, The limit of detection (S/N=3) and limit of quantification (S/N=10) were 2.5 mg/kg and 6.25 mg/kg, respectively. At spiked levels of 40, 80, and 120 mg/kg, the recoveries of the marker peptides were 95.5%-101.9%, while the relative standard deviations (RSDs) of the results for parallel analyses at various spiked levels were 1.1%-3.2%. The developed method is simple, rapid, sensitive, and specific, and it can be used for the identification of Bothrops atrox venom species and determination of the svTLE content. The findings of this study would help ensure the quality of hemocoagulase products from the relevant source and provide a reference for the quality control of other snake venom products.

[Determination of dimethyl sulfate genotoxic impurities in tertiary amine drugs by ultra-high performance liquid chromatography-tandem mass spectrometry].

Dimethyl sulfate is an important chemical raw material that is widely used in the synthesis of drugs, dyes, spices, and pesticides. The highly toxic and corrosive dimethyl sulfate residue in medicines is harmful to the human body, and hence, the residue level should be strictly controlled. Traditional detection methods use high-purity acetonitrile and anhydrous as the solvents, which limits the choice of detection solvents and degrades the versatility and accuracy of detection. Therefore, a simple and accurate method for the determination of dimethyl sulfate residues is urgently needed. Dimethyl sulfate is usually detected by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with pyridine as the methylation substrate. In this study, a new method for the detection of dimethyl sulfate was established using tertiary amines such as aminophenazone, which has many advantages over pyridine, as the methylation substrate. For example, the hybrid orbital and electron cloud of the N atom are different, resulting in stronger nucleophilicity of aminophenazone. High temperatures that are detrimental to the stability of dimethyl sulfate are not required when using aminophenazone, and the aliphatic quaternary ammonium salt product is more stable, with good stability, low interference, good ionization properties, and high response. The separation was performed on a Waters Atlantis HILIC C18 column (100 mm×2.1 mm, 3.0 µm) using a mobile phase consisting of 10 mmol/L ammonium acetate solution-0.1% formic acid methanol solution (50∶50, v/v) at a flow rate of 0.3 mL/min. The column temperature was set at 40 ℃, and the sample size was 1 µL. Dimethyl sulfate was determined in the electrospray positive ionization (ESI+) and multiple reaction monitoring (MRM) modes. Dimethyl sulfate showed good linear relationships within the range of 0.9935 to 7.9480 ng/mL (r=0.9997). The limit of detection and limit of quantification for dimethyl sulfate were 0.50 ng/mL and 1.15 ng/mL, respectively. The recoveries (n=3)of dimethyl sulfate were 94.9% to 106.4%. The relative standard deviations (RSDs) were 1.44% to 5.51%. The RSD of the methylated aminophenazone peak area was 4.32%, indicating good stability of the reaction product. Dimethyl sulfate genotoxic impurities were not detected in 9 batches of aminophenazone, caffeine, and tegafur samples, which indicated that the drug manufacturers paid attention to the control of these impurities. The proposed method is advantageous over the existing techniques in terms of the better ion peak shape and higher molecular weight, without interference from other fragments. The method is specific, sensitive, simple, rapid, and accurate, and it can be used for the determination of dimethyl sulfate genotoxic impurities in aminophenazone and other medicines.

A Species-Specific Strategy for the Identification of Hemocoagulase Agkistrodon halys pallas Based on LC-MS/MS-MRM.

Hemocoagulase Agkistrodon halys pallas is a complex mixture composed of snake venom thrombin-like enzymes (svTLEs) and small amounts of thrombokinase-like enzymes. It has been widely used as a hemostatic with rapidly growing marketing due to its advantage of localized clotting fibrinogen other than systemic coagulation. However, svTLEs from different species have various structures, functions, and hemostatic mechanisms. To ensure the efficacy and safety of Hemocoagulase Agkistrodon halys pallas, an exclusive and sensitive method has been developed to identify specific marker peptides based on liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (LC-MS/MS-MRM) mode. By combining transcriptomics and proteomics, a series of species-specific peptides of Agkistrodon halys pallas were predicted and examined by LC-MS/MS. After reduction, alkylation, and tryptic digestion were performed on Hemocoagulase Agkistrodon halys pallas, a target peptide TLCAGVMEGGIDTCNR was analyzed by LC-MS/MS-MRM. It offers a new and effective approach for the quality control of Hemocoagulase Agkistrodon halys pallas products. This method is superior to the current assays in terms of sensitivity, specificity, precision, accuracy, and throughput. The strategy can also be applied in studying other important protein-based medicines.

[Determination of donkey skin ingredients in Asini Corii Colla by ultra-high performance liquid chromatography-tandem mass spectrometry].

In recent years, due to the shortage of donkey skin resources, the price of Asini Corii Colla has seen a rapid increase. Consequently, fake gelatin prepared from horse, mules, pig, and cow skin has appeared in the market, resulting in unreliable quality of Asini Corii Colla. Therefore, there is an urgent need to develop an efficient and accurate method for improving the quality of Asini Corii Colla. Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to determine the donkey skin components in Asini Corii Colla. Accordingly, 0. l g of the evenly mixed sample was weighed and placed in a 50 mL volumetric flask; then, 1% ammonium bicarbonate solution was added to dissolve the sample, and the solution was diluted to the scale. Precisely 1.00 mL of the solution was extracted into a 5 mL volumetric flask, followed by the addition of 1.0 mL trypsin solution and 100 µL mixed internal standard working solution. This mixture was diluted to the scale using 1% ammonium bicarbonate solution, shaken, and placed in an incubator for 16 h to induce enzymolysis at a constant temperature of 37 ℃. The mixture was subsequently removed from the incubator, cooled to ambient temperature, filtered through a 0.22 µm membrane, and analyzed by LC-MS. Separation was performed on an UPLC system with a BEH C18 column (100 mm×2.1 mm, 2.5 µm) under gradient elution using acetonitrile containing 0.1% (v/v) formic acid (B) and water containing 0.1% (v/v) formic acid (A) as the mobile phases at a flow rate of 0.3 mL/min. The column temperature was 30 ℃, and the sample size was 2 µL. The gradient elution conditions are: 0-1 min, 10%B; 1-5 min, 10%B-30%B; 5-5.1 min, 30%B-70%B; 5.1-7 min, 70%B; 7-7.1 min, 70%B-10%B; 7.1-10 min, 10%B. The marker peptides were determined in positive electrospray ionization (ESI +) and multiple reaction monitoring (MRM) modes using the isotopic internal standard method. The optimized enzymolysis conditions were as follows: enzymolysis temperature, 37 ℃; enzymolysis time, 16 h; and amount of enzyme, 1 mL. The two marker peptides showed good linearities in the range of 50 to 1250 mg/L; the correlation coefficients (r) were greater than 0.996, and the limits of quantitation (S/N=10) were 20 mg/kg. At spiked levels of 300 mg/kg, 600 mg/kg, and 900 mg/kg, the average recovery ratios of the two marker peptides were 103.2% to 108.3%, while the relative standard deviations (RSDs) of 1.0%-3.0%. This method was favorable for testing actual samples. Asini Corii Colla from 29 production companies was detected by this method, and the sum contents of the two marker peptides was different because the production process and raw materials were different. The sum contents of the samples were 0.096% to 0.180% with an average of 0.151%. The developed method is simple, reliable, and reproducible, and it is suitable for detecting the donkey hide components Asini Corii Colla.

[Determination of the ingredients of horse, ox, sheep, pig, camel and deer skin in colla corii asini by ultra performance liquid chromatography-tandem mass spectrometry].

A method was established for detecting the ingredients of horse, ox, sheep, pig, camel and deer skin in colla corii asini using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).Theoretical marker peptides were selected by comparing the collagen sequences in the skins of donkeys, horses, oxen, sheep, pigs, camels, and deer.Marker peptides were identified by protease cutting techniques and high resolution mass spectrometry and then analyzed by UPLC-MS/MS.The separation was performed on a UPLC system with a BEH C18 column via the gradient elution of acetonitrile containing 0.1%(v/v) acetic acid and water containing 0.1%(v/v) acetic acid.The marker peptides were identified in the modes of electrospray positive ionization (ESI+) and multiple reaction monitoring (MRM).The marker peptides for the horse, ox, sheep, pig and camel skin were detected in 15 samples.The method is simple, highly reproducible, and suitable for the identification of mixed skin ingredients in colla corii asini.It has been successfully used to detect the authenticity of colla corii asini.

Species-specific identification of collagen components in Colla corii asini using a nano-liquid chromatography tandem mass spectrometry proteomics approach.

Colla corii asini (CCA) is a protein-based traditional Chinese medicine made from donkey skins. Because it has the ability to nourish blood, its demand is increasing rapidly. The shortage of donkey skins increases the risk of the adulteration of CCA products with other animal skins. To ensure the drug efficacy and safety of CCA products, a proteomics technique was applied to reveal proteins in the skins of donkey, horse, cattle, and pig. Species-specific peptides for each animal species were predicted using bioinformatics, and their presence in the skins and gelatin samples was examined by nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS). One unique marker peptide for each animal species was selected to develop an LC-MS/MS multiple reaction monitoring method. The capability of this method to identify donkey, horse, cattle, and pig materials was demonstrated by analyzing in-house-made donkey gelatins containing different amounts of other animal skins and commercial CCA products. The adulteration of non-donkey species could be sensitively detected at a low level of 0.5%. Hybrid animals, such as mules and hinnies, were also differentiated from donkeys. We provide a practical tool for the quality control of CCA products. The strategy can also be used to study other important traditional Chinese medicines which contain animal proteins.

Improving the productivity of S-adenosyl-l-methionine by metabolic engineering in an industrial Saccharomyces cerevisiae strain.

S-Adenosyl-l-methionine (SAM) is an important metabolite having prominent roles in treating various diseases. In order to improve the production of SAM, the regulation of three metabolic pathways involved in SAM biosynthesis were investigated in an industrial yeast strain ZJU001. GLC3 encoded glycogen-branching enzyme (GBE), SPE2 encoded SAM decarboxylase, as well as ERG4 and ERG6 encoded key enzymes in ergosterol biosynthesis, were knocked out in ZJU001 accordingly. The results indicated that blocking of either glycogen pathway or SAM decarboxylation pathway could improve the SAM accumulation significantly in ZJU001, while single disruption of either ERG4 or ERG6 gene had no obvious effect on SAM production. Moreover, the double mutant ZJU001-GS with deletion of both GLC3 and SPE2 genes was also constructed, which showed further improvement of SAM accumulation. Finally, SAM2 was overexpressed in ZJU001-GS to give the best SAM-producing recombinant strain ZJU001-GS-SAM2, in which 12.47g/L SAM was produced by following our developed pseudo-exponential fed-batch cultivation strategy, about 81.0% increase comparing to its parent strain ZJU001. The present work laid a solid base for large-scale SAM production with the industrial Saccharomyces cerevisiae strain.

The Improvement of SAM Accumulation by Integrating the Endogenous Methionine Adenosyltransferase Gene SAM2 in Genome of the Industrial Saccharomyces cerevisiae Strain.

S-Adenosyl-L-methionine (SAM) plays important roles in trans-methylation, trans-sulfuration, and polyamine synthesis in all living cells, and it is also an effective cure for liver disease, depressive syndromes, and osteoarthritis. The increased demands of SAM in pharmaceuticals industry have aroused lots of attempts to improve its production. In this study, a multiple-copy integrative plasmid pYMIKP-SAM2 was introduced into the chromosome of wild-type Saccharomyces cerevisiae strain ZJU001 to construct the recombined strain R1-ZJU001. Further studies showed that the recombinant yeast exhibited higher enzymatic activity of methionine adenosyltransferase and improved its SAM biosynthesis. With a three-phase fed-batch strategy in 15-liter bench-top fermentor, 8.81 g/L SAM was achieved after 52 h cultivation of R1-ZJU001, about 27.1 % increase over its parent strain ZJU001, whereas the SAM content was also improved from 64.6 mg/g DCW to 91.0 mg/g DCW. Our results shall provide insights into the metabolic engineering of SAM pathway in yeast for improved productivity of SAM and subsequent industrial applications.

Efficient Production of Hydroxylated Human-Like Collagen Via the Co-Expression of Three Key Genes in Escherichia coli Origami (DE3).

Mature collagen is abundant in human bodies and very valuable for a range of industrial and medical applications. The biosynthesis of mature collagen requires post-translational modifications to increase the stability of collagen triple helix structure. By co-expressing the human-like collagen (HLC) gene with human prolyl 4-hydroxylase (P4H) and D-arabinono-1, 4-lactone oxidase (ALO) in Escherichia coli, we have constructed a prokaryotic expression system to produce the hydroxylated HLC. Then, five different media, as well as the induction conditions were investigated with regard to the soluble expression of such protein. The results indicated that the highest soluble expression level of target HLC obtained in shaking flasks was 49.55 ± 0.36 mg/L, when recombinant cells were grew in MBL medium and induced by 0.1 mM IPTG at the middle stage of exponential growth phase. By adopting the glucose feeding strategy, the expression level of target HLC can be improved up to 260 mg/L in a 10 L bench-top fermentor. Further, HPLC analyses revealed that more than 10 % of proline residues in purified HLC were successfully hydroxylated. The present work has provided a solid base for the large-scale production of hydroxylated HLC in E. coli.

Cloning of a novel arylamidase gene from Paracoccus sp. strain FLN-7 that hydrolyzes amide pesticides.

The bacterial isolate Paracoccus sp. strain FLN-7 hydrolyzes amide pesticides such as diflubenzuron, propanil, chlorpropham, and dimethoate through amide bond cleavage. A gene, ampA, encoding a novel arylamidase that catalyzes the amide bond cleavage in the amide pesticides was cloned from the strain. ampA contains a 1,395-bp open reading frame that encodes a 465-amino-acid protein. AmpA was expressed in Escherichia coli BL21 and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. AmpA is a homodimer with an isoelectric point of 5.4. AmpA displays maximum enzymatic activity at 40°C and a pH of between 7.5 and 8.0, and it is very stable at pHs ranging from 5.5 to 10.0 and at temperatures up to 50°C. AmpA efficiently hydrolyzes a variety of secondary amine compounds such as propanil, 4-acetaminophenol, propham, chlorpropham, dimethoate, and omethoate. The most suitable substrate is propanil, with K(m) and k(cat) values of 29.5 µM and 49.2 s(-1), respectively. The benzoylurea insecticides (diflubenzuron and hexaflumuron) are also hydrolyzed but at low efficiencies. No cofactor is needed for the hydrolysis activity. AmpA shares low identities with reported arylamidases (less than 23%), forms a distinct lineage from closely related arylamidases in the phylogenetic tree, and has different biochemical characteristics and catalytic kinetics with related arylamidases. The results in the present study suggest that AmpA is a good candidate for the study of the mechanism for amide pesticide hydrolysis, genetic engineering of amide herbicide-resistant crops, and bioremediation of amide pesticide-contaminated environments.

SulE, a sulfonylurea herbicide de-esterification esterase from Hansschlegelia zhihuaiae S113.

De-esterification is an important degradation or detoxification mechanism of sulfonylurea herbicide in microbes and plants. However, the biochemical and molecular mechanisms of sulfonylurea herbicide de-esterification are still unknown. In this study, a novel esterase gene, sulE, responsible for sulfonylurea herbicide de-esterification, was cloned from Hansschlegelia zhihuaiae S113. The gene contained an open reading frame of 1,194 bp, and a putative signal peptide at the N terminal was identified with a predicted cleavage site between Ala37 and Glu38, resulting in a 361-residue mature protein. SulE minus the signal peptide was synthesized in Escherichia coli BL21 and purified to homogeneity. SulE catalyzed the de-esterification of a variety of sulfonylurea herbicides that gave rise to the corresponding herbicidally inactive parent acid and exhibited the highest catalytic efficiency toward thifensulfuron-methyl. SulE was a dimer without the requirement of a cofactor. The activity of the enzyme was completely inhibited by Ag(+), Cd(2+), Zn(2+), methamidophos, and sodium dodecyl sulfate. A sulE-disrupted mutant strain, ΔsulE, was constructed by insertion mutation. ΔsulE lost the de-esterification ability and was more sensitive to the herbicides than the wild type of strain S113, suggesting that sulE played a vital role in the sulfonylurea herbicide resistance of the strain. The transfer of sulE into Saccharomyces cerevisiae BY4741 conferred on it the ability to de-esterify sulfonylurea herbicides and increased its resistance to the herbicides. This study has provided an excellent candidate for the mechanistic study of sulfonylurea herbicide metabolism and detoxification through de-esterification, construction of sulfonylurea herbicide-resistant transgenic crops, and bioremediation of sulfonylurea herbicide-contaminated environments.

Catellibacterium nanjingense sp. nov., a propanil-degrading bacterium isolated from activated sludge, and emended description of the genus Catellibacterium.

A novel facultatively anaerobic, non-spore-forming, non-motile, catalase- and oxidase-positive, Gram-negative and rod-shaped bacterial strain, designated Y12(T), was isolated from activated sludge of a wastewater bio-treatment facility. The strain was able to degrade about 90% of added propanil (100 mg l(-1)) within 3 days of incubation. Growth occurred in the presence of 0-4.5% (w/v) NaCl (optimum 0.5%), at 10-40 °C (optimum 28 °C) and at pH 5.5-10.0 (optimum pH 7.0). Vesicular internal membrane structures and photoheterotrophic growth were not observed. The major respiratory quinone was ubiquinone-10 and the major cellular fatty acid was summed feature 8 (C(18:1)ω6c and/or C(18:1)ω7c). The genomic DNA G+C content of strain Y12(T) was 63.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that strain Y12(T) was a member of the genus Catellibacterium, as it showed highest sequence similarities to Catellibacterium caeni DCA-1(T) (99.1%) and <96.0% similarities with other species of the genus Catellibacterium. Strain Y12(T) showed low DNA-DNA relatedness values with C. caeni DCA-1(T). Based on phenotypic, genotypic and phylogenetic properties, strain Y12(T) represents a novel species of the genus Catellibacterium, for which the name Catellibacterium nanjingense sp. nov. is proposed. The type strain is Y12(T) (=CCTCC AB 2010218(T) =KCTC 23298(T)). An emended description of the genus Catellibacterium is also presented.

Sphingobium jiangsuense sp. nov., a 3-phenoxybenzoic acid-degrading bacterium isolated from a wastewater treatment system.

A non-sporulating, non-motile, catalase- and oxidase-positive, Gram-negative, rod-shaped bacterial strain, designated BA-3T, was isolated from activated sludge of a wastewater treatment facility. The strain was able to degrade about 95 % of 100 mg 3-phenoxybenzoic acid l(-1) within 2 days of incubation. Growth occurred in the presence of 0-2 % (w/v) NaCl [optimum, 0.5 % (w/v) NaCl], at pH 5.5-9.0 (optimum, pH 7.0) and at 10-37 °C (optimum, 28 °C). Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain BA-3T was a member of the genus Sphingobium; it showed highest gene sequence similarity to Sphingobium qiguonii X23T (98.2 %), and similarities of <97.0 % with strains of other Sphingobium species. The polar lipid pattern, the presence of spermidine and ubiquinone Q-10, the predominance of summed feature 8 (C18:1ω6c and/or C18:1ω7c) in the cellular fatty acid profile and the DNA G+C content also supported affiliation of the isolate to the genus Sphingobium. Strain BA-3T showed low DNA-DNA relatedness values (21.3±0.8 %) with Sphingobium qiguonii X23(T). Based on phenotypic, genotypic and phylogenetic data, strain BA-3T represents a novel species of the genus Sphingobium, for which the name Sphingobium jiangsuense sp. nov. is proposed; the type strain is BA-3T (=CCTCC AB 2010217T= KCTC 23196T=KACC 16433T).

Degradation of cyhalofop-butyl (CyB) by Pseudomonas azotoformans strain QDZ-1 and cloning of a novel gene encoding CyB-hydrolyzing esterase.

Cyhalofop-butyl (CyB) is a widely used aryloxyphenoxy propanoate (AOPP) herbicide for control of grasses in rice fields. Five CyB-degrading strains were isolated from rice field soil and identified as Agromyces sp., Stenotrophomonas sp., Aquamicrobium sp., Microbacterium sp., and Pseudomonas azotoformans; the results revealed high biodiversity of CyB-degrading bacteria in rice soil. One strain, P. azotoformans QDZ-1, degraded 84.5% of 100 mg L(-1) CyB in 5 days of incubation in a flask and utilized CyB as carbon source for growth. Strain QDZ-1 could also degrade a wide range of other AOPP herbicides. An esterase gene, chbH, which hydrolyzes CyB to cyhalofop acid (CyA), was cloned from strain QDZ-1 and functionally expressed. A chbH-disrupted mutant dchbH was constructed by insertion mutation. Mutant dchbH could not degrade and utilize CyB, suggesting that chbH was the only esterase gene responsible for CyB degradation in strain QDZ-1. ChbH hydrolyzed all AOPP herbicides tested as well as permethrin. The catalytic efficiency of ChbH toward different AOPP herbicides followed the order quizalofop-P-ethyl ≈ fenoxaprop-P-ethyl > CyB ≈ fluazifop-P-butyl > diclofop-methyl ≈ haloxyfop-P-methyl; the results indicated that the chain length of the alcohol moiety strongly affected the biodegradability of the AOPP herbicides, whereas the substitutions in the aromatic ring had only a slight influence.

[Characteristics of a 3-phenoxybenzoic acid degrading-bacterium and the construction of a engineering bacterium].

A bacterium capable of utilizing 3-phenoxybenzoic acid (3-PBA) as sole carbon source was isolated from petroleum-contaminated soil. This bacterium, designated as BA3, was identified as Sphingobium sp. according to its physiological & biochemical characteristic and the similarity analysis of its 16S rDNA sequence. Strain BA3 was able to degrade 99% of 100 mg x L(-1) 3-phenoxybenzoic acid within 60 h. The optimal pH and temperature for the degradation were 7.0 and 30 degrees C, respectively. The degradation efficiency was related positively to initial inoculum size. The pyrethroid hydrolase gene (pytH) gene was amplified from the genomic DNA of Sphingobium sp. JZ-2 by PCR. Recombinant plasmids pPYTH was constructed by ligating pytH gene into the broad host vector pBBRMCS- 5. Under the help of plasmid RK600, pPYTH was transferred into Sphingobium sp. BA3 to construct engineering strain BA3-pytH; Fenpropathrin degradation experiments showed that strain JZ-2 was able to degrade only 60% of 50 mg x L(-1) fenpropathrin in 48 h while engineering strain BA3-pytH was able to degrade over 95% of 50 mg x L(-1) fenpropathrin under the same conditions. Even more, BA3-pytH could rapidly degrade 3-PBA, metabolic products of pyrethroid insecticides, eliminating the inhibition of 3-PBA to pyrethroid hydrolase. Therefore, in contrast to strain JZ-2, engineering strain BA3-pytH had more advantages in bioremediation of pyrethroid insecticides contaminated environment.

Rhodanobacter xiangquanii sp. nov., a novel anilofos-degrading bacterium isolated from a wastewater treating system.

A Gram-staining-negative, oxidase-positive, catalase-positive, non-motile, non-spore-forming, and rod-shaped bacterium, designated BJQ-6(T), was isolated from activated sludge of a waste-water treatment plant in Jiangsu Province, China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain BJQ-6(T) belonged to the genus Rhodanobacter, and shared 98.7% similarity with Rhodanobacter lindaniclasticus LMG 18385(T) and <97% similarities with other Rhodanobacter species. The major fatty acids were iso-C15:0 (17.6%), iso-C16:0 (19.3%), and Summed feature 9 (isoC17:1 ω9c and/or C16:0 10-methyl) (25.8%). The DNA G+C content of strain BJQ-6(T) was 64.8 mol%. Based on the phenotypic and phylogenetic considerations, strain BJQ-6(T) represents a novel species of the genus Rhodanobacter, for which the name Rhodanobacter xiangquanii sp. nov. is proposed. The type strain is BJQ-6(T) (=CCTCC AB 2010106(T) =KCTC 23100(T)).

Sphingobium wenxiniae sp. nov., a synthetic pyrethroid (SP)-degrading bacterium isolated from activated sludge in an SP-manufacturing wastewater treatment facility.

A synthetic pyrethroid (SP)-degrading bacterial strain, designated JZ-1(T), was isolated from activated sludge of a SP-manufacturing wastewater treatment facility and studied using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JZ-1(T) belonged to the genus Sphingobium, showing highest sequence similarities to Sphingobium faniae DSM 21829(T) (98.6 %), Sphingobium cloacae JCM 10874(T) (98.5 %), Sphingobium vermicomposti DSM 21299(T) (97.4 %) and Sphingobium ummariense CCM 7431(T) (96.9 %). The polar lipid pattern, the presence of spermidine and ubiquinone Q-10, the predominance of the cellular fatty acids C(18 : 1)ω7c, C(19 : 0) cyclo ω8c, 11 methyl C(18 : 1)ω7c, C(16 : 0) and C(14 : 0) 2-OH, and the G+C content of the genomic DNA also supported the affiliation of the strain with the genus Sphingobium. Strain JZ-1(T) showed low DNA-DNA relatedness values with S. faniae DSM 21829(T) (30.2 %), S. cloacae JCM 10874(T) (23.3 %), S. vermicomposti DSM 21299(T) (10.9 %) and S. ummariense CCM 7431(T) (7.9 %). Based on its phylogenetic position and its phenotypic and genotypic properties, strain JZ-1(T) represents a novel species of the genus Sphingobium, for which the name Sphingobium wenxiniae sp. nov. is proposed. The type strain is JZ-1(T) ( = CGMCC 1.7748(T)  = DSM 21828(T)).

Methylopila jiangsuensis sp. nov., an aerobic, facultatively methylotrophic bacterium.

The taxonomic status was determined of an aerobic, facultatively methylotrophic strain, JZL-4(T), isolated from activated sludge. The cells were gram-negative, asporogenous, colourless, motile, short rods. The strain utilized methanol, methylamine, formate and a variety of polycarbon compounds, but not methane, dichloromethane or CO(2)/H(2), as carbon and energy sources. C(1) compounds were assimilated via the isocitrate lyase-negative serine pathway. Optimal growth occurred at 30 °C, pH 6.5-7.5 and 0.5 % (w/v) NaCl. The major cellular fatty acids were C(18 : 1)ω7c and C(18 : 0). The major phospholipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and phosphatidylmonomethylethanolamine (PME); PME, the main phospholipid of strain JZL-4(T), was absent or present in only minor amounts in Methylopila capsulata IM1(T), Methylopila helvetica DM9(T) and Albibacter methylovorans DM10(T). The major ubiquinone was Q-10. The DNA G+C content of strain JZL-4(T) was 70.4 mol% (T(m)). Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain showed high sequence similarities to M. capsulata IM1(T) (97.2 %), A. methylovorans DM10(T) (94.9 %) and M. helvetica DM9(T) (94.1 %), and showed less than 94 % similarity to strains of other species with validly published names. Strain JZL-4(T) had a low level of DNA-DNA relatedness (34 %) with M. capsulata IM1(T). On the basis of phenotypic, genetic and phylogenetic data, strain JZL-4(T) is proposed to represent a novel species of the genus Methylopila, with the name Methylopila jiangsuensis sp. nov. The type strain is strain JZL-4(T) ( = ACCC 05406(T)  = DSM 22718(T)  = VKM B-2555(T)).

Flavobacterium haoranii sp. nov., a cypermethrin-degrading bacterium isolated from a wastewater treatment system.

A Gram-negative, non-spore-forming, yellow-pigmented bacterium, strain LQY-7(T), was isolated from activated sludge treating synthetic pyrethroid-manufacturing wastewater. The taxonomic status of the strain was determined using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain LQY-7(T) was a member of the genus Flavobacterium but had low similarities with other species of this genus (95.0 % similarity with Flavobacterium indicum GPTSA100-9(T) and <94 % similarities with other Flavobacterium species). On the basis of phenotypic, genetic and phylogenetic data, strain LQY-7(T) should be classified as a representative of a novel species of the genus Flavobacterium, for which the name Flavobacterium haoranii sp. nov. is proposed; the type strain is LQY-7(T) (=ACCC 05409(T) =KCTC 23008(T)).

Sphingobium faniae sp. nov., a pyrethroid-degrading bacterium isolated from activated sludge treating wastewater from pyrethroid manufacture.

A bacterial strain capable of degrading pyrethroid, designated JZ-2(T), was isolated from activated sludge treating pyrethroid-contaminated wastewater. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JZ-2(T) belongs to the genus Sphingobium. It showed the highest levels of 16S rRNA gene sequence similarity to Sphingobium cloacae JCM 10874(T) (98.3 %) and Sphingobium ummariense CCM 7431(T) (97.1 %), and 94.8-96.9 % similarity to the type strains of other members of the genus Sphingobium. Strain JZ-2(T) contained C(18 : 1)omega7c as the predominant fatty acid, C(14 : 0) 2-OH as the major 2-hydroxy fatty acid, ubiquinone Q-10 as the main respiratory quinone, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylmonomethylethanolamine, phosphatidylethanolamine and two sphingoglycolipids as the predominant polar lipids and spermidine as the major polyamine. DNA-DNA hybridization results showed that strain JZ-2(T) had low genomic relatedness with S. cloacae JCM 10874(T) (34 %) and S. ummariense CCM 7431(T) (23 %). Based on the phenotypic, genotypic and phylogenetic data presented, strain JZ-2(T) is considered to represent a novel species of the genus Sphingobium, for which the name Sphingobium faniae sp. nov. is proposed. The type strain is JZ-2(T) (=CGMCC 1.7749(T) =DSM 21829(T)).